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4,630 | https://bio-protocol.org/en/bpdetail?id=4630&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
A Tumor-admixture Model to Interrogate Immune Cell–dependent Tumorigenesis
JN Jordan T. Noe
CD Chuanlin Ding
AG Anne E. Geller
BR Beatriz E. Rendon
JY Jun Yan
RM Robert A. Mitchell
Published: Vol 13, Iss 5, Mar 5, 2023
DOI: 10.21769/BioProtoc.4630 Views: 1204
Reviewed by: Ivan ShapovalovXiaoyu Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Advances Nov 2021
Abstract
A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.
Keywords: Cancer Immunology Immunotherapy Macrophage Mouse models
Background
Evasion of anti-tumor immunity is now a well-established hallmark of cancer progression (Hanahan and Weinberg, 2011). Translation of fundamental concepts in tumor immunity has resulted in the development of immune checkpoint inhibitors that are highly efficacious in select oncological types (Xin et al., 2019; Robert, 2020). However, several types of malignancies do not respond, have intrinsic resistance, or develop secondary adaptive resistance to these therapies (Sharma et al., 2017; Jenkins et al., 2018; Schoenfeld and Hellmann, 2020). These issues have led to an outpouring of basic science and translational projects aimed at identifying additional molecular mechanisms by which cancer cells or infiltrating immune cells evade anti-tumor immunity, with the goal of developing novel immunotherapeutics (Fares et al., 2019; Karasarides et al., 2022). Here, we detail a published tumor/immune cell–admixture model (Liu et al., 2020; Noe et al., 2021), using tumor-associated macrophages as an example, that can serve as a useful tool to investigate the relative contributions of tumor-infiltrating immune cells in promoting cancer progression.
Tumor-associated macrophages (TAMs) are myeloid-derived immune cells that infiltrate into the tumor microenvironment to affect tumorigenesis (Mantovani et al., 1992; Noy and Pollard, 2014; Pan et al., 2020; Pittet et al., 2022). TAMs have both pro- and anti-tumor effector functions—their phagocytosis of cancer cells (Kamber et al., 2021), presentation of tumor-associated antigens (Asano et al., 2011), and activation of anti-tumor adaptive immunity play a critical role in the elimination of cancer cells (Modak et al., 2022). In contrast, TAMs can also promote tumorigenesis by suppressing phagocytic and antigenic innate immune responses (Barkal et al., 2019; Demaria et al., 2019; Zhou et al., 2020), as well as by inducing adaptive immune cell exhaustion (DeNardo and Ruffell, 2019; Singhal et al., 2019). Understanding the TAM-dependent contribution to tumor progression or regression is critical for identifying novel tumor immunotherapeutic strategies.
Several different tumor models have been developed to interrogate the effects of specific immune cell types in tumorigenesis. These protocols traditionally include the targeted depletion (Xiao and Jiang, 2014) or transgenic manipulation (McCubbrey et al., 2017) of the desired immune cell in the context of orthotopic or heterotopic tumors in xenograft or allograft models. The tumor-admixture model described herein is a heterotopic, allograft murine model using ex vivo differentiated/polarized bone marrow–derived macrophages (BMDMs) injected with tumor cell lines into congenic recipient mice. This admixture model has been shown to be an advantageous strategy to relatively easily determine gene-specific contributions to TAM-dependent tumor progression (Liu et al., 2020; Noe et al., 2021).
While this protocol was developed to investigate the ability of ex vivo interleukin-4 (IL-4) polarized pro-tumor TAMs to suppress anti-tumor immunity leading to enhanced tumor growth (Liu et al., 2020; Noe et al., 2021), this model can be used with other TAM phenotypes, different immune cell types, and additional experimental manipulations. A potential critique of this model includes the relative contribution of host-derived, tumor-infiltrating TAMs, which can be addressed by incorporating a targeted depletion strategy (Xiao, and Jiang, 2014). Adapting this model using anti-tumor TAMs (Liu et al., 2020; Noe et al., 2021) offers an interesting approach to understanding the mechanisms by which TAMs promote tumor regression. With rigorous optimization, other immune cell types that are critical regulators of tumorigenesis, such as neutrophils (Shaul, and Fridlender, 2019), dendritic cells (Wculek et al., 2020), or myeloid-derived suppressor cells (Veglia et al., 2021), could be adapted for use. Lastly, this model is highly amendable to the post-injection administration of small molecules or biologic inhibitors to further validate their importance as a clinically viable therapeutic (Zanoni et al., 2012; Noe and Mitchell, 2020). Altogether, this tumor-admixture model is a relatively straightforward, highly adaptable protocol to investigate immune cell–dependent cancer progression.
Materials and Reagents
0.70 μm filter
2 mL syringe
28 gauge needle
Falcon conical centrifuge tubes, 15 mL (Corning, catalog number: 352095)
Microcentrifuge tubes, 1.5 mL (Sigma Aldrich, catalog number: HS4323)
C57BL/6J mice, CD45.2+ donor (Jackson Laboratory, strain #: 000664)
B6.SJL mice, CD45.1+ congenic recipient (Jackson Laboratory, strain #: 002014)
Matrigel matrix basement membrane (Corning, catalog number: 354234)
CellStripper dissociation solution (Corning, catalog number: 25-056-CI)
Trypsin-EDTA, 0.05% (Gibco, catalog number: 25300062)
Phosphate-buffered saline (PBS) (Corning, catalog number: 21-040)
RPMI 1640 medium (Gibco, catalog number: 11-875-093)
Fetal bovine serum (FBS), heat-inactivated (Gibco, catalog number: 10-082-147)
10 cm tissue culture–treated plates (Corning, catalog number: CLS430167)
Trypan Blue solution (Gibco, catalog number: 15250061)
Lewis lung carcinoma cells (ATCC, catalog number: CRL-1642)
Note: As an example, we engrafted Lewis lung carcinoma cells; see Procedure section B for more information. Please utilize the previously described Bio-Protocol manuscripts for BMDM differentiation and culture of C57BL/6J CD45.2+ donor mice: Zanoni et al. (2012), Haag and Murthy (2021).
Equipment
Tabletop centrifuge (e.g., Kendro Sorval legend RT)
Microcentrifuge (e.g., Eppendorf refrigerated microcentrifuge 5415R)
Water bath at 37 °C (e.g., Thermo Scientific PRECISION water bath)
Incubator at 37 °C with 5% CO2 (e.g., Thermo Scientific HERACell 150i CO2 incubator)
Laminar flow hood (e.g., The Baker Company, SterilGARD class II biological safety cabinet)
Cell lifter (VWR, catalog number: 76036-006)
Digital calipers (VWR, catalog number: 62379-531)
Procedure
Preparation
Pre-warm RPMI 1640 medium, PBS, FBS, trypsin-EDTA, and CellStripper dissociation solution in a water bath set to 37 °C.
Thaw Matrigel matrix basement membrane on ice.
Tip: Matrigel can prematurely solidify at room temperature, so care must be taken to ensure it is maintained in a liquid state until immediately before use.
Make wash media: RPMI 1640 medium + 5% FBS.
Make injection solution: PBS + 5% FBS.
Tumor cell line collection
Note: Most murine tumor cell lines are likely amendable to this model, but this should be predetermined by optimization studies using B6.SJL recipient mice. On the day of injection, tumor cells should be in 10 cm tissue culture plates and in log-growth phase (approximately 5.0 × 106–7.0 × 106). Although 5.0 × 105 tumor cells per injection is our recommended baseline, this number should be optimized based upon the cell line’s in vivo proliferation rate and desired time of tumor growth.
Wash plates twice with 10 mL of PBS and aspirate PBS after each wash.
Add 3 mL of trypsin-EDTA to each plate and place in incubator for 3–5 min.
Transfer cells to fresh Falcon tubes.
Wash plate twice with 5 mL of wash media and add wash to Falcon tubes.
Centrifuge at 350 × g for 5 min and aspirate supernatant.
Resuspend cell pellet with 10 mL of wash solution.
Repeat steps B5 and B6.
Filter cell suspension through a 0.70 μm filter into a fresh Falcon tube.
Count cells in Trypan Blue solution using a cell counter or other standard method.
Centrifuge at 350 × g for 5 min and aspirate supernatant.
Resuspend cells to 4 × 106 cells/mL in the Falcon tubes and place tumor cells on ice.
Tip: The tumor cell collection can be performed simultaneously with the BMDM collection (Section C) to increase efficiency and reduce the length of time the tumor cells are kept on ice.
Bone marrow–derived macrophage (BMDM) collection
Note: BMDMs need to be derived from C57BL/6J CD45.2+ donor mice. The length of BMDM polarization prior to injection should be optimized based upon polarization method and experimental investigations (i.e., M1 vs. M2 polarization, small molecule inhibitor treatments, or transgenic manipulations). For example: we have used a transgenic model of BMDMs from C57BL/6J CD45.2+ donor mice that were polarized with IL-4 for 24 h (Noe et al., 2021). On the day of injection, BMDMs should have high viability (>90%), as determined by Trypan Blue exclusion, and 2.0 × 105 BMDMs per injection is needed.
In a laminar flow hood, wash plates twice with 10 mL of PBS and aspirate PBS after each wash.
Add 3 mL of CellStripper dissociation solution to each plate and place in the incubator for 3–5 min.
Gently lift BMDMs with a cell lifter and transfer cells to Falcon tubes.
Tip: Avoid aggressive scraping of BMDMs or using trypsin instead of cell stripper, as this causes reduced BMDM viability.
Wash plate twice with 5 mL of wash media and add wash to Falcon tubes.
Centrifuge at 350 × g for 5 min and aspirate supernatant.
Resuspend cell pellet with 10 mL of wash solution.
Repeat steps C5 and C6.
Filter cell suspension through a 0.70 μm filter into a fresh Falcon tube.
Count cells in Trypan Blue using a cell counter or other standard method.
Centrifuge at 350 × g for 5 min and aspirate supernatant.
Resuspend cells to 1.6 × 106 cells/mL in the Falcon tubes and place BMDMs on ice.
Tumor-BMDM admixture
Note: A limited optimization study should be performed beforehand to determine the preferred tumor cell:BMDM ratio. As a baseline, we have found that a 2.5:1 ratio provides meaningful data (Liu et al., 2020; Noe et al., 2021), but results may vary depending on tumorigenic potential, immunogenicity, and other experimental conditions. Each 2 mL tube can hold three tumor injections (1.5 mL injection + 0.5 mL slop; the required volume needed for tumor injections should be determined beforehand). For best results, the following steps should be performed immediately prior to injections. Currently, we describe a heterotopic model utilizing subcutaneous injections. In some cancer subtypes, different treatment responses occur depending on whether an orthotopic or heterotopic model is used (Erstad et al., 2018). We anticipate that this admixture model could be adapted to an orthotopic approach, but this will likely require thorough optimization beforehand.
Per 2 mL microcentrifuge tube: three injections (500 μL/injection) + 500 μL extra volume:
Mix tumor cell suspension, aliquot 500 μL to fresh microcentrifuge tube, and place on ice.
Mix BMDM suspension, aliquot 500 μL to the same microcentrifuge tube, mix well, and place on ice.
Immediately prior to injection, aliquot 1 mL Matrigel to the microcentrifuge tube, mix well, and place on ice.
Draw 500 μL of the admixture into a 2 mL syringe and place a 28 gauge needle.
Tip: Again, Matrigel can prematurely solidify at room temperature. Pre-chilling syringes on ice before drawing admixture and placing syringe on ice in between injections may be needed.
Administer a 500 μL subcutaneous injection into the flank of a B6.SJL recipient mouse.
Tip: The flank can be shaved prior to injection to increase visibility during injection or subsequent measurements. Following injection, leave the needle in place for 1–3 s to prevent leakage from the insertion site. Any significant leakage should be noted and may represent a failed injection.
Using a new syringe, repeat steps D4 and D5 for each sequential mouse.
Downstream analyses
Note: Following injections, several experimental manipulations and/or downstream analyses are possible. The effect of a small molecule inhibitor or a biologic can be interrogated through the administration of these agents at predetermined time points. Once tumor endpoints are achieved, immunophenotyping with flow cytometry is possible by obtaining the spleen, blood, and tumor through previously described protocols (Almeida et al., 2021). At a minimum, tumor growth and end-point weight should be measured to determine gross changes in tumorigenesis, which are described below.
Monitor mice for tumor outgrowth to indicate a successful injection.
Once tumors are palpable (approximately day 5–7), measure the tumor length and width every 2–3 days with external calipers.
Record the length and width to calculate the tumor volume using the following formula: (length × width2)/2.
Once the tumor endpoint is reached, euthanize mice, resect tumors, and measure weight (Figure 1).
Proceed with downstream analyses, if desired.
Figure 1. Tumor-admixture model enhances tumor outgrowth. Gross dissection of tumors from mice 16 days post-injection with Lewis lung carcinoma (LLC) cells plus M2-polarized bone marrow–derived macrophages (BMDMs) (above) or LLC cells alone (below). Size differences represent variability of tumor size within each cohort. Please refer to Noe et al. (2021) for a more comprehensive analysis of tumor growth curves and endpoint tumor weight.
Acknowledgments
This work was supported by National Institutes of Health (NIH) Predoctoral Fellowship Award F30CA232550 (to J.T.N.), NIH Research Grant R01CA186661 (to R.A.M.), NIH Research Grant GB130096P3 (to R.A.M.), and NIH Project Grant P20GM135004 (to J.Y. and R.A.M.). This protocol is derived from the original research paper (Noe et al., 2021; DOI: 10.1126/sciadv.abi860).
Competing interests
The authors declare no competing interests.
Ethics
Animals were maintained under specific pathogen-free conditions and handled in accordance with the Association for Assessment and Accreditation of Laboratory Animals Care international guidelines. The Institutional Animal Care and Use Committee at the University of Louisville approved the experiments.
References
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Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Immunology > Animal model > Mouse
Cancer Biology > Tumor immunology > Tumor formation
Cell Biology > Cell isolation and culture > Co-culture
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This protocol has been corrected. See the correction notice.
Peer-reviewed
Establishment of an in vitro Differentiation and Dedifferentiation System of Rat Schwann Cells
YZ Ying Zou
Published: Vol 13, Iss 5, Mar 5, 2023
DOI: 10.21769/BioProtoc.4631 Views: 695
Reviewed by: Gal HaimovichPhilipp Wörsdörfer Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Molecular Neurobiology Jan 2022
Abstract
In the peripheral nervous system, Schwann cells are the primary type of glia. This protocol describes an in vitro differentiation and dedifferentiation system for rat Schwann cells. These cultures and systems can be used to investigate the morphological and biochemical effects of pharmacological intervention or lentivirus-mediated gene transfer on the process of Schwann cell differentiation or dedifferentiation.
Graphical abstract
Keywords: Peripheral nerve Schwann cells Differentiation Dedifferentiation In vitro
Background
Schwann cells are the primary glial cells of the peripheral nervous system. During axonal sorting and myelination in the peripheral nerves, Schwann cells originate from neural crest cells that differentiate into mature phenotypes (Cristobal and Lee, 2022). Moreover, Schwann cells’ incredible plasticity is one of the most important characteristics following nerve damage or demyelination (Nocera and Jacob, 2020). Schwann cells undergo dedifferentiation after injury and redifferentiate to promote nerve regeneration and complete functional recovery (Jessen and Mirsky, 2019). Therefore, it is important to study Schwann cells’ differentiation and dedifferentiation status to understand their role in nerve development and injury. As reported previously, dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) induces Schwann cells to acquire a differentiated phenotype (Yamauchi et al., 2011). After stimulation with 1 mM dbcAMP, Schwann cells from rats change from bipolar or tripolar to flat within 24 h. By contrast, mouse Schwann cells retain their bipolar or tripolar morphology after dbcAMP treatment (Arthur-Farraj et al., 2011). And a previous study described in vitro assays of scalable differentiation and dedifferentiation of Schwann cell in the absence of neurons (Monje PV., 2018). Based on previous studies, we established a detailed and simplified system for differentiation and dedifferentiation of Schwann cells. In order to clearly distinguish morphological changes, we performed the differentiation and dedifferentiation assay using rat Schwann cells. In brief, the first step was to obtain Schwann cells from the spinal nerves of rats based on a previous study (Wen et al., 2017). During the second step, differentiation and dedifferentiation assays were conducted. Schwann cells were purified and passaged, and experiments were conducted on the third passage. Finally, Schwann cell status was determined by morphological examination, western blotting analysis, and immunofluorescence detection.
Materials and reagents
Cell culture dish (3.5 and 6 cm) (JET BIOFILT, catalog numbers: CD000035, TCD-010-060)
50 mL centrifuge tubes (Corning, catalog number: 430828)
15 mL centrifuge tubes (Corning, catalog number: 430790)
1.5 mL tube (JET BIOFILT, catalog number: CFT000015)
Polyvinylidene fluoride membrane (PVDF) (Bio-Rad, catalog number: 1620177)
Cell glass coverslips (diameter: 12 mm, thickness: 0.13–0.17 mm) (Fisherbrand, catalog number: FIS12-545-80)
Neonatal Sprague-Dawley (SD) rat [postnatal 1–2 days (P1–P2)]
Distilled water
75% ethanol
Phosphate buffered saline (PBS) (Gibco, catalog number: 10010023)
Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1274)
0.25% trypsin-EDTA (Gibco, catalog number: 25200072)
Fetal bovine serum (FBS) (Corning, catalog number: 35-076-CV)
DMEM/F12 (Gibco, catalog number: 11330057)
Cytosine arabinoside (Ara-C) (Sigma-Aldrich, catalog number: C1768)
Recombinant human heregulin β-1 (PeproTech, catalog number: 100-03)
Forskolin (Sigma-Aldrich, catalog number: F6886)
4% paraformaldehyde (PFA) (Biosharp, catalog number: BL539A)
Dimethyl sulfoxide (DMSO), suitable for cell culture (Beyotime, catalog number: ST038)
Dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) (Sigma-Aldrich, catalog number: D0627)
Triton X-100 (Sigma-Aldrich, catalog number: V900502)
Tween-20 (Sigma-Aldrich, catalog number: P1379)
Phalloidin (Abcam, catalog number: ab176759)
RIPA lysis buffer (FUDE Biological Technology, catalog number: FD009)
Rabbit monoclonal (EP1039Y) anti-p75 (Abcam, catalog number: ab52987)
Rabbit anti-Krox20 (Novus Biologicals, catalog number: 13491-1-AP)
Mouse monoclonal anti-c-Jun (BD Biosciences, catalog number: 610326)
Mouse monoclonal (9-9-3) anti-Sox2 (Abcam, catalog number: ab79351)
Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, catalog number: A11001)
Alexa Fluor® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, catalog number: A11011)
4’,6-diamidino-2’-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D8417)
Penicillin–streptomycin (Gibco, catalog number: 15140-122)
Omni-ECLTM Light Chemiluminescence kit (EpiZyme, catalog number: SQ201)
10% dodecyl sulfate sodium salt-polyacrylamide gel (EpiZyme, catalog number: PG112)
5% non-fat milk (Solarbio, catalog number: D8340)
Tris-buffered saline (Sigma-Aldrich, catalog number: 93318)
Horseradish peroxidase (HRP)-conjugated secondary antibody (Invitrogen, catalog numbers: 31430, 31460)
1,000× Ara-C (10 mM in distilled water, see Recipes)
1,000× dbcAMP (1,000 mM in DMSO, see Recipes)
30 mM forskolin stock solution (see Recipes)
Complete growth medium of rat Schwann cells (see Recipes)
10% FBS (see Recipes)
3% FBS (see Recipes)
1% FBS (see Recipes)
1 mM dbcAMP (see Recipes)
0.1% Triton X-100 (see Recipes)
5% gelatin (see Recipes)
PBST (see Recipes)
Blocking buffer (see Recipes)
Equipment
Pipettes (Thermo Fisher Scientific, 10, 200, and 1,000 μL)
CO2 incubator (Thermo Fisher Scientific, model: Heracell 150i)
Surgical scissors and forceps (Shenzhen RWD, catalog numbers: S14014 and F12029-09)
Spring scissors (Shenzhen RWD, catalog number: S11001)
Superfine forceps (Shenzhen RWD, catalog number: F13002)
Stereomicroscope (Jiangnan Novel, model: SZ6060)
Centrifuge (Eppendorf, model: Micro21)
Phase contrast microscope (Zeiss, model: Primovert)
Fluorescence microscope (Zeiss, model: Axio Imager A2)
Software
ImageJ (Version 1.8.0, https://imagej.en.softonic.com/)
Procedure
Preparation for primary Schwann cell cultures from neonatal rat
Prepare the culture plate: coat 3.5 cm cell culture dishes with 1 mL of PLL solution (0.1 mg/mL). Incubate overnight at 37 °C in a CO2 incubator. This step is only used to increase cell adhesion to the culture dish. Therefore, the CO2 content is not relevant at this point.
The next day, remove the PLL solution, rinse the dish surface thoroughly with distilled water, and air dry before use. Add 1.5 mL of cold PBS to each dish and place on ice.
Note: Do not use PBS to clean the dishes as this will cause salt crystallization after drying and disrupt cell adhesion.
Prepare culture media and solutions:
Medium for cell proliferation (culture medium to expand Schwann cells) (Medium I): DMEM/F12 containing 3% FBS, 3 μM forskolin, 10 ng/mL heregulin β-1, and 100 mg/mL penicillin–streptomycin.
Medium for cell starvation (Medium II): DMEM/F12 containing 1% FBS and 100 mg/mL penicillin–streptomycin.
Medium for Schwann cell differentiation (Medium III): DMEM/F12 containing 1% FBS, 100 mg/mL penicillin–streptomycin, and 1 mM dbcAMP.
Medium for Schwann cell dedifferentiation (Medium IV): DMEM/F12 containing 1% FBS and 100 mg/mL penicillin–streptomycin.
Culture of primary Schwann cells from rat spinal nerves
Note: This part refers to a previous study (Wen et al., 2017).
Prepare surgical equipment (see Figure 1).
Figure 1. Surgical equipment used in this protocol
Tissue collection: anesthetize the neonatal rat by putting it on ice for 2–3 min, sterilize them with 75% ethanol, remove the head with surgical scissors and forceps, and collect the sciatic nerves and spinal nerves with superfine forceps.
Tissue digestion: prepare one 1.5 mL tube, add 1 mL of PBS, and cool on ice. Transfer these nerves to a 1.5 mL tube and use spring scissors to cut them into 1-mm-longsegments. Then, add 1 mL of pre-warmed 0.25% trypsin-EDTA to the tube. Cap the tube and incubate at 37 °C in a CO2 incubator for 30 min with occasional shaking every 10 min.
Collect cells by centrifugation: stop digestion by adding 100 μL of FBS, then prepare a single cell suspension by gently digesting the cell sample 30 times with a 1 mL pipette. Then, centrifuge the cell suspension at 100× g for 5 min at room temperature and discard the supernatant. Resuspend the cell sediment in 200 μL of DMEM/F12 containg 10% FBS, place the cell suspension in a 3.5 cm PLL-coated culture dish, and culture the cells in a CO2 incubator for 1–2 h to ensure cell adhesion. Add 1 mL of 10% FBS to the culture plated and culture the cells for 24 h.
Note: At this time, the cells suspension is a mixture of Schwann cells and fibroblasts (see Figure 2A).
Cell purification: replace the culture medium with DMEM/F12 containing 10% FBS and 10 μM Ara-C (add 1 μL of 10 mM Ara-C stock solution per 1 mL of medium) to eliminate the fibroblasts. After 48 h, replace the culture medium with Medium I of rat Schwann cells.
Note: After purification, approximately 1 × 104 Schwann cells per rat can be obtained. When 100% confluence is reached after expansion, the number of Schwann cells per dish will be approximately 1 × 105–2 × 105 (seeFigure 2B).
Cell passage: when the culture reaches 90% confluence, wash the cells with 1 mL of PBS, discard the PBS wash, add 1 mL of 0.25% trypsin-EDTA to the cell dish, and leave at room temperature for 2 min. Observe the cells under a phase contrast microscope and gently shake the dish. When the cells begin to detach, add 1 mL of 10% FBS to stop digestion, collect the cells in a 1.5 mL tube, centrifuge at 100× g for 5 min at room temperature, discard the supernatant, and gently resuspend the cells in 1 mL complete growth medium of rat Schwann cells.
Note: Schwann cells are passaged and expanded at a ratio of 1:3–1:4, and the third passage cells are used for further experiments (see Figure 2C). At this time, cells are actively proliferating and fibroblasts are absent.
Figure 2. Purification and expansion of rat Schwann cells. A. 24 h after cell dissection, the culture should consist of Schwann cells and fibroblasts. Schwann cells have an elongated spindle-like morphology, whereas fibroblasts are very flat. B. On day 3, purified rat Schwann cells became confluent. C. After 2 days of growth in Medium I, the culture is routinely passaged to passage 3 in complete growth medium in the presence of forskolin and heregulin β-1, and the culture is filled with Schwann cells (>95% confluence) with typical bipolar spindle cell bodies.
Differentiation and dedifferentiation of Schwann cells (Figure 3)
Seed and starvation: seed 5,000 Schwann cells in a 3.5 cm dish (covered with glass coverslips, for immunofluorescence analysis) or seed 1 × 105 Schwann cells in a 6 cm dish (for western blotting analysis) in Medium I to allow a fast proliferation of rat Schwann cells. After one day, remove the complete growth medium and immediately replace it with an equal volume of Medium II overnight to adapt to the lack of mitotic stimulation.
Note: Complete growth medium (Medium I) of rat Schwann cells containing forskolin can induce Schwann cells to acquire a differentiated phenotype, and 10% FBS will induce Schwann cell proliferation but not differentiation. Therefore, before inducing Schwann cell differentiation, we starved the cells by culturing them in Medium II at low serum concentrations and without forskolin.
Differentiation: remove the starvation medium (Medium II) from each dish, replace with an equal volume of Medium III, and culture the cells in a CO2 incubator for 72 h. Before the dedifferentiation process, observe the cultures of dbcAMP-induced differentiated Schwann cells under a phase contrast microscope to confirm the expected morphological changes.
Note: After about 6 h, the rat Schwann cells began to show morphological changes. We recommended starting with cultures without forskolin and dbcAMP as controls. To extend the differentiation time of Schwann cells, simply replace the culture medium with fresh differentiation conditioned medium (Medium III).
Dedifferentiation: after 72 h, Schwann cells differentiated under the influence of dbcAMP. To induce dedifferentiation, simply change the culture medium to Medium IV, and culture the cells in a CO2 incubator for 24 h. Continue collecting or analyzing cultures the day after dbcAMP withdrawal, or as required by the experimental design.
Note: dbcAMP-induced differentiated Schwann cells does not dedifferentiate upon exposure to growth factors such as forskolin and heregulin β-1. Thus, we recommend using a medium that does not contain forskolin and heregulin β-1 throughout the differentiation and dedifferentiation experiments. Approximately 6 h after removal of dbcAMP, almost half of the rat Schwann cells return to typical bipolar or tripolar shape. You can add drugs or perform virus transfections before dedifferentiation experiment.
Figure 3. Differentiation and dedifferentiation system of rat Schwann cells. Schwann cells are cultured in Medium I for several days, then the third-passage cells are plated at an appropriate density and starved overnight in Medium II. The next day, the Schwann cells are treated with 1 mM dbcAMP, which induces morphological change and upregulation of differentiation markers, which could be examined by phase contrast microscope observation, immunofluorescence, or western blotting. After 72 h, Schwann cell dedifferentiation can be induced by simply changing the culture medium to Medium IV. Undiff: Undifferentiated; Diff: Differentiated; De-diff: Dedifferentiated.
Data analysis
Phase contrast microscopy (Figure 4A)
To identify the phenotype of Schwann cells, observe their morphology under a phase contrast microscope. Schwann cells were treated with dbcAMP to obtain a differentiated phenotype with morphological transition from an elongated spindle-like shape to a flattened shape; simple dbcAMP removal can reverse the differentiated Schwann cells into an elongated bipolar morphology.
Immunofluorescence (Figure 4B)
Cell fixation: fix cells with 4% PFA for 30 min at room temperature.
Permeabilization and blocking: penetrate cells with 0.1% Triton X-100 (see Recipe 8) for 30 min and incubate with 5% gelatin (see Recipe 9) at room temperature for 1 h.
Primary antibodies: prepare a 1:200 dilution of mouse anti-c-Jun and a 1:100 solution of rabbit anti-Krox20 in 5% gelation solution. Incubate the cells with primary antibodies at 4 °C overnight.
Secondary antibodies: prepare a 1:400 dilution of Alexa Fluor® 488 goat anti-mouse IgG (H+L) or Alexa Fluor® 568 goat anti-rabbit IgG (H+L) in PBST (see Recipe 10) secondary antibodies. Incubate cells with the corresponding secondary antibodies at room temperature for 2 h, wash cells with PBST twice, and then incubate cells with 1 μg/mL DAPI for 5 min at room temperature to stain nuclei.
Western blotting (Figure 4C, 4D)
Cells lysis: add 100 μL of RIPA lysis buffer to the cell dish, blow, and collect lysates into a 1.5 mL tube.
Protein electrophoresis: prepare 10% dodecyl sulfate sodium salt-polyacrylamide gel, pipette 10 μg of protein into each well, and run the electrophoresis using the following parameters: 80 V and 300 mA for 1.5 h. When the electrophoresis is completed, remove the gel carefully and transfer proteins to a PVDF membrane.
Incubation of primary and secondary antibodies: after blocking with blocking buffer for 2 h at room temperature, prepare a 1:800 dilution of mouse anti-Sox2 and a 1:500 solution of rabbit anti-p75 in blocking buffer, incubate the membrane in primary antibody at 4 °C overnight, and incubate Horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h.
Visualization and calculation: visualize the membrane using Omni-ECLTM Light Chemiluminescence kit and calculate protein quantity using ImageJ.
Figure 4. Identification of rat Schwann cells’ phenotype. (A) Cell morphology and cytoskeleton stained with phalloidin were observed under a microscope. (B) Immunofluorescence of c-Jun (indicates immature or dedifferentiated Schwann cells) and Krox20 (indicates mature or differentiated Schwann cells). (C, D) Western blotting analysis and data statistics of Sox2 and p75 (indicates immature or dedifferentiated Schwann cells). Undiff: Undifferentiated; Diff: Differentiated; De-diff: Dedifferentiated.
Recipes
1,000× Ara-C
Dissolve 4.86 mg of Ara-C in 2 mL of distilled water to make a 1,000× stock solution of 10 mM and sterilize the solution by filtration. Store at -20 °C.
1,000× dbcAMP
Dissolve 50 mg of dbcAMP in 101.756 μL of distilled DMSO to make a 1,000× stock solution of 1,000 mM and store at -20 °C.
30 mM forskolin stock solution
Dissolve 10 mg of forskolin in 812 μL of distilled DMSO to prepare a 30 mM forskolin stock solution. Store at -20 °C.
Complete growth medium of rat Schwann cells
48.5 mL of DMEM/F12 containing 1.5 mL of FBS, 3 μM forskolin, 10 ng/mL heregulin- β-1, and 100 mg/mL penicillin–streptomycin.
10% FBS
45 mL of DMEM/F12 containing 5 mL of FBS supplemented with 1% penicillin–streptomycin.
3% FBS
48.5 mL of DMEM/F12 containing 1.5 mL of FBS supplemented with 1% penicillin–streptomycin.
1% FBS
49.5 mL of DMEM/F12 containing 0.5 mL of FBS supplemented with 1% penicillin–streptomycin.
1 mM dbcAMP
Add 1 μL of 1,000× dbcAMP (1,000 mM) to 1 mL of 1% FBS.
0.1% Triton X-100
Dilute 1 mL of Triton X-100 into 1,000 mL of PBS.
5% gelatin
Dissolve 0.5 g of gelatin in 10 mL of PBS. Add 300 μL of Triton X-100 (0.3%) to the buffer.
PBST
Add 1 mL of Tween-20 to 1,000 mL of PBS. Store at room temperature.
Blocking buffer
5% non-fat milk in Tris-buffered saline containing 0.5% Tween-20
Acknowledgments
This protocol is adapted from the previous published papers (Zou et al., 2022), (Wen et al., 2017) and (Monje PV., 2018). Thanks to Professor Jiasong Guo (Southern Medical University, Guangzhou, China).
Competing interests
There are no conflicts of interest or competing interests.
Ethics
All animal procedures were performed in accordance with the guidelines for the ethical treatment of animals.
References
Arthur-Farraj, P., Wanek, K., Hantke, J., Davis, C. M., Jayakar, A., Parkinson, D. B., Mirsky, R. and Jessen, K. R. (2011). Mouse schwann cells need both NRG1 and cyclic AMP to myelinate. Glia 59(5): 720-733.
Cristobal, C. D. and Lee, H. K. (2022). Development of myelinating glia: An overview. Glia 70(12): 2237-2259.
Jessen, K. R. and Mirsky, R. (2019). The Success and Failure of the Schwann Cell Response to Nerve Injury. Front Cell Neurosci 13: 33.
Nocera, G. and Jacob, C. (2020). Mechanisms of Schwann cell plasticity involved in peripheral nerve repair after injury. Cell Mol Life Sci 77(20): 3977-3989.
Wen, J., Tan, D., Li, L. and Guo, J. (2017). Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat. Bio Protoc 7(20): e2588.
Yamauchi, J., Miyamoto, Y., Hamasaki, H., Sanbe, A., Kusakawa, S., Nakamura, A., Tsumura, H., Maeda, M., Nemoto, N., Kawahara, K., Torii, T. and Tanoue, A. (2011). The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination. J Neurosci 31(35): 12579-12592.
Zou, Y., Zhang, J., Liu, J., Xu, J., Fu, L., Ma, X., Xu, Y., Xu, S., Wang, X. and Guo, J. (2022). SIRT6 Negatively Regulates Schwann Cells Dedifferentiation via Targeting c-Jun During Wallerian Degeneration After Peripheral Nerve Injury. Mol Neurobiol 59(1): 429-444.
Article Information
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© 2023 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 > Peripheral nervous system > Schwann cell
Cell Biology > Cell isolation and culture > Cell differentiation
Cell Biology > Cell imaging > Fixed-cell imaging
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Fast Detection and Quantification of Interictal Spikes and Seizures in a Rodent Model of Epilepsy Using an Automated Algorithm
KJ Kyle J. Jackson *
SS Shaunik Sharma *
GT Grant Tiarks
SR Saul Rodriguez
AB Alexander G. Bassuk
(*contributed equally to this work)
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4632 Views: 822
Reviewed by: Prashanth N SuravajhalaJayaraman ValadiWenyang Li
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Abstract
The electroencephalogram (EEG) is a powerful tool for analyzing neural activity in various neurological disorders, both in animals and in humans. This technology has enabled researchers to record the brain’s abrupt changes in electrical activity with high resolution, thus facilitating efforts to understand the brain’s response to internal and external stimuli. The EEG signal acquired from implanted electrodes can be used to precisely study the spiking patterns that occur during abnormal neural discharges. These patterns can be analyzed in conjunction with behavioral observations and serve as an important means for accurate assessment and quantification of behavioral and electrographic seizures. Numerous algorithms have been developed for the automated quantification of EEG data; however, many of these algorithms were developed with outdated programming languages and require robust computational hardware to run effectively. Additionally, some of these programs require substantial computation time, reducing the relative benefits of automation. Thus, we sought to develop an automated EEG algorithm that was programmed using a familiar programming language (MATLAB), and that could run efficiently without extensive computational demands. This algorithm was developed to quantify interictal spikes and seizures in mice that were subjected to traumatic brain injury. Although the algorithm was designed to be fully automated, it can be operated manually, and all the parameters for EEG activity detection can be easily modified for broad data analysis. Additionally, the algorithm is capable of processing months of lengthy EEG datasets in the order of minutes to hours, reducing both analysis time and errors introduced through manual-based processing.
Keywords: EEG Automated Algorithm Interictal Spikes Seizures MATLAB
Background
The emphasis of translational research in epilepsy is now shifting from the development of anti-seizure therapies to anti-epileptogenic and disease-modifying treatments. Long-term electroencephalographic recordings during the acute and chronic phases of disease are an important part of the drug-testing paradigm for these therapies. Manual analysis of prolonged electroencephalography (EEG) recordings can be time-consuming, laborious, and expensive. Further, manual EEG interpretation risks the introduction of human error and implicit bias, which can negatively impact data quantification and subsequent findings (Arends et al., 2017; Benbadis et al., 2017; Barger et al., 2019). Improper EEG analysis generates inconsistent data, which skews interpretation of the efficacy of interventional therapies at preventing the advent or progression of disease. Additionally, the limited signal-to-noise ratio makes manual analysis of seizures, seizure patterns, epileptiform spikes, and detection of other epileptiform events extremely challenging (Kaplan et al., 2005; Liu et al., 2019). These issues present a sizeable roadblock to rapid and unbiased scoring of electrographic spikes and seizure analysis. An automated, algorithm-based EEG analysis program is an ideal solution to meet this goal.
Previous work towards the development of automated EEG analysis tools has led to the production of algorithms that are capable of detecting and quantifying interictal activity and seizures, both in real-time and post data collection (Bergstrom et al., 2013;Tieng et al., 2016 and 2017). Unfortunately, many of these algorithms have been produced in programming languages that are no longer widely used or require substantial computational power to operate effectively (Harner et al., 2009). As a result of the steady evolution in the capabilities of computer processing equipment, higher levels of computational power can now be achieved. However, these new computational technologies are expensive, and often require additional hardware upgrades to be utilized in computing systems. Therefore, we developed an algorithm that detects and quantifies interictal spikes and seizures, using a programming language that requires minimal computational power and a computer with average processing hardware.
We evaluated our novel spike detection algorithm—a Basic Function Algorithm programmed in MATLAB—using large-scale EEG data obtained from a mouse model of epilepsy. We utilized EEG traces from mice with traumatic brain injury–induced epilepsy. The interictal spikes and seizures were manually detected using commercially available software (Neuroscore, Data Science International), and automatically detected using our EEG algorithm. All seizures detected using the automated algorithm were cross-referenced with Neuroscore for verification. Our comparative analysis revealed that the automated, algorithmic scoring of electrographic spikes and seizures is faster, more accurate, and less laborious than manual EEG analysis. Moreover, the algorithm is more dependable, resulting in a lower risk of introducing bias than manual, visual-based analysis.
Materials and Reagents
Materials for electrode implantation
Forceps, scalpel handles, scissors (Stoelting, catalog numbers: 5210883P, 52171P, 5210002P)
26 Gauge needles (Fisher Scientific, catalog number: BD 305111)
1 mL syringes (Fisher Scientific, catalog number: BD 309659)
Cotton tip applicators (Puritan, catalog number: 806 WC)
Burr drill tips (Stoelting, catalog number: 514552)
Surgical clips (Cellpoint Scientific, catalog number: 203-1000)
Surgical sutures, size 5-0 (Ethicon, catalog number: J493G)
Dental cement (Co-Oral-ite MFG Co, catalog number: 525000)
HD-X02 implants with two biopotential channels [Data Sciences International (DSI) (division of Harvard Bioscience, Inc)], catalog number: 270-0172-001)
Reagents
70% ethanol
Chlorhexidine scrub (Mölnlycke, catalog number: 0234-0575-08)
0.9% saline solution
Artificial tears ointment (Aventix, catalog number: 13585)
Meloxicam (Norbrook, catalog number: 0342.90A)
Baytril (Bayer Pharmaceuticals LLC, catalog number: AH039GH)
5% Haemo-Sol regular (Haemo-Sol, catalog number: 026-050)
Equipment
EEG acquisition system
Ponemah Software System (DSI, catalog number: PNM-P3P-CFG)
Included in system:
Analysis Module – Electroencephalogram Analysis Software Module (PNM-EEG100W)
Video Module – Noldus media recorder (PNM-VIDEO-008)
Ponemah Software 6.51 (PNM-P3P-651)
New Ponemah Acquisitions Software (PNM-P3P-TELELV8)
Analysis Module – Electromyogram Analysis Software module (PNM-EMG100W)
Computer – Lenovo ThinkPad T490 64-bit Windows 10 with docking station (271-0112-031)
Small Business Router – Cisco RV160 VPN router (DSI, catalog number: RV160)
Network Switch – 183w Network Switch (DSI, catalog number: 271-0115-003)
MX2 – Matrix 2.0 with USB Port for Signal Interface Support (DSI, catalog number: 271-0119-002)
RPC-1 – Receiver Pad for Plastic Cages with 4.5 m cable (DSI, catalog number: 272-6001-001)
Neuroscore software (DSI, 271-0165-CFG)
Included in system:
NS 3.3 Core – Neuroscore v3.3 Core software (271-0165-330)
Seizure Module – Neuroscore Seizure module (271-0167-SEIZURE)
Video Module – Neuroscore Video Synchronization module (271-0168-VIDEO)
Video Camera – Axis M1145-L Network Camera Kit with Axis T8120 Midspan (AXIS, catalog number: 275-0204-001)
Computational hardware
One of the primary goals of this project was to develop an autonomous algorithm that could rapidly process large volumes of EEG data using minimal computational power. To do so, an HP Spectre x360 laptop (Manufacturer: HP, Model: 15-eb1043dx) was used to process all EEG data. The CPU utilized was an Intel i7-10510U running at 2.30 GHz base clock speed, with a maximum clock speed of 4.30 GHz on all four cores. A total of 16 GB of GDDR4 memory was operated at 2667 MHz. The laptop had an additional 32 GB of Intel Optane memory available; however, this was disabled during data processing, and thus the maximum memory available at any given time was 16 GB. The laptop contained an Nvidia MX250 graphics card; however, since MATLAB is a CPU-intensive program, GPU processing power was not strongly considered. EEG data was written on an internal M.2 solid-state drive, after increased processing times were observed when using a standard USB 2.0 hard disk drive.
Software
Windows 10 Operating System (Microsoft, https://www.microsoft.com/en-us/software-download/windows10)
Neuroscore, Version 3.3 (Data Sciences International, https://www.datasci.com/products/software/neuroscore)
Ponemah Software 6.51 (https://www.datasci.com/products/software/ponemah)
MATLAB R2021a (MathWorks, https://www.mathworks.com/products/matlab.html)
MATLAB Signal Processing Toolbox (MathWorks, https://www.mathworks.com/products/signal.html)
Procedure
In the following sections, we describe the procedures for electrode implantation, EEG acquisition, and analysis of interictal spikes and seizures. For more information on electrode implantation and EEG acquisition procedures, we direct readers to the original articles (Puttachary et al., 2015; Sharma et al., 2018 and 2021).
Surgical preparation and electrode implantation
Clean surgical equipment with 5% Haemo-Sol solution for 48–72 h and dry at room temperature prior to autoclaving. On the day of surgery (electrode implantation), shave each mouse’s head (under anesthesia), and clean the surgical site in an onion shell pattern with 70% ethanol and a chlorhexidine scrub. Administer analgesic (Meloxicam, 1–2 mg/kg, subcutaneous) and eye ointment prior to surgery. Using a scalpel, make a small midline incision at the mid-dorsal aspect of the head and neck. Insert a transmitter into the flank region, by tunneling between the skin and the fascia using blunt dissection. Then, drill bilateral holes into the skull of each hemisphere, and place electrodes epidurally into the cerebral hemispheres. Completely cover and secure the electrodes in place using dental cement. Close the incision using sterile surgical sutures (Puttachary et al., 2015; Sharma et al., 2018 and 2021).
A video of the electrode implantation protocol has been provided (Video 1).
Video 1. Mouse electrode implantation procedure
EEG acquisition and setup
PhysioTelTM radio telemetry devices (transmitters) use oscillators controlled by voltage that convert biopotentials into high-frequency signals. All PhysioTelTM HD radiotelemetric devices are equipped with serial numbers. Once turned on, these devices send signals to receiver pads that record the serial number from the implanted device. These high-frequency signals are then transferred to the data exchange matrix that forwards output signals to the computer. Eight receiver pads can be connected to a single data exchange matrix, and multiple video-synchronized EEG traces can be monitored simultaneously through Ponemah Software 6.51, with a sampling frequency of 1,000 Hz. All information regarding the receiver pads, animal identification number, transmitter serial number, jack location in the data matrix, signal strength, and transmitter information, such as EEG and temperature calibration, are recorded to each matrix under hardware settings in the acquisition window. The acquisition of the EEG signal is performed at a sampling rate of 1,000 Hz, filter cutoff at 100 Hz, and full scale at 10 mV, by the Ponemah Acquisition Software. The frequency range of the telemetry transmitter is 1–100 Hz. Shortly after the implantation of the transmitters, start EEG recording to obtain baseline activity tracings for each animal. Approximately 5 GB of video (at 20 frames/s) and EEG data can be collected per day from eight animals simultaneously (Puttachary et al., 2015; Sharma et al., 2018). Once data acquisition is complete, export the EEG data as EDF files, to be used within MATLAB.
Installation of MATLAB and toolboxes
MATLAB can be installed from the MathWorks product webpage for Windows, Linux, and Macintosh operating systems (OS). For our purposes, a Windows OS was used, and the following instructions pertain to installation of MATLAB on a Windows 64-bit OS. However, additional information regarding MATLAB installation on a Linux OS can be found in the Notes section. First, create a MathWorks account and navigate to https://www.mathworks.com/products/matlab.html to view available products. Select the version of MATLAB you wish to download and click “Download for Windows.” Open the installation file by double clicking on it once it has downloaded. Sign in to your MathWorks account when prompted and follow the on-screen instructions to finish installation. During installation, select the MathWorks Signal Processing Toolbox and install the supplemental software package when prompted, to select add-on applications.
Manual EEG Analysis
Electrographic spikes and seizures are first detected manually using Neuroscore 3.3 software, which is commercially available from DSI. Spikes are analyzed using the “spike train protocol,” which identifies epileptiform spikes and spike duration, using dynamic and absolute threshold values. Dynamic thresholding allows a user to define a threshold based on a multiplication of the root mean square value of the EEG signal approximately one minute prior to the time point being analyzed, while the absolute threshold allows the user to define a fixed amplitude threshold (voltage) that is applied in both the positive and negative directions. Dynamic threshold values include threshold ratio as well as maximum and minimum ratios for epileptiform spike detection–criteria, which prevent false positives from being detected in low noise settings. The absolute threshold values include minimum and maximum amplitudes for EEG spikes, which are kept between 200 and 1,500 μV. Therefore, all spikes above 200 μV are considered epileptiform spikes. These parameters are specified to extract epileptiform spikes from the raw EEG data. These parameters also consider the minimum and maximum time interval between spikes, which is used to determine possible members of a spike train. Lastly, the duration of a spike train is also used to filter true epileptiform activity from background noise. Once the settings are finalized, load the raw EEG data via the EDF file, and specify the required signals (power bands and activity counts). Once complete, the raw EEG file can be analyzed (Puttachary et al., 2015). Interictal spikes and seizures are differentiated based on the EEG spike characteristics, such as amplitude, inter-spike interval, duration, and power spectral analysis. Manually remove EEG signals associated with mouse behavior (exploratory and grooming) and electrical artifacts, so they do not interfere with the analysis. These behavioral activities can be verified via video EEG recordings (Tse et al., 2014; Puttachary et al., 2015).
For seizure analysis, we set the duration for seizures (CS) at >10 s. CS episodes can last from 10–120 s, with inter-spike intervals of 100–300 ms. Spike amplitude often varies from 500–1,500 μV, with a spike frequency/min of 180–720 within an episode (Puttachary et al., 2015). Additionally, the power band spectrum is known to be a reliable measure for the detection of seizures (Puttachary et al., 2015; Sharma et al., 2018). The power spectrum varies with the type of seizure, and the spectrum frequently changes within stage-specific seizures (Tse et al., 2014; Puttachary et al., 2015).
Algorithm-based (automated) EEG analysis
File path and data import (Block 1)
The MATLAB EEG algorithm was programmed using three grouped blocks of code. The first block of the algorithm establishes a file path to the working folder and imports all the EDF files present within that folder using the edfread function. This allows the user to extract every EDF file using a single button click. Once imported, the EEG data from the EDF files is stored within cell arrays, using the default units of seconds and microvolts (μV). The algorithm then converts the EEG amplitude values into millivolts (mV). Then, the recording date is converted into a date-number format, while the time values are converted from a frequency value to a whole number in seconds.
Baseline EEG activity determination (Block 2)
The second block of code contains the data processing elements, allowing the algorithm to autonomously locate and record the location of interictal spikes and seizures. First, the algorithm calculates the baseline EEG activity value via a “running while” loop. This is done by iterating from a set minimum of 100 μV by increments of 5 μV, until 97% of the baseline EEG data points in the first hour of data are less than the value of the current iteration. Once this condition is satisfied, the algorithm saves the current amplitude iteration, outputs the value on-screen to the user, and proceeds with the calculation of the upper and lower spike thresholds. The time window (default ~1 h) for determining baseline activity can be readily changed from 1 h to any value that is suitable for the user, as long as it is shorter than the total length of the EEG tracing. This modification allows users to analyze EEG traces that are less than 1 h long and provides the opportunity to achieve a more accurate baseline by extending the time window beyond 1 h, if needed.
Lower threshold and upper threshold determination (Block 2)
The lower threshold variable incorporated in the algorithm is the value at which a true interictal spike is differentiated from background EEG noise and normal baseline activity. Relevant literature has revealed that a lower threshold of twice the baseline activity value is often sufficient for differentiating true interictal activity from the baseline EEG signal (Anjum et al., 2018). After determining baseline EEG activity, as described in the previous section, the algorithm calculates the lower threshold for interictal differentiation by doubling the baseline activity value. Thus, the lower threshold value for interictal spiking is not fixed, but rather depends on the baseline activity value of each individual EEG tracing. The upper threshold for interictal spiking is also pre-specified, to differentiate true interictal spikes from background electrical noise or animal movement. As reported in the literature, an upper threshold value of 1,500 μV often accurately captures true interictal activity, while removing EEG artifacts due to noise and animal behavior (Tse et al., 2014; Puttachary et al., 2015; Casillas-Espinosa et al., 2019). The upper threshold value is fixed, for every EEG trace, but can be readily adjusted as needed.
Interictal spike detection (Block 2)
Following determination of these key parameters, the remainder of the second block of code detects interictal spikes and seizures. The algorithm detects EEG spikes using the findpeaks function that is part of the MathWorks Signal Processing Toolbox, alongside a variety of associated input arguments used to clarify interictal activity. The minimum distance between interictal spikes, MinPeakDistance, is set at a value of 100 ms, to ensure that poly-spikes and other abnormal spiking patterns are not counted as multiple spikes (Puttachary et al., 2015). The minimum peak prominence, MinPeakProminence, better described as relative peak amplitude, is set at a value of 200 μV, which is also used to reduce the detection of poly-spikes and abnormal activity, such as grooming and mouse movement. Minimum peak amplitude, MinPeakHeight, is specified at the lower threshold value to ensure that only spikes with twice the baseline activity value are counted as interictal spikes. Lastly, a maximum peak width, MaxPeakWidth, of 200 ms is specified to reduce quantification of abnormal spikes and EEG artifacts. As with other quantification parameters, all these values are easily modified to filter the EEG trace being analyzed. After completing interictal spike detection, the algorithm records the date, time, and amplitude of every interictal spike that has met all the prespecified constraints, and provides the option to generate on-screen figures, as in Figure 1. Then, the algorithm removes any spike that fails to meet all the specified criteria (Table 1) from further quantification.
Figure 1. Representative example of interictal spikes determined using the automated algorithm. (A) MATLAB-generated output showing interictal activity detection (positive and negative spikes marked by red circles). The blue horizontal lines represent the automatically determined baseline activity value and the red horizontal lines represent the lower threshold value (2X baseline activity). (B) An enlarged image of an individual inter-ictal spike.
Table 1. Criteria for determining the presence of interictal spikes for algorithmic analysis
Interictal Spike Classification Criteria
Lower Detection Threshold 2X Baseline Activity Value
Upper Detection Threshold ≤1,500 μV
Minimum Peak Prominence ≥200 μV
Maximum Peak Width 200 mS
Inter-spike Interval ≥100 mS
Seizure detection (Block 2)
The last section of code in the second block contains the seizure detector, which analyzes the EEG trace subsequent to the interictal spike quantification process described in the previous section. Seizures are defined as an electrographic seizure with high amplitude frequency discharges, lasting for at least 10 seconds (Guo et al., 2013; Goodrich et al., 2013), with three times the baseline activity value, and an inter-spike interval of less than 5 seconds (Puttachary et al., 2015). The criteria for quantifying seizures are based on published literature quantifying spike clusters and grouped ictal activity for the evaluation of epileptogenesis (Tse et al., 2014; Puttachary et al., 2015; Anjum et al., 2018; Casillas-Espinosa et al., 2019). First, using the data from the previous section of code, the algorithm calculates the time elapsed between adjacent electrographic spikes. The algorithm then determines the starting and ending points of clusters of spiking activity. Any interval that is greater than the prespecified 5-second inter-seizure interval will break the chain of spiking activity, and automatically restart quantification of the next cluster of activity. Once complete, the code processes each spike cluster and removes any cluster that fails to meet the predefined seizure criteria (Table 2). If all the criteria are met, the algorithm stores these ictal activity clusters as well as the corresponding date, time of onset, duration, and number of ictal spikes within the cluster, which can be plotted for on-screen analysis (Figure 2).
Table 2. Criteria for determining the presence of a seizure during algorithmic analysis
Seizure Classification Criteria
Minimum Spike Amplitude 3X Baseline Activity Value
Maximum Spike Amplitude ≤1,500 μV
Minimum Seizure Duration >10 s
Maximum Inter-Spike Interval <5 s
Figure 2. Visual representation of spike and seizure detection within MATLAB. (A) Segment of the EEG trace showing interictal events and seizures on day 21 post-injury. Three episodes of spontaneous seizures were observed on the same day in one mouse (shown in rectangular blocks marked with triangles). (B) Expanded EEG trace of one of the seizures from 'A'. (C) Increased EMG activity is shown below the seizure trace. (D) Further expanded EEG trace of the seizure shown in C. Abbreviations: IIE, interictal events; EEG, electroencephalogram; EMG, electromyogram; SRS, spontaneous recurrent seizures.
Data Processing and Export (Block 3)
The third and final block of code is responsible for terminal data processing, and exporting compiled data to Microsoft Excel, for further data manipulation or comparison. First, there are instructions for plotting data so the data can be viewed on-screen, if desired. To reduce the computational workload and promote time savings, the plotting instructions are suppressed by default. However, in the semi-automated version of the code, plotting is displayed on-screen, allowing the user to manually review each interictal spike and seizure. The remaining code creates individual data tables containing the interictal spike data and seizure data for each EEG file present in the current working folder. Thus, when the code terminates, the interictal spike counts and seizure quantification data for all EEG traces are written to separate Excel files and stored within the current folder. This allows the user to rapidly process multiple EEG traces and have the data readily available for further review, or further analysis outside the MATLAB programming environment. Figure 3 provides a simplified, graphical representation of the various algorithm blocks and processing steps.
Figure 3. Schematic diagram outlining the processing steps of the autonomous MATLAB algorithm created for the detection of interictal spikes and seizures. Block 1 of the algorithm is dedicated to data import and unit conversion. Block 2 contains the logic statements that quantify interictal spikes and seizures using the inclusion criteria highlighted in red. Block 3 is responsible for finalizing data, plotting figures, and exporting finalized spike and seizure data.
Data analysis
To verify the capabilities of this algorithm, interictal spike counts and seizure counts from the automated algorithmic analysis were compared head-to-head against a visually-based Neuroscore quantification (referred to as the “manual” analysis). Identical threshold values and spike detection parameters were used in both quantitative methods. Average interictal spike counts were calculated using eight individual mice over three days. In total, approximately 170 h of EEG data were analyzed and used for this comparison. The interictal spike counts averaged 1.5% higher when using the automated algorithm than with manual quantification, with a standard deviation of 4.7%. Table 3 outlines the interictal spike detection data for each individual animal, and the detection ratio of algorithmic analysis using MATLAB to manual analysis using NeuroScore. For animals S22 and S24 with ratios less than 1.00, it is important to highlight that the lower threshold value for interictal spike detection was based on brief visual inspection, rather than defined criteria. That being said, although lower than 1.00, the MATLAB analysis provided a more consistent and reliable analysis than random assignment with visual inspection.
Table 3. Interictal spike counts for MATLAB and NeuroScore quantification, and the ratio of MATLAB to NeuroScore counts indicate increased interictal spike detection accuracy for most animals, when using the automated algorithm
Mouse MATLAB Interictal Spikes NeuroScore Interictal Spikes Ratio
S20 6344 6174 1.0275
S21 2756 2703 1.0196
S22 1688 1813 0.9311
S23 5435 5373 1.0115
S24 729 776 0.9394
S25 16482 16130 1.0218
S26 6676 6327 1.0552
S27 7624 7286 1.0464
Following automated quantification, the interictal spike counts for all eight mice were manually verified within the MATLAB workspace, to ensure that extraneous EEG activity was not being quantified as true interictal activity. The manual verification confirmed that the algorithm successfully identified true interictal activity and did not quantify non-interictal events or extraneous EEG activity, such as normal mouse movement. Thus, these results reveal that the autonomous MATLAB algorithm has nearly the same interictal spike detection capacity, if not higher, than manual, visual-based quantification using NeuroScore software.
Seizure detection was also quantified using the autonomous algorithm. The manual quantification was error-prone, i.e., involved a substantial amount of user error, and this issue prevented the calculation of an average and standard deviation. However, the autonomous MATLAB algorithm was able to accurately identify all seizures that were manually recorded on NeuroScore. Additionally, the algorithm was able to identify numerous seizures that were missed during the visual-based quantification, substantially reducing the component of human error associated with manual data analysis. Once again, all seizures were manually reviewed within the MATLAB workspace to ensure that the identified events were, in fact, true seizures that met our prespecified criteria, and not artifacts. Thus, the algorithm was determined to have a substantially higher detection capacity for seizures than the manual, visual-based quantification method, which missed numerous seizures.
Our most significant finding is the amount of time saved by using the automated algorithm. The manual, visual-based quantification in NeuroScore took approximately four months to completely analyze EEG data recorded from eight animals over an observation period of three months. In contrast, the autonomous MATLAB-based algorithm was able to quantify all three months’ worth of data in approximately 60 min. In sum, comparing the algorithm to manual analysis, we observed over a 2,000-fold decrease in the time required for the algorithm to complete the EEG quantification. At the same time, the automated algorithm maintained a high spike detection accuracy and showed improved seizure detection compared to the manual process.
Computational load was assessed using the built-in Task Manager Program within Windows 10. CPU load averaged 50% utilization at a clock speed of 4.3 GHz. Maximum memory utilization peaked at 8.7 GB, but 5.0 GB of RAM was in use for operating background processes. Therefore, while actively running, the algorithm utilized a maximum of 3.7 GB of RAM during data analysis. Integrated GPU utilization reached 14%, but this usage was the result of MATLAB generating on-screen text and the background graphical user interface, rather than from data processing and quantification.
Notes
The code for these analyses is available at: https://github.com/Jackson-Kyle-CCOM/Automated-EEG-Algorithm. Of note, all EEG analysis parameters can be easily adjusted by reading comments left within the algorithm code inside the MATLAB program. This allows a user to tailor the algorithm to best-approximate their own EEG data. It should be noted that EEG data with substantial background EEG activity and noise is likely to reduce the reliability of the algorithm, despite proper adjustment.
Although this algorithm was developed in MATLAB operating on a Windows operating system (OS), we are aware that some users may prefer to operate on a Linux OS instead. MATLAB is available for Linux-based systems and there are instructions available from MathWorks regarding setup and operation at https://www.mathworks.com/help/matlab/matlab_env/start-matlab-on-linux-platforms.html. Once the program has been installed, MATLAB can be launched using the matlabroot/bin/matlab command and the algorithm code can be operated using the following command ssh local.foo.com matlab -nodisplay -nojvm < hello.m, via the command window.
Acknowledgments
The work was supported by NIH 5R01NS098590 and a UIDM/Tross Family grant to Dr. Alexander G. Bassuk. The authors would also like to thank Michael Rebagliati for editing the manuscript.
Competing interests
All authors have nothing to disclose and declare no conflict of interest in this study.
Ethics
All animal experiments were performed under the guidelines of the Institutional Animal Care and Use Committee (IACUC), University of Iowa, USA, following prior approval.
References
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Arends, J. B., van der Linden, I., Ebus, S. C., Debeij, M. H., Gunning, B. W. and Zwarts, M. J. (2017). Value of re-interpretation of controversial EEGs in a tertiary epilepsy clinic. Clin Neurophysiol 128(4): 661-666.
Barger, Z., Frye, C. G., Liu, D., Dan, Y. and Bouchard, K. E. (2019). Robust, automated sleep scoring by a compact neural network with distributional shift correction.PLoS One 14(12): e0224642.
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Casillas-Espinosa, P. M., Sargsyan, A., Melkonian, D. and O'Brien, T. J. (2019). A universal automated tool for reliable detection of seizures in rodent models of acquired and genetic epilepsy. Epilepsia 60(4): 783-791.
Goodrich, G. S., Kabakov, A. Y., Hameed, M. Q., Dhamne, S. C., Rosenberg, P. A. and Rotenberg, A. (2013). Ceftriaxone treatment after traumatic brain injury restores expression of the glutamate transporter, GLT-1, reduces regional gliosis, and reduces post-traumatic seizures in the rat. J Neurotrauma 30(16): 1434-1441.
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Puttachary, S., Sharma, S., Tse, K., Beamer, E., Sexton, A., Crutison, J. and Thippeswamy, T. (2015). Immediate Epileptogenesis after Kainate-Induced Status Epilepticus in C57BL/6J Mice: Evidence from Long Term Continuous Video-EEG Telemetry.PLoS One 10(7): e0131705.
Sharma, S., Carlson, S., Gregory-Flores, A., Hinojo-Perez, A., Olson, A. and Thippeswamy, T. (2021). Mechanisms of disease-modifying effect of saracatinib (AZD0530), a Src/Fyn tyrosine kinase inhibitor, in the rat kainate model of temporal lobe epilepsy. Neurobiol Dis 156: 105410.
Sharma, S., Carlson, S., Puttachary, S., Sarkar, S., Showman, L., Putra, M., Kanthasamy, A. G. and Thippeswamy, T. (2018). Role of the Fyn-PKCdelta signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy.Neurobiol Dis 110: 102-121.
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4,634 | https://bio-protocol.org/en/bpdetail?id=4634&type=0 | # Bio-Protocol Content
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Isolation of RNA from the Murine Colonic Tissue and qRT-PCR for Inflammatory Cytokines
IS Ibrahim M. Sayed
KI Kaili Inouye
SD Soumita Das
LA Laura Crotty Alexander
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4634 Views: 1775
Reviewed by: Masahiro MoritaSandhya Ganesan Anonymous reviewer(s)
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Bio-protocol journal peer-reviewed
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Jul 28, 2022
Original Research Article:
The authors used this protocol in eLIFE Apr 2022
Abstract
E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs, including the brain, lung, heart, and colon. The effect of flavored fourth-generation pod-based E-cigs (JUUL) on murine gut inflammation is modulated by both flavor and exposure period. Exposure of mice to JUUL mango and JUUL mint for one month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after one month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after three months of exposure. In this protocol, we detail the process of RNA isolation from the mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon.
Keywords: E-cigarette Flavored JUUL Mouse colon RNA Inflammation
Background
E-cigarettes (E-cigs) were first introduced to the international market in the mid-2000s as an alternative to conventional tobacco smoking (O'Loughlin et al., 2016). E-cigs produce an aerosol (commonly called vapor) upon heating and the aerosolization of vehicle solvents propylene glycol and vegetable glycerin. In addition, a large variety of flavors are added to E-cigs to add appeal to people of all kinds, including women, children, and minorities (Zhu et al., 2014).
JUUL is one of the most popular pod-based E-cig brands. They sell pods containing e-liquids in different flavors, such as mint and mango (Huang et al., 2019). The effect of chronic inhalation of the aerosols produced from these devices on health is not yet understood.
Moshensky et al. (2022) observed in vivo mouse exposures of daily JUUL aerosol inhalation with different flavors (mango and mint) for one and three months to evaluate the effects of JUUL aerosol inhalation on the function and inflammation of different organs (Moshensky et al., 2022). The authors found that the JUUL aerosol inhalation induced inflammation in the brain, gut, and heart (Moshensky et al., 2022). A recent study using a stem cell–based approach of 3D gut organoids derived from healthy individuals revealed that E-cig induced inflammation in the gut epithelium and damaged epithelial tight junctions (Sharma et al., 2021).
Extraction of RNA from the murine colon is the most important step in the relative quantification of colon transcripts. Several factors affect the quality of extracted RNA used in qRT-PCR, such as the method used in the extraction, RNA purity and concentration, and the presence of other impurities, including guanidinium isothiocyanate, phenolic compounds, ethanol, tissue DNA, and protein. The extraction procedure should be performed efficiently, since the purity of the extracted RNA affects downstream processing, such as cDNA synthesis and PCR (Toni et al., 2018). In addition, tissue DNA and protein are other contaminants to the extracted RNA. In this protocol, we describe the detailed steps of efficient isolation of RNA from the colon of mice exposed to E-cigs with minimized levels of contamination that affect downstream processing. Also, we describe the process of quantitative measurement of the transcripts of inflammatory cytokines in the extracted RNA. For complete details of this paper and additional methods, please refer to Moshensky et al. (2022).
Materials and Reagents
Biological materials
Female C57BL/6 mice (6–8 weeks)
Materials
E-cigarette devices, Kanger Mini ProTank glassomizer, https://www.vaporauthority.com/products/genuine-kanger-mini-protank-3-glassomizer
Juul mango and mint pods, JUUL, https://www.juul.com/shop
Microcentrifuge tube (1.5 mL) (Fisher Scientific, catalog number: 07 200 534)
50 mL centrifuge tubes (Genesee Scientific, catalog number: 28-108)
15 mL centrifuge tubes (Genesee Scientific, catalog number: 28-103)
FisherbrandTM wood handled cotton swabs and applicators (Fisher Scientific, catalog number: 22-363-173)
Petri dishes, stackable (Genesee Scientific, catalog number: 32-107G)
MicroAmpTM optical 96-well reaction plate (Thermo Fisher Scientific, catalog number: N8010560)
Optical adhesive covers GPLE (Thermo Fisher Scientific, catalog number: A49767).
BrandTechTM BRANDTM thin wall 0.2 mL PCR tubes with attached caps (Fisher Scientific, catalog number: 13-882-58)
Reagents
qScript cDNA SuperMix (Quanta Biosciences, catalog number: 95048)
2× SYBR Green qPCR Master Mix (Bimake, catalog number: B21203)
Direct-zolTM RNA Miniprep kit (Zymo Research, catalog number: R2053)
TRI reagent (Zymo Research, catalog number: R2050-1-200)
GibcoTM PBS, pH 7.4 (Fischer Scientific, catalog number:10-010-023)
UltraPureTM DNase/RNase-free distilled water (Invitrogen, catalog number: 10-977-015)
Ethanol, pure for molecular biology (Sigma-Aldrich, catalog number: E7023)
RNAlater (Sigma-Aldrich, catalog number: R0901-500ML)
DNase I treatment preparation (this enzyme is provided with Direct-zolTM RNA Miniprep kit) (see Recipes)
Synthesis of cDNA from RNA using qScript cDNA Synthesis kit (see Recipes)
qPCR reaction mixture (see Recipes)
Equipment
Noyes spring scissors, angled (Fine Science Tools, catalog number: 15013-12)
Fine forceps (Fine Science Tools, catalog number: 11254-20)
Micropipette (Eppendorf, catalog number: M22873H)
E-cigarette InExpose system (SCIreQ)
Balance (METTLER TOLEDO, Balance XPR204S)
FisherbrandTM 150 handheld homogenizer motor (Thermo Fisher Scientific, catalog number: 15-340-168)
ST8 microcentrifuge (Thermo Scientific, catalog number: 75-667-200)
MiniAmp Plus thermal cycler (Applied Biosystems, catalog number: A37835)
QuantStudioTM 3 real-time PCR system, 96-well, 0.2 mL (Thermo Fisher Scientific, catalog number: A28137)
Software
GraphPad Prism, RRID: SCR_002798
QuantStudio Design & Analysis Software
Procedure
Isolation of colon from the exposed mice (Figure 1)
Expose female C57BL/6 mice (6–8 weeks) to E-cig aerosols produced from JUUL mango or JUUL mint or to air for one and three months using the InExpose system.
Note: A detailed protocol on mouse exposure and composition of JUUL pods is present in the original article (Moshensky et al., 2022). Briefly, C57BL/6 mice (6–8 weeks of age) were placed in an exposure chamber and exposed to E-cig aerosol for 20 min three times daily, for a total of 60 min per day, for 4–12 weeks. All experiments were conducted with the approval of the UCSD Institutional Animal Care and Use Committee (IACUC protocol S16021).
At the end of exposure, euthanize mice by regulated slow (~50% flow rate) carbon dioxide inhalation followed by cervical dislocation.
Using scissor and forceps, cut the whole colon of a mouse from the anus to the cecum (Figure 1).
Figure 1. Flow of the design experiment. Mice are exposed either to air or Juul pods (mango or mint) through the InExpose system. After three months of exposure, mice are sacrificed and colons are collected, cleaned from any remaining stool, and then stored in RNAlater solution. The colon is either processed immediately or stored at -80 °C until processing.
Put the colon in a Petri dish containing PBS to rinse away any blood. Remove any attached adipose tissue from the colon.
Use a cotton swab to empty the colon from remaining stool.
Cut the colon into small pieces and transfer part of the colon into a 1.5 mL Eppendorf containing 400–500 μL of RNAlater.
Notes:
Saving the whole colon in RNAlater solution is an option if no other procedures/techniques are required, such as processing of the mouse colon into formalin-fixed paraffin-embedded blocks. Therefore, step A6 is optional.
In case histology is required for the colon, cut it into three parts: proximal, mid, and distal colon. The distal colon is used to assess the transcripts of inflammatory cytokines. Mid and proximal colons are used for histology and other purposes.
The amount of RNAlater can be increased (600 μL to 1 mL) if a larger part or the whole colon will be saved. The RNAlater should cover the part/whole colon saved as shown in Figure 1.
Store the colon samples at -80 °C.
Note: If RNA extraction is done on the same day of euthanasia, step A7 is not required. Saving the tissue in RNAlater is important when the downstream processing will be performed later.
Extraction of RNA from the colon tissues (Figure 2)
Thaw the colon samples on ice; then, use scissors and forceps to cut the colon tissues into small pieces.
Note: Ensure that no precipitated salts are around the tissue. Wash the colon with PBS to remove any precipitated salts.
Weigh the colon piece using a scale/balance and ensure that the weight is ≤ 20 mg/piece.
Notes:
It is very important to weigh the mouse colon; ideally, tissues between 5 and 20 mg give good RNA yield. Using larger tissue pieces gives a poor RNA yield due to column blockage during the RNA extraction process.
It is recommended to mince/cut the colon into small pieces after weighing to speed up the homogenization step.
Transfer the colon piece into a 1.5 mL Eppendorf containing 600 μL of TRI reagent solution.
Homogenize the colon piece in TRI reagent solution using the 150 handheld homogenizer for 1–2 min until complete disruption of tissue.
Notes:
It is important to clean and disinfect the homogenizer using 70% ethanol and water between samples to avoid cross contamination.
Make sure that the weighted piece is completely disintegrated and no visible tissue remains.
In case of tough tissue, you can increase the volume of TRI reagent to 800 μL and/or increase the homogenization time.
The homogenizer has three different speeds (low, middle, and high). It is recommended to use the middle speed, since the highest could be destructive to tissues if it is continued for a long time and the lowest could take a long period of time to disintegrate the tissue.
Centrifuge the tissue suspension at a maximum speed of 13,000 × g for 2 min.
After the centrifugation step, you can see a precipitated debris at the bottom of the Eppendorf and a clear supernatant solution above it (Figure 2). Collect the supernatant into a new 1.5 mL Eppendorf and discard the tube containing the precipitated debris.
Mix the supernatant from step B6 with an equal volume (approximately 600 μL) of ethanol (95–100%).
Transfer the previous mixture into a Zymo-SpinTM IICR column in a collection tube (this column is a part of Direct-zolTM RNA Miniprep kit).
Note: The maximum capacity of Zymo-SpinTM IICR is 700 μL and the volume of each sample is approximately 1,200 μL (600 μL TRI/tissue mix + 600 μL ethanol). Therefore, step B8 is repeated twice on the same column.
Centrifuge the mixture from step B8 at 13,000 × g for 1 min. Then, discard the flow through waste into 15 or 50 mL falcon tubes and discard the collection tube.
Note: At this step, the flow through waste can be used to purify the protein from the mouse colon.
Transfer the Zymo-SpinTM IICR column to a new collection tube.
Add RNA wash buffer (a part of Direct-zolTM RNA Miniprep kit) (400 µL/sample) to the column and centrifuge at 13,000 × g for 30 s to 1 min. Discard the flow through and transfer the column to a new collection tube.
Add 80 µL of DNase I treatment preparation (Recipe 1) per column. Incubate the mixture with the column at room temperature for 15 min.
Notes:
DNase I (part of Direct-zolTM RNA Miniprep kit) is provided as lyophilized powder. Reconstitute it using with DNase/RNase-free water according to the manufacturer’s instructions. Mix well and then store the aliquots at -20 °C until use.
This step is highly recommended to get rid of DNA present in the sample.
Add 400 µL of Direct-zolTM RNA PreWash buffer (part of Direct-zolTM RNA Miniprep kit) to the column and centrifuge at 13,000 × g for 1 min. Discard the flow through and repeat this step one more time.
Note: RNA PreWash buffer is provided as a concentrate buffer. You should dilute it with ethanol according to the manufacturer’s instructions.
Add 700 µL of RNA wash buffer to the column and centrifuge at 13,000 × g for 1 min to remove the wash buffer.
Note: RNA wash buffer is provided as a concentrate buffer. You should dilute it with ethanol according to the manufacturer’s instructions.
Centrifuge the empty column present in a new collection tube at 13,000 × g for 5 min to ensure complete removal of the ethanol that is present in RNA PreWash buffer and RNA wash buffer.
Note: It is important to get rid of ethanol since any residual amount can impact the downstream qPCR steps and/or the yield and purity of extracted RNA.
Transfer the column carefully into an RNase-free tube, add 50 µL of DNase/RNase-free water directly to the column matrix, incubate at room temperature for 2–5 min, and then centrifuge at 13,000 × g for 1 min.
Repeat step B16 using the RNA eluted from the column. Transfer the eluted RNA into the same column, incubate at room temperature for 2 min, and then centrifuge at 13,000 × g for 1 min.
Figure 2. Steps of RNA isolation from the colon. Colon samples are cut into small pieces, weighted, and then homogenized. The homogenized samples are centrifuged to remove the tissue debris and the supernatants are processed for RNA extraction. The extracted RNA is used for quantification of the transcripts of inflammatory cytokines.
RT-qPCR for the inflammatory transcript in the mouse colon
Conversion of RNA into cDNA
Convert the extracted RNA (approximately 500–750 ng) into cDNA using qScript cDNA SuperMix according to manufacturer’s instructions.
To a 0.2 mL thin-walled PCR tube placed on ice, add the components as shown in Recipe 2. The volume of RNA added depends on the concentration. Total RNA added should be between 500 and 750 ng. The volume of free water added depends on the volume of RNA, according to Recipe 2.
Note: When comparing samples, it is preferred to use the same amount of RNA. For example, to compare the transcripts from mouse colon derived from air-exposed groups and from JUUL mango pods, use the same amount of RNA to form cDNA; either 500 or 750 ng for all samples or in between concentrations.
Vortex the mixture gently and then centrifuge for 10 s to collect contents.
Place the PCR tubes in a MiniAmp Plus thermal cycler using the following program (Table 1):
Table 1. PCR program for synthesis of cDNA using qScript cDNA synthesis kit
Number of cycles Temperature Time
1 25 °C 5 min
1 42 °C 30 min
1 85 °C 5 min
4 °C Hold
Dilute the cDNA product into 1/5 or 1/10 using nuclease-free water and store the diluted cDNA at -20 °C until analysis.
qRT-PCR reaction system
Use the synthesized cDNA as a template for qPCR reaction with 2× SYBR Green qPCR Master Mix.
Using a MicroAmpTM optical 96-well reaction plate, add the PCR reaction mixtures according to Recipe 3.
Add optical adhesive covers GPLE to the plate, put in the machine, and run the following PCR program (Table 2):
Table 2. PCR program
Step Hold PCR (40 cycles) Melt curve (1 cycle)
Denaturation Anneal Extend
Temperature 95.0 °C 95.0 °C 60.0 °C 72.0 °C 95.0 °C 60.0 °C 95.0 °C
Time 10 min 15 s 30 s 30 s 15 s 60 s 15 s
Data analysis
High-yield purified RNA extraction is possible from mouse colon tissue. Using the RNAlater solution helps preserve tissues and/or the RNA for a longer period at -80 °C (Figure 1). The previous step aids in the extraction of many samples at the same time to minimize the inter-assay procedure. It is important to weigh the colon piece undergoing the RNA extraction process (Figure 2). Since each column has a maximum capacity, including too large samples could lead to blockage of the column, and therefore negatively impact all the downstream process of RNA extraction. The addition of TRI reagent stops RNase enzymes and neutralizes any infectious agents in the samples; therefore, it helps in improving the quality and stability of extracted RNA. Following the previously described procedure, you can assess the concentration and purity of RNA using a Nanodrop spectrophotometer. For the concentration, you may obtain ~100 ug of total RNA from the previous procedure. The purity of extracted RNA can be assessed by the measurement of absorbance (A260/280 and A260/230). The acceptable value for pure RNA in terms of A260/280 is 1.8–2.2 or a value >1.8. A value lower than 1.8 indicates the presence of contaminants that absorb at 280 nm, such as protein and phenol. Similarly, 260/230 values >1.8 indicate pure RNA, while values lower than 1.8 indicate contamination with TRI reagent that absorbs at 230 nm.
For the qRT-PCR, the cycle threshold (Ct) value for the target gene was calculated using QuantStudio Design & Analysis Software, and then normalized to the housekeeping gene (∆CT). For comparison of the relative transcript expression between the different groups of mice, we used the formula ∆∆CT. For example, to compare the relative IL-6 transcript expression, we determined:
∆CT for IL-6 in JUUL exposed mice = CT IL6 - CT housekeeping gene)
∆CT for IL-6 in air-exposed mice = CT IL6 - CT housekeeping gene)
∆∆CT = ∆CT for IL-6 in JUUL exposed mice - ∆CT for IL-6 in air-exposed mice
Further data analysis for relative gene expression can be done using GraphPad Prism.
Notes
This method is suitable for RNA extraction from different mouse organs
Although the previous protocol focused mainly on the mouse colon, it can be applied to extract RNA from different mouse organs such as the spleen, liver, brain, lung, etc. We previously used this protocol to assess the inflammatory responses in the liver, spleen, cecum, and intestine of mice infected with Salmonella (Sayed et al., 2021). The liver and spleen are thicker than the intestine; therefore, the volume of TRI reagent should be increased to 800 μL and the homogenization time should be increased to 3–5 min.
The quality of RNA and PCR products is important in the interpretation of the results
The quality of extracted RNA plays an important role in PCR reactions. The presence of impurities, which can be determined by the measurement of absorbance A260/280 and A260/230, could impact the downstream processing. Also, the primer design is important in the qPCR reaction. It is important to check the melting curve for each gene and confirm it is a single peak. The presence of several peaks indicates poor primer design and the possibility of primer dimer that affects the final interpretation of the results.
Recipes
DNase I treatment preparation
Reagent Volume
DNase I (1 U/µL) 5 µL
DNA digestion buffer 75 µL
These components are parts of Direct-zolTM RNA Miniprep kit. DNase I is provided as lyophilized powder. Reconstitute it using with DNase/RNase-Free Water. The volume of added water depends on the unit in DNase I powder to reach the final concentration of 1 U/µL. For example, add 250–275 µL water for 250 U of DNase I powder and 50–55 µL water for 50 U of DNase I powder. Mix well and then store the aliquots at -20 °C until use.
Synthesis of cDNA from RNA using qScript cDNA synthesis Kit
Reagent Volume to add
qScript reaction mix (5×) 4 μL
RNA (0.5–0.75 µg) Variable
Nuclease-free water Variable
Total volume 20 μL
qPCR reaction mixture
Component Volume per 10 μL reaction Final concentration
2× SYBR
Green qPCR Master Mix including ROX a
5 μL 1×
cDNA (50–100 ng) b 2 μL 10–20 ng b / reaction (10 μL)
Forward primer (4 μM) c 1 μL 0.4 μM
Reverse primer (4 μM) c 1 μL 0.4 μM
Water 1 μL
Total volume d 10 μL
a Add ROX reference dye 2 (low concentration) to the Green Master Mix according to the manufacturer's instructions, to reach a 1× final concentration.
b The amount of cDNA formed in Recipe 2 is 500 ng, and dilution (1/5 or 1/10) is done to synthesized cDNA to become 50–100 ng. In general, 5-100 ng cDNA per reaction is acceptable.
c For the sequences of primers used in the amplification of inflammatory transcripts in the colon please refer to Moshensky et al. (Moshensky et al., 2022)
d Regarding the evaluation of the transcripts of inflammatory cytokines, 10 μL of reaction is sufficient since the abundance of these genes in the colon is high. For low-abundance genes, it is recommended to use a higher-volume reaction system (20–50 μL).
Acknowledgments
This study was supported by the National Institutes of Health R01HL137052, American Heart Association 16BGIA27790079, University of California, San Diego RS169R, American Thoracic Society Foundation Award for Outstanding Early Career Investigator, and U.S. Department of Veterans Affairs 1I01B x 004767 to Laura E Crotty Alexander (LCA). Soumita Das (SD) was supported by Tobacco-Related Disease Research Program 28IP-0024 and both IMS and SD were supported by NIDDK- R01DK107585. This protocol is derived from the original research paper (Moshensky et al., 2022; https://doi.org/10.7554/eLife.67621.sa0).
Competing interests
The authors declare there is no conflict of interest.
Ethics
This animal study was approved at the University of California San Diego Institutional Animal Care and Use Committee (IACUC protocol S16021).
References
Huang, J., Duan, Z., Kwok, J., Binns, S., Vera, L. E., Kim, Y., Szczypka, G. and Emery, S. L. (2019). Vaping versus JUULing: how the extraordinary growth and marketing of JUUL transformed the US retail e-cigarette market. Tobacco Control 28(2): 146-151.
Moshensky, A., Brand, C. S., Alhaddad, H., Shin, J., Masso-Silva, J. A., Advani, I., Gunge, D., Sharma, A., Mehta, S., Jahan, A., et al. (2022). Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice. Elife 11: e67621.
O'Loughlin, J., Wellman, R. J. and Potvin, L. (2016). Whither the e-cigarette? Int J Public Health 61(2): 147-148.
Sayed, I. M., Ibeawuchi, S. R., Lie, D., Anandachar, M. S., Pranadinata, R., Raffatellu, M. and Das, S. (2021). The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses. Gut Microbes 13(1): 1991776.
Sharma, A., Lee, J., Fonseca, A. G., Moshensky, A., Kothari, T., Sayed, I. M., Ibeawuchi, S. R., Pranadinata, R. F., Ear, J., Sahoo, D., et al. (2021). E-cigarettes compromise the gut barrier and trigger inflammation. iScience 24(2): 102035.
Toni, L. S., Garcia, A. M., Jeffrey, D. A., Jiang, X., Stauffer, B. L., Miyamoto, S. D. and Sucharov, C. C. (2018). Optimization of phenol-chloroform RNA extraction. MethodsX 5: 599-608.
Zhu, S.-H., Sun, J. Y., Bonnevie, E., Cummins, S. E., Gamst, A., Yin, L. and Lee, M. (2014). Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation. Tob Control 23(suppl 3): iii3-iii9.
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Multiphoton Microscopy of FITC-labelled Fusobacterium nucleatum in a Mouse in vivo Model of Breast Cancer
LP Lishay Parhi
AS Amjad Shhadeh
NM Naseem Maalouf
TA Tamar Alon-Maimon
VS Viviana Scaiewicz
GB Gilad Bachrach
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4635 Views: 525
Reviewed by: Ivan Shapovalov Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Communications Jun 2020
Abstract
Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.
Keywords: Multiphoton microscopy Breast cancer Fusobacterium nucleatum Bacterial colonization
Background
Fusobacterium nucleatum is an oral anaerobic bacterium that is prevalent in periodontal disease (Nozawa et al., 2020) and preterm birth (Hill, 1993, 1998; Han et al., 2009; Gonzales-Marin et al., 2013). Recently, F. nucleatum was found to colonize colorectal adenocarcinoma (Castellarin et al., 2012; Kostic et al., 2012), esophageal cancer (Yamamura et al., 2016), pancreatic cancer (Mitsuhashi et al., 2015), and breast cancer (Parhi et al., 2020). The presence of F. nucleatum in colon, pancreatic, and esophageal cancer has been associated with poor prognosis (Mima et al., 2016; Yamamura et al., 2016; Yamaoka et al., 2018; Mitsuhashi et al., 2015). In a mouse model, F. nucleatum colonizes breast cancer and promotes tumor growth and metastatic progression (Parhi et al., 2020).
In order to identify Fusobacterium nucleatum colonization in human and mouse cancer tissues, the methods previously used were microbiome analysis, PCR, fluorescence in situ hybridization, and culture (Castellarin et al., 2012; Kostic et al., 2012; Parhi et al., 2020; Abed et al., 2016; Abed et al., 2020). Specifically directing bacteria to a target cancer as a future therapy strategy requires visualization tools for imaging and monitoring bacterial trafficking in animal models. Here, we show a simple method that requires minimal time, equipment, materials, and resources to quickly validate bacterial localization in cancer tissue in an in vivo model. This is a direct method to visualize the bacteria in the tissue with minimal intermediate steps that may affect the results.
Materials and Reagents
1 mL syringe (BD PlastipakTM, catalog number: 303084)
27G and 26G needle (BD PlastipakTM, catalog numbers: 305109 and 303176)
Sterile surgical pad
10 cm tissue culture dish (Corning Incorporated, Falcon, catalog number: 353003)
11 mL (NuncTM catalog number: 347856), 15 and 50 mL tubes (Miniplast, catalog numbers: 835-015-40-111 and 835-050-21-111)
1.5 mL Eppendorf microtubes (Bar Naor Ltd., catalog number: BN022RL)
Scalpel (Bar Naor Ltd., catalog number: BN400-21-JH)
Whatman syringe filter 0.2 µm (Bar Naor Ltd., catalog number: BNFCA206030H)
Coverslip (Bar Naor Ltd., catalog number: BN1001-18-1CN)
Superfrost Plus glass slides (Thermo Fisher Scientific, catalog number: J1800AMNT)
Pipette tips [Axygen, catalog numbers: TF-200-R-S (20–200 μL), TF-1000-R-S (100–1,000 μL)]
Glass vials (Bar Naor Ltd., catalog number: BN1228WH)
Animals: 5–7-weeks-old female C57BL/6 mice (Envigo, Israel)
AT3 cell line (Sigma SCC178)
Dulbecco’s modified eagle medium (DMEM), 500 mL (Biological Industries, catalog number: 01-050-1A)
Accutase solution normal human, 100 mL (Biological Industries, catalog number: C-41310)
Dulbecco’s PBS, 500 mL (Biological Industries, catalog number: 02-020-1A)
Heat-inactivated fetal bovine serum (FBS), 500 mL (Biological Industries, catalog number: 04-127-1A)
L-glutamine in saline solution, 100 mL (Biological Industries, catalog number: 03-020-1B)
Penicillin–streptomycin solution, 100 mL (Biological Industries, catalog number: 03-031-1B)
MEM non-essential amino acids solution, 100 mL (Biological Industries, catalog number: 01-340-1B)
Fusobacterium nucleatum strain of interest (for example ATCC 23726)
Wilkins-Chalgren broth (Oxoid, catalog number: CM0643)
Fluorescein isothiocyanate (FITC), 1 g (Sigma-Aldrich, catalog number: F7250-1G)
Ethanol (Sigma-Aldrich, catalog number: E7023)
Trypan blue solution (Biological Industries, catalog number: 03-102-1B)
Sodium carbonate (Sigma-Aldrich, catalog number: 497-19-8)
Sodium hydroxide (Sigma-Aldrich, catalog number: 1310-73-2)
Isoflurane, USP TerrellTM (Piramal, catalog number: VED1350)
Double deionized water (DDW)
Ice
Wilkins medium (see Recipes)
Sodium carbonate buffer (0.1 M, pH 9.0) (see Recipes)
FITC solution (see Recipes)
Equipment
Pipettes
Centrifuge (Eppendorf 5810 R)
Autoclave (Tuttnauer 2540MK)
Anesthesia induction chamber
Hemocytometer
Spectrophotometer (600 nm wavelength)
Orbital shaker
Nikon multiphoton A1MP
Stereo microscope (Nikon SMZ2-)
Scalpel handle
Scissors
Forceps
Tail vein injection platform
Heat lamp
Anaerobic chamber (Bactron I–II Shellab, USA)
Caliper
Vortex
Software
NIS-Elements (Nikon Instruments Inc.) https://www.microscope.healthcare.nikon.com/products/software/nis-elements)
Procedure
Prepare cells
Seed 5 × 105 AT3 cells in a 10 cm tissue culture dish in 10 mL of DMEM medium containing 10% heat-inactivated FBS, 1% L-glutamine, 1% penicillin–streptomycin, and 1% MEM non-essential amino acids solution. Incubate the cells at 37 °C in a humidified incubator with 5% CO2 for 3–4 days. Ensure that the cells are 80%–100% confluent on the day of the experiment.
Change media (DMEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, 1% penicillin–streptomycin, and 1% MEM non-essential amino acids solution) the day before implantation.
To detach the tumor cells from the tissue culture dish, aspirate the culture medium, wash the cells with 5 mL of PBS, aspirate the PBS, and add 2 mL of accutase solution. Incubate the cells for 5–8 min with accutase at 37 °C. Trypsin/EDTA is an acceptable alternative to accutase.
Add 3 mL of DMEM to neutralize the accutase and pipette up and down until all cells have been detached. Transfer the cells suspension to a 15 mL conical centrifuge tube and add 10 mL of medium (the same as in step A1).
Centrifuge the cells at 1,000 rpm (200 × g) for 5 min at room temperature (RT). Aspirate the supernatant, leaving 500 μL of medium above the cell pellet. Resuspend the cells in the residual volume and add 10 mL of PBS. Invert the tube 2–3 times to get a homogeneous cell suspension and take 10 μL to count the cells in a hemocytometer. Use trypan blue to distinguish between live and dead cells.
Centrifuge the cell suspension at 200 × g for 5 min at RT. Aspirate the PBS and resuspend the cells in PBS at a final concentration of 2 × 106 cells/mL. Ensure that the single cell suspension has no clumping cells. Transfer the cells to an Eppendorf tube and keep on ice until use.
Orthotopical injection of AT3 tumor cells into mammary fat pad
Anesthetize the mice in an induction chamber receiving a slow flow rate of isoflurane (3%–5% in oxygen).
Place the mouse on a sterile surgical pad and make sure the head is properly placed inside the isoflurane nose cone. Confirm proper anesthesia by pinching the paw. Disinfect the injection site with 70% ethanol. Consider applying eye gel to prevent vision deterioration while the mouse is anesthetized.
Inject subcutaneously 1 × 106 AT3 cells in 50 μL of PBS orthotopically into the inguinal mammary fat pad using a 1 mL syringe with a 26G needle, by raising the nipple with forceps and inserting the needle underneath and perpendicular to the nipple.
Monitor the mice until they regain consciousness; make sure they regain full consciousness before joining other mice.
Bacteria growth, staining, and injection to mice
Perform bacterial injection when tumor size reaches 500 mm3 measured externally by caliper.
Grow F. nucleatum strain of interest in 5 mL of sterile Wilkins medium (see Recipes) overnight for 12–18 h in an anaerobic chamber in an atmosphere of 90% N2, 5% CO2, and 5% H2 at 37 °C.
Centrifuge at 2,057 × g for 5 min at 4 °C and discard supernatant. Resuspend the pellet with 1 mL of PBS, then add 4 mL of PBS and centrifuge again under the same conditions.
Discard the supernatant and resuspend the pellet with 1 mL of a fresh FITC solution (see Recipes). Ensure that the single cell suspension has no clumps.
Incubate in the dark with orbital rotation for 30 min at RT.
Add 4 mL of PBS and suspend. Centrifuge at 2,057 × g for 5 min at 4 °C and repeat the washing until the PBS in the supernatant is clear.
Discard the supernatant, resuspend the bacteria with 1 mL of PBS, and bring the bacteria to an OD600 of 1 (approximately 1 × 109 bacteria/mL). Keep bacteria on ice and in the dark until use.
Restrain the mouse in a tail vein injection platform. Position the tail such that the vein is facing upwards.
Prior to injection, warm the tail using an overhead heat lamp for a few minutes to dilate the veins.
Using a 1 mL syringe and a 27G needle, insert the needle gently, while the bevel is up, into the lateral or dorsal tail vein towards the direction of the head. Keep the needle and syringe parallel to the tail. Inject intravenously 5 × 107 fluorescein-5-isothiocyanate (FITC)-labeled F. nucleatum ATCC 23726. The contents of the needle should inject easily without resistance. In order to confirm correct intravenous injection, the piston should easily move and the vein color should change to a whiter color momentaneously.
Mice sacrifice and tissue harvesting
Twenty-four hours after bacterial injection, transfer mice to a chamber containing 2% isoflurane in oxygen for approximately 30 s.
Verify that mice are fully anesthetized and perform cervical dislocation. Another option is performing CO2 euthanasia followed by confirmation by cervical dislocation.
Clean tumor area with 70% ethanol. Cut the skin with a pair of sterilized scissors in the ventral middle of the mice, near the tumor. Hold the skin near the tumor with sterile forceps and remove tumor carefully using a sterile scalpel.
Hold the skin on the other side of the mice’s body near the healthy (control) breast tissue with sterile forceps and remove tissue carefully using a sterilized scalpel.
Place tumor and healthy breast tissue in a glass vial containing PBS, on ice.
Multiphoton microscopy of FITC-labeled F. nucleatum
Cut a slice from the fresh tissue using a scalpel and photograph using a Nikon SMZ25. Representative images are shown in Figure 1.
Figure 1. Nikon SMZ25 images of breast cancer tissue and adjacent mammary tissue. Representative microscopy images of tumor and normal adjacent mammary samples harvested 24 h post intravenous injection of 5 × 107 FITC-labeled F. nucleatum ATCC 23726 or with PBS vehicle into AT3 cancer-bearing mice.
Place the tissue on a slide with a drop of water and cover with a coverslip.
Observe and photograph immunofluorescent images using the Nikon multiphoton A1MP microscope set to 740 nm wavelength using a 25× objective. Representative images are shown in Figure 2.
Figure 2. Nikon multiphoton A1MP microscope images of breast cancer tissue and adjacent mammary tissue. Representative multiphoton microscopy images of tumor and normal adjacent mammary samples harvested 24 h post intravenous injection of AT3 cancer-bearing mice with 5 × 107 FITC-labeled F. nucleatum ATCC 23726. Images were taken from Parhi et al. (2020). No changes were made.
Data analysis
Images obtained from the fluorescence microscope are converted to 8-bit images by NIS-Elements software (see Figures 1 and 2).
Recipes
Wilkins medium
100 mL of DDW
3.1 g of Wilkins-Chalgren broth
Mix until dissolved
Autoclave at 121 °C for 30 min and aliquot 5 mL to 11 mL tube
Reduce in an anaerobic chamber overnight
Sodium carbonate buffer (0.1 M, pH 9.0)
84 mg of sodium carbonate
10 mL of DDW
Adjust pH to 9.0 with sodium hydroxide
Sterilize by filtering through 0.2 μm Whatman filter
FITC solution
Dissolve 1 mg of FITC in 10 mL of sodium carbonate buffer by vortexing
Keep in the dark until used
Acknowledgments
The authors thank Dr. Zakhariya Manevitch for his valuable help in microscopy. This work was supported by the Israel Cancer Research Fund Project grant and the Israel Science Foundation Moked grant. This protocol was derived from Parhi et al. (2020). Nature Communications. https://doi.org/10.1038/s41467-020-16967-2
Competing interests
The authors declare no competing interests.
Ethics
Experiments were carried out under protocol MD-17-15239-5 approved by the Hebrew University of Jerusalem Ethics Committee and signed by the chairperson Prof. Sara Eyal.
References
Abed, J., Emgard, J. E., Zamir, G., Faroja, M., Almogy, G., Grenov, A., Sol, A., Naor, R., Pikarsky, E., Atlan, K. A., et al. (2016). Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc. Cell Host Microbe 20(2): 215-225.
Abed, J., Maalouf, N., Manson, A. L., Earl, A. M., Parhi, L., Emgard, J. E. M., Klutstein, M., Tayeb, S., Almogy, G., Atlan, K. A., Chaushu, S., Israeli, E., Mandelboim, O., Garrett, W. S. and Bachrach, G. (2020). Colon Cancer-Associated Fusobacterium nucleatum May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System. Front Cell Infect Microbiol 10: 400.
Castellarin, M., Warren, R. L., Freeman, J. D., Dreolini, L., Krzywinski, M., Strauss, J., Barnes, R., Watson, P., Allen-Vercoe, E., Moore, R. A., et al. (2012). Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res 22(2): 299-306.
Gonzales-Marin, C., Spratt, D. A. and Allaker, R. P. (2013). Maternal oral origin of Fusobacterium nucleatum in adverse pregnancy outcomes as determined using the 16S-23S rRNA gene intergenic transcribed spacer region. J Med Microbiol 62(Pt 1): 133-144.
Han, Y. W., Shen, T., Chung, P., Buhimschi, I. A. and Buhimschi, C. S. (2009). Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth. J Clin Microbiol 47(1): 38-47.
Hill, G. B. (1993). Investigating the source of amniotic fluid isolates of fusobacteria. Clin Infect Dis 16 Suppl 4: S423-424.
Hill, G. B. (1998). Preterm birth: associations with genital and possibly oral microflora. Ann Periodontol 3(1): 222-232.
Mima, K., Nishihara, R., Qian, Z. R., Cao, Y., Sukawa, Y., Nowak, J. A., Yang, J., Dou, R., Masugi, Y., Song, M., et al. (2016). Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis. Gut 65(12): 1973-1980.
Mitsuhashi, K., Nosho, K., Sukawa, Y., Matsunaga, Y., Ito, M., Kurihara, H., Kanno, S., Igarashi, H., Naito, T., Adachi, Y., et al. (2015). Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis. Oncotarget 6(9): 7209-7220.
Nozawa, A., Oshima, H., Togawa, N., Nozaki, T. and Murakami, S. (2020). Development of Oral Care Chip, a novel device for quantitative detection of the oral microbiota associated with periodontal disease. PLoS One 15(2): e0229485.
Kostic, A. D., Gevers, D., Pedamallu, C. S., Michaud, M., Duke, F., Earl, A. M., Ojesina, A. I., Jung, J., Bass, A. J., Tabernero, J., et al. (2012). Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res 22(2): 292-298.
Parhi, L., Alon-Maimon, T., Sol, A., Nejman, D., Shhadeh, A., Fainsod-Levi, T., Yajuk, O., Isaacson, B., Abed, J., Maalouf, N., et al. (2020). Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun 11(1): 3259.
Yamamura, K., Baba, Y., Nakagawa, S., Mima, K., Miyake, K., Nakamura, K., Sawayama, H., Kinoshita, K., Ishimoto, T., Iwatsuki, M., et al. (2016). Human Microbiome Fusobacterium nucleatum in Esophageal Cancer Tissue Is Associated with Prognosis. Clin Cancer Res 22(22): 5574-5581.
Yamaoka, Y., Suehiro, Y., Hashimoto, S., Hoshida, T., Fujimoto, M., Watanabe, M., Imanaga, D., Sakai, K., Matsumoto, T., Nishioka, M., et al. (2018). Fusobacterium nucleatum as a prognostic marker of colorectal cancer in a Japanese population. J Gastroenterol 53(4): 517-524.
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Biological Sciences > Microbiology > Microbial communities
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Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4636 Views: 575
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Original Research Article:
The authors used this protocol in Plant Physiology Sep 2022
Abstract
Ethylene is an important plant hormone that is involved in the regulation of numerous processes in plant development. It also acts as a signaling molecule in response to biotic and abiotic stress conditions. Most studies have investigated ethylene evolution of harvested fruit or small herbaceous plants under controlled conditions, but only a few explored ethylene release in other plant tissues, such as leaves and buds, particularly those of subtropical crops. However, in light of increasing environmental challenges in agriculture (such as temperature extremes, droughts, floods, and high solar radiation), studies on these challenges and on potential chemical treatments for mitigating their effects on plant physiology have become more and more important. Thus, adequate techniques for the sampling and analysis of tree crops are needed to ensure accurate ethylene quantification. As part of a study on ethephon as a mitigating agent to improve litchi flowering under warm winter conditions, a protocol was developed for ethylene quantification in leaf and bud tissue of litchi following ethephon application, taking into account that these plant organs release lower ethylene concentrations than fruit. At sampling, leaves and buds were placed in glass vials of appropriate sizes for the respective plant tissue volumes and allowed to equilibrate for 10 min to release possible wound ethylene before incubating the samples for 3 h at ambient temperature. Thereafter, ethylene samples were aspirated from the vials and analyzed using a gas chromatograph with flame ionization detection, the TG-BOND Q+ column for separation of ethylene, and helium as the carrier gas. Quantification was achieved based on a standard curve derived from an external standard gas calibration with certified ethylene gas. This protocol will also be appropriate for other tree crops with similar plant materials as study foci. It will enable researchers to accurately determine ethylene production in various studies investigating the role of ethylene in general plant physiology or stress-induced plant responses following a range of treatment conditions.
Keywords: Ethylene Gas chromatography Flame ionization detector Headspace Leaves Apical buds Subtropical crops
Background
Ethylene is a gaseous plant hormone that plays an important role in the regulation of numerous physiological and developmental plant processes, such as seed germination, root and shoot development, cellular growth regulation, carbon assimilation, senescence of leaves and flowers, organ abscission, and fruit ripening (Abeles et al., 1992; Pierik et al., 2006; Olsen, 2010; Wang et al., 2013; Dubois et al., 2018). Ethylene also acts as a signaling molecule in responses to biotic and abiotic environmental stresses, such as temperature extremes, droughts, floods, shading, radiation, nutrient deficiency, and mechanical and chemical damage (Yang and Hoffman, 1984; Iqbal et al., 2013; Dubois et al., 2018). The amount of ethylene production depends on the plant species, developmental stage of the plant, and organ type (Cristescu et al., 2013). Apart from endogenous or stress-induced ethylene production, production in plant organs can also be induced by exogenous application of the plant growth regulator ethephon (2-chloroethylphosphonic acid). Ethephon has been widely used in agriculture for different applications, including fruit ripening and coloration, organ abscission to aid harvesting, bloom delay, and suppression of vegetative growth (Nickell, 1994).
Accurate quantification of ethylene evolution from ethephon as well as endogenous ethylene production following different treatments is necessary to correlate production with respective plant responses and elucidate the functions and role of ethylene. According to Cristescu et al. (2013), there are three main methods to detect ethylene in plants: gas chromatography detection, electrochemical detection, and optical detection. These can be used for periodic or continuous measurement depending on the type of plant material and experimental design. Gas chromatography (GC) is most widely used for separation and analysis of ethylene, due to its small sample requirement, high selectivity, and fast analysis. Although more sensitive detection techniques have been developed, such as a laser-based sensing technique (Cristescu et al., 2013; Gwanpua et al., 2018), GC detection is still the most applicable method for tree crops, since non-destructive and/or continuous sampling is not possible. This applies particularly to studies that focus on trees in orchard systems and that are subject to climatic effects.
In fruit tree crops, the quantification of ethylene has mainly been used to assess the effect of endogenous ethylene production in developing fruit, e.g., on fruit coloration (Yin et al., 2001; Chervin et al., 2005; Wang et al., 2007), or in harvested fruit, e.g., on fruit quality and shelf life after harvest (Tseng et al., 2000; Gwanpua et al., 2018). However, few studies investigated ethylene production in leaves, buds, flowers, or other vegetative material from tree crops. Examples are GC detection of ethylene in leaves of citrus (Tudela and Primo-Millo, 1992), in shoot portions of apple (Sanyal and Bangerth, 1998), and in floral buds and leaves of mango (Bindu et al., 2017), peach (Liu et al., 2021) and litchi (Cronje et al., 2022) trees. However, most of these studies only provided limited information on the sampling and analysis techniques used to successfully reproduce the techniques for application in other crops with similar plant organs. For this purpose, we developed a protocol to easily determine ethylene evolution in leaves and apical buds of litchi as part of a recent study, which used ethephon to induce bud dormancy and delay panicle emergence for more consistent floral initiation in litchi (Cronje et al., 2022). To determine the mode-of-action of ethylene inside the leaf and bud tissue, as well as the downstream processes, such as relative expression of ethylene-, dormancy-, and flowering-related genes, ethylene concentration needed to be quantified reliably and accurately in these plant organs to correlate the results with those obtained from corresponding biochemical and molecular analyzes (Cronje et al., 2022). The sampling technique and incubation period was adapted specifically for litchi to account for low overall ethylene production and wound ethylene release after detaching of the respective plant organs. The quantification protocol was derived from a protocol for the quantification of ethylene evolution in tomato leaves developed by Kim et al. (2016) and used a porous layer open tubular (PLOT) TG-Bond Q+ column (Thermo Scientific) for direct separation of ethylene. While several related studies used packed alumina (Al2O3) columns that are recognized for the separation of hydrocarbons from C1 to C5, the use of the mentioned PLOT column is considered advantageous since it is specifically developed for selective separation of acetylene (C2H2), ethylene (C2H4) and ethane (C2H6) to baseline. Additionally, the use of a capillary column in conjunction with a flame ionization detector has proven to be highly sensitive with a linear calibration range of three orders of magnitude, i.e., 0.08 nL to 80 nL of injected C2H4. Although the protocol was specifically developed for leaves and buds of litchi, it can be equally applied to plant material from other fruit tree crops by making appropriate changes to container sizes.
Materials and Reagents
100 μL gas tight syringe with needle: 50 mm length, 23 gauge, point style 5, side hole (Thermo Scientific, catalog number: 36520050)
2,500 μL gas tight syringe with needle: 65 mm length, 23 gauge, point style 5, side hole (Thermo Scientific, catalog number: 365Q2131)
20 mL crimp top headspace vials (Thermo Scientific, catalog number: CHCV20-14)
20 mm Si/PTFE septa (Cronus, catalog number: VCS-2004-1000)
20 mm Al crimp cap (Cronus, catalog number: VCC-2002CS-1000)
20 mm magnetic crimp cap (Cronus, catalog number: VCC-2002BM-500)
20 mm hand crimper (Cronus, catalog number: VTC-20)
1.8 mL, 9 mm screw top vials (Cronus, catalog number: VZS-0209C-100)
9 mm PTFE/red rubber screw thread closure (Cronus, catalog number VKB-0203-09CB-5000)
51 mm, 23 gauge Hamilton needles, point style 5, side hole (Hamilton, catalog number: 7729-06)
17 mm injection port septum (Thermo Scientific, catalog number: 31303211)
3 ml disposable plastic syringes (generic)
Plastic 2-way valves with luer locks and needle adaptor for syringes (Vernier, catalog number: PS-2WAY)
High purity helium (He) gas, 99.999% (Afrox, Baseline 5.0, catalog number: 524203-SE-C)
79 μL·L-1 C2H4 gas standard, balance nitrogen (N2) gas (Afrox, catalog number: GOC mix 3292)
TG-BOND Q+ porous layer open tubular capillary column (Thermo Scientific, catalog number: 26005-6030)
0.32 mm graphite encapsulated ferrules (Thermo Scientific, catalog number: 29053487)
5 mm glass split liner (Thermo Scientific, catalog number: 45350030)
Graphite liner seal (Thermo Scientific, catalog number: 29033406)
Equipment
Long nose pliers (generic)
Straight Iris scissors (generic)
Gas chromatograph with a flame ionization detector (Thermo Scientific, model: Trace GC Ultra)
Autosampler (Thermo Scientific, model: Triplus RSH)
Headspace tool (Thermo Scientific, catalog number: 1R77010-1125)
Air generator (Peak Scientific, model: Precision Zero Air)
N2 generator (Peak Scientific, model: Precision Nitrogen)
H2 generator (Peak Scientific, model: Precision Hydrogen)
2-stage gas regulator with gauges and inlet stem with 5/8’’ left hand BSP nut (Afrox, catalog number: W019220)
Analytical balance (Adam Precision, model: PW184)
Software
Thermo Xcaliber 3.0.63.3 (Thermo Fisher Scientific Inc.)
Excel (Microsoft Office 2016)
Procedure
Hardware setup of gas chromatograph (GC)
Follow the manufacturer’s instruction manual to perform the following setup of the GC:
Install a 5 mm glass split liner together with a graphite seal into the split/splitless injector.
Place a 17 mm coated septum between the septum support and septum holder and hand tighten the injector cap. A diagram together with a photograph of the layout and components that form part of the injector is presented in Figure 1.
Figure 1. Components and layout of the GC’s split/splitless injector. A. Diagram reprinted from Thermo Scientific (2010). B. Photograph of components.
Connect the TG-BOND Q+ capillary column between the flame ionization detector (FID) and split liner using 0.32 mm graphite encapsulated ferrules. The column insertion depths are 40 mm and 94 mm into the split liner and FID, respectively. Figure 2 illustrates the installed column inside the oven, as well as how to determine the correct column insertion depths into the injector and FID.
Figure 2. Installation of the TG-BOND Q+ capillary column. A. View of the column as installed in the GC’s oven between the split/splitless injector and the flame ionization detector (FID). B. Measuring the insertion depth of the column for the FID. C. Details of the position of the ferrule when gauging the column insertion depth for the injector.
Confirm that a supply of high purity He gas at a pressure of 800 kPa is connected to the GC.
Check operation of the Peak Scientific gas generators for providing fuel and make up gas to the FID at pressures and flow rates as specified in Table 1.
Table 1. Pressure and flow specifications of gas supply from gas generators to FID
Gas Pressure (kPa) Flow range (mL·min-1)
H2 420 30–50
Dry air 420 300–600
N2 420 10–60
Follow Table 2 for a summary of chromatography measurement parameters to be configured for the GC in the “Instrument Setup” page of the Xcaliber software.
Table 2. List of GC measurement parameters
Variable Unit Value
Carrier gas - He
Inlet mode - Split
Injector base temperature °C 100
Liner mm 5
Split flow mL·min-1 40
Septum purge mL·min-1 5
Carrier mode - Constant pressure
Pressure kPa 140
Column - TG-BOND Q+
Stationary phase - Divinyl benzene homopolymer
Column length m 30
Column diameter mm 0.32
Column film thickness μm 10
Oven starting temperature °C 60
Heating rate °C·min-1 0
Oven final temperature °C 60
FID base temperature °C 200
H2 flow rate mL·min-1 35
Air flow rate mL·min-1 350
N2 flow rate mL·min-1 30
FID range - 1
FID gain - 1
Analog filter status - On
Sampling depth mm 25
Syringe filling speed mL·min-1 50
Injection speed mL·min-1 50
Injection depth mm 50
Needle penetration speed mm·s-1 25
Configuration of a processing setup in Thermo Xcaliber software
Use the “Processing Setup” option to import a “.raw” file for peak identification. The “.raw” file should represent a GC trace from a gas injection consisting of a low concentration C2H4 gas.
Follow Table 3 for the set of variables to be configured into the software.
Table 3. Parameters for the GC’s software processing in terms of peak identification
Page Variable Value
Identification Detector type Analog
Peak detect ICIS
Expected time (min) 1.64
Window (sec) 5.00
View width 0.75
Detection Smoothing points 1
Baseline window 60
Area noise factor 5
Peak noise factor 10
ICIS peak detection Highest peak
Minimum peak height (S/N) 3.0
Calibration Component type Target compound
Weighting Equal
Calibration curve Linear
Units nL
Origin Ignore
Response Area
Levels 1 0.075
2 0.150
3 0.301
4 0.752
5 1.505
6 3.010
7 8.809
8 16.709
9 24.609
10 44.359
11 83.859
An example of a part of the processing setup is depicted in Figure 3 illustrating a retention time of 1.64 min.
Figure 3. Example of the Xcaliber Processing Setup for peak identification
Creation of sequence setups in Xcaliber software for calibration standards and samples
Create a sequence file using the “Sequence Setup” window in Xcaliber consisting of 11 entries for the calibration standards. It is important to reference the instrument method file under heading “Inst Meth” and the processing method under “Proc Meth”.
Give a file path and name for the calibration file in the sequence table under the heading “Cal File”.
Create a sequence file for the planned number of samples and enter the name of the calibration file to be used under the heading “Cal File”.
Note that the tray holder and slot position is only important for calibration standards 1 to 6 since the remainder of injections are done manually.
Details to create sequence files are further illustrated in Figure 4.
Figure 4. Steps to follow in the Xcaliber software to create sequence files
Collection of plant material
Detach two leaflets (one leaflet each from two terminal shoots) from the compound leaf closest to the terminal bud by manually breaking them off at the natural abscission layer. Remove up to 20 terminal buds (4–8 mm in length, depending on bud stage and size) with straight sharp Iris scissors. Insert the leaflets and buds into the 20 and 2 mL vials, respectively, as illustrated in Figure 5. This will yield minimum masses for buds and leaves of 0.2 and 1.1 g, respectively. The actual mass of plant material is not critical because the calculated rate of C2H4 release is normalized to unit mass of plant material. Refer to equation 5.
Figure 5. Leaves and buds sealed in 20 mL (left) and 2 mL (right) vials, respectively
Document the time of harvesting of each individual sample as reference for the standardization of the 10 min duration required for the release of wound ethylene (see next step).
Leave vials open for 10.0 ± 0.5 min after insertion to allow for release of wound ethylene. This can readily be achieved at the location of harvesting under ambient conditions.
After the 10-min equilibration time, seal the 20 mL vials with 20 mm Si/PTFE septa and 20 mm aluminum crimp caps using the 20 mm hand crimper.
Ensure that the septa inside the crimp caps have the PTFE face downwards as illustrated in Figure 6, i.e., the PTFE face will contact the beveled edge of the headspace vial.
Figure 6. Position of a Si/PTFE septum inside a crimp cap. A. Top view. B. Bottom view.
Close the screw caps on the 2 mL vials hand-tight following the 10-min equilibration time.
Document the time when each individual vial is sealed as starting time of the 3-h incubation period (see step H1).
Preparation and GC measurements of C2H4 calibration standards by headspace vial dilution
For calibration standards 1 to 6, seal 6 × 20 mL headspace vials containing ambient air with 20 mm magnetic crimp caps and 20 mm Si/PTFE septa.
With a 2-stage regulator connected to a 79 μL·L-1 C2H4 gas cylinder, seal the low-pressure outlet with a custom sized Si/PTFE septum inside a nut.
Set the outlet pressure of the regulator to 50 kPa. It is important not to exceed 100 kPa as it presents a danger to equipment and personnel due to the nature of the configuration of the outlet. The initial pressure setting is done as follows:
The cylinder shut-off valve is in the closed position.
Turn the control knob on the regulator fully counterclockwise to have the regulator outlet in the closed position.
Open the cylinder shut-off valve and close it immediately again; this will charge the inlet stem and regulator with the cylinder gas.
The reading on the regulator’s high-pressure gauge now indicates the cylinder pressure.
Turn the regulator’s control knob slowly clockwise until the low-pressure gauge reads 50 kPa.
Purge the regulator three times with the analytical gas from the valve side to the low-pressure outlet of the regulator as follows:
It is important to have the cylinder shut-off valve closed at the start of the procedure.
Insert a 23 gauge needle through the septum on the low-pressure outlet.
Monitor the pressure on the high-pressure gauge of the regulator until it decreases close to the zero reading. Remove the needle just prior to the gauge reaching zero. This implies that the needle should be removed before the low-pressure gauge decreases from 50 kPa. A small decrease in outlet pressure from the 50 kPa setting can be tolerated as long as the gauge pressure remains positive to prevent air from flowing back into the outlet of the regulator.
Open the cylinder valve and close it again to charge the inlet stem and regulator once more.
Insert the needle through the septum again and repeat steps 4a–4d three times. The necessity of purging in triplicate is to ensure that all air inside the inlet stem and regulator is replaced by gas from the cylinder and thereby ensuring that the actual analytical gas is aspirated when preparing standards.
Use the 100 μL and 2,500 μL syringes to aspirate volumes of analytical gas from the low-pressure outlet of the regulator that is connected to the 79 μL·L-1 C2H4 gas cylinder. Figure 7 presents the technique for extracting gas from a cylinder using a gastight syringe.
Figure 7. Aspirating a volume of C2H4 through a septum from a pressure regulator
The required volumes, Vsyringe, for injection into the 20 mL headspace vials are specified in column 3 of Table 4, with a photographic illustration of the use of a 100 μL syringe given in Figure 8. A needle insertion depth of 30 mm is sufficient.
Figure 8. Use of a 100 μL syringe to dilute C2H4 gas in a headspace vial
The syringe technique should consist of a volume extraction that exceeds the specified volume. The syringe plunger can then be pushed back to the required volume to eliminate pressure effects inside the syringe.
Inject 1 mL volumes of standards 1 to 6, Vinj, into the GC using the Triplus autosampler with the configurations as described for the instrument method, as well as processing and sequence setups. An automated injection step using a headspace tool is presented by the photograph in Figure 9.
Figure 9. Automated injection with a headspace tool
Since an FID is a mass sensitive detector, the calibration is performed based on the quantity of analytical gas, i.e., C2H4, injected into the split liner, V’inj. The values presented in Table 4 can be obtained by means of equations 1 to 3:
(1),
(2),
(3).
Definition of the variables are as follows:
Ccylinder = concentration of C2H4 in the gas cylinder (μL·L-1),
Vvial = volume of headspace vial (mL),
Vsyringe = volume aspirated into syringe (μL),
v’ = volume C2H4 in the syringe (μL),
C’vial = concentration C2H4 in headspace vial (nL·L-1),
Vinj = total volume gas injected into the GC liner (mL), and
V’inj = volume of C2H4 injected into the GC liner (nL).
Table 4. Required vial and syringe volumes to prepare standards 1 to 6 in headspace vials
Ccylinder
(μL·L-1)
Vvial
(mL)
Vsyringe
(μL)
v’
(μL)
C’vial
(nL·L-1)
Vinj
(mL)
V’inj
(nL)
79 20 20 1.580 × 10-3 79 1 0.075
79 20 40 3.160 × 10-3 158 1 0.150
79 20 80 6.320 × 10-3 316 1 0.301
79 20 200 1.580 × 10-2 790 1 0.752
79 20 400 3.160 × 10-2 1,580 1 1.505
79 20 800 6.320 × 10-2 3,160 1 3.010
Preparation and GC measurements of C2H4 calibration standards by in-situ syringe dilution
Standards 7 to 11 are prepared by means of in-situ dilution in a 2,500 μL gastight syringe.
Aspirate volumes of gas, Vsyringe, from the cylinder as indicated in column 1 of Table 5. Apply again the technique of extracting volumes in excess of the required and then moving the plunger to the required volume.
Table 5. Required volumes to prepare standards 7 to 11 using in situ syringe dilution
Vsyringe
(μL)
Vdead
(μL)
Ccylinder
(μL·L-1)
V’inj-IS
(nL)
50 61.5 79 8.809
150 61.5 79 16.709
250 61.5 79 24.609
500 61.5 79 44.359
1000 61.5 79 83.859
Thereafter “dilute” the analytical gas with air by setting the plunger to the 1000 μL mark.
Inject 1 mL of standards 7 to 11 manually into the GC during a programmed calibration sequence in the Xcaliber software.
Limit the insertion depth of the needle into the split liner to 50 mm.
Observe that a dead volume, Vdead, of the 2,500 μL syringe that contributes to the volume of analytical gas is indicated in Table 5. This value is syringe-specific.
Calculate the volume of C2H4, V’inj-IS, injected into the liner in nL units by means of equation 4:
(4).
Preparation and GC measurements of blanks and C2H4 quality control (QC) standards
For the 20 mL headspace QC vials, seal 4 × 20 mL headspace vials, containing only ambient air, with 20 mm aluminum crimp caps and 20 mm Si/PTFE septa.
For the 2 mL screw top QC vials, close the screw tops of 4 × 2 mL vials. These vials also only contain ambient air.
For the C2H4 QC standards, use a 100 μL gastight syringe to aspirate 20 μL and 80 μL of the C2H4 standard to prepare three technical replicates of the 2 mL and 20 mL vials, respectively.
To sample the respective gas volumes for QC samples, aspirate from the regulator in excess of the required volume, move the plunger back to the exact required volume and then inject into the respective vials.
Refer to Table 6 for data on the prepared C2H4 QC samples.
Table 6. Required vial and syringe volumes to prepare C2H4 QC samples
Ccylinder
(μL·L-1)
Vvial
(mL)
Vsyringe
(μL)
v’
(μL)
C’vial
(nL·L-1)
Vinj
(mL)
V’inj
(nL)
79 2 20 1.580 × 10-3 790 0.5 0.316
79 20 80 6.320 × 10-3 316 1 0.301
The total number of prepared QC vials consists of 1 × 2 mL blank (containing ambient air), 1 × 20 mL blank (containing ambient air), a 3 × 2 mL C2H4 QC standards (containing 20 μL of C2H4 standard), and a 3 × 20 mL C2H4 QC standards (containing 80 μL of C2H4 standard).
Perform gas sampling from the QC vials using disposable 3 mL syringes with luer valves, each connected to a 23 gauge needle of 51 mm length. An example of a syringe with a luer valve can be seen in Figure 10.
Figure 10. Disposable 3 mL syringe with luer lock and attached needle
Aspirate 1 mL and 0.5 mL from the 20 mL and 2 mL vials, respectively.
An advisable syringe technique is to cycle the required volume two times with the needle stationary through the septum prior to aspirating the required volume.
Take care to close the luer valve while holding the plunger in position for the required aspiration volume. This is specifically relevant when sampling 0.5 mL from a 2 mL vial, i.e., air pressure will restore the plunger to a volume less than extracted, which is prevented by ensuring that the valve is closed prior to releasing the syringe plunger. The technique to achieve this is demonstrated by the sequence of photographs in Figure 11.
Figure 11. Technique to aspirate a gas sample from a small volume (2 mL) vial with a disposable syringe. A. Positioning the needle through the septum with the luer lock open. B. Withdrawing of a gas sample by moving the plunger outwards. C. Closing the valve’s luer lock while holding the plunger in position at the required volume.
Use a clean 3 mL syringe to flush needles that are alternating between vials to ensure any C2H4 gas is expelled from the needles.
Inject the QC samples manually into the GC liner during a programmed sequence.
The syringe can be inserted with its full length through the injector’s septum, i.e., 51 mm, to agree with the programmed injection depth setting of 50 mm. The manual injection process is illustrated by the sequence of photographs in Figure 12.
Figure 12. Sequence of steps for manual injection. A. Positioning the needle on top of the injector. B. Inserting the needle through the septum. C. Opening the valve’s luer lock while maintaining control of the syringe’s plunger to prevent the head pressure from pushing the plunger upwards. D. Pressing the plunger down to inject the gas sample into the injector.
GC measurements of C2H4 evolved from leaves and buds
Perform gas sampling 3.00 ± 0.05 h after sealing vials using disposable 3 mL syringes with luer valves connected to 23 gauge needles of 51 mm length. The 3-h incubation period for each vial consists of the total time the plant material is allowed to release C2H4 under sealed conditions. It includes time between sealing of the vials at the location of collection (orchard), transport to the laboratory, and monitored time on the laboratory bench pending gas sampling. The 3-h incubation period is effectively terminated once gas is sampled from a vial using a syringe with a luer valve.
An advisable syringe technique is to cycle the required volume twice with the needle stationary through the septum prior to aspirating the final required volume.
The volumes to aspirate are 1 mL and 0.5 mL from the 20 mL and 2 mL vials, respectively. Piercing of the plant material should not occur. In the case of leaves, this is prevented by the technique with which the leaves are inserted into the vial, i.e., predominantly against the wall of the vial. In the case of the buds, the insertion of the needle is visibly controlled to avoid contact with the plant material. Moreover, the needle has a dome tip with a side hole.
Take care to close the luer valve while holding the plunger in position for the required aspiration volume as described under G10.
Document the sampling time of each vial.
Use a clean 3 mL syringe to flush needles that are alternating between sample vials to ensure any C2H4 gas is expelled from the needles.
Once a headspace volume is aspirated into a syringe and locked, the syringe can be stored for the duration of the sequence of chromatography measurements.
Inject gas samples manually into the GC in accordance with a programmed sequence file.
The needle can be inserted with its full length through the injector’s septum as described under G13.
Weighing of plant material
Following chromatography analysis, remove the 20 mL vial caps with a pair of pliers and unscrew the caps of the 2 mL vials for weighing the vials individually with their contents.
Remove the contents and weigh the masses of the empty 20 mL and 2 mL vials as associated with each sample number.
Data analysis
Use the “Quan Browser” function in the Xcaliber software to retrieve quantitative reports of calibration standards and samples as exemplified in Figures 13 and 14. Note from Figure 14 that the peak status of the “blank” is indicated as “not found” by the software. This confirms that C2H4 is absent, hence no retention time can be identified, or peak area calculated.
Figure 13. Example of quantitative data of calibration standards from the Xcaliber “Quan Browser” software
Figure 14. Example of quantitative data of samples from the Xcaliber “Quan Browser” software
In the “Quan Browser” window, use the file menu “Export data to Excel” to create an Excel document with the measured data for individual sequences.
Perform data processing in Excel to calculate the specific C2H4 production rate, R, in μL·kg-1·h-1, using equation 5. Assignment of variables is as follows:
GCC2H4 = GC analyzed C2H4 volume, i.e., calculated amount in Quan Browser (nL),
Vvial = volume of vial (mL),
Vinj = total volume of gas injected with syringe (mL),
mvc = mass of vial and contents (g),
mv = mass of empty vial (g) and
t = difference between time of sealing vial and, ts, time of gas aspirated from vial (h), ta.
(5).
Notes
Depending on the size of the plant material to be analyzed, the number of plant organs and/or vial sizes can be adjusted.
A minimum of five biological replicates, with sub-samples per replicate, is advisable as considerable variation in C2H4 readings can be expected after exogenous C2H4 application, e.g., through ethephon application.
The duration for wound ethylene release and incubation period can differ when plant tissues of other crops and vial volumes, respectively, are used as study material. It is advisable to determine these periods before the experiment is conducted.
As a cautionary point, it is important to perform timing accurately to ensure that the incubation time is the same for all samples.
It is advisable to perform ad-hoc qualitative chromatography of typical samples prior to systematic quantitative measurements to assess the expected maximum and minimum levels of C2H4. The observed peak areas can serve as a guide to the required calibration ranges.
Table 3 lists the retention time of C2H4 as 1.64 min. Slight deviation from the expected 1.64 min may still occur even if the protocol is reproduced in its totality. The retention time for C2H4 (or any compound of interest) can be identified or confirmed by an increase in peak area following the measurement of GC traces for a sequence of standards with increasing C2H4 concentration.
Calibration curves with a correlation coefficient >99.0% can be easily attained with the described technique.
It is worthwhile to include blank and QC injections intermittently during a sequence to calculate precision data and to validate the measurements of unknowns.
Acknowledgments
This work was supported by funding from the South African Litchi Growers’ Association and the Agricultural Research Council of South Africa. This protocol was adapted from previous work (Kim et al., 2016; Cronje et al., 2022).
Competing interests
The authors declare no financial and non-financial competing interests.
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4,637 | https://bio-protocol.org/en/bpdetail?id=4637&type=0 | # Bio-Protocol Content
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Live Imaging of Phagoptosis in ex vivo Drosophila Testis
DK Diana Kanaan
BS Boris Shklyar
LP Lilach Porat-Kuperstein
HT Hila Toledano
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4637 Views: 753
Reviewed by: Gal HaimovichRafael Sênos DemarcoRajesh D Gunage
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Original Research Article:
The authors used this protocol in Science Advances Jun 2022
Abstract
Phagoptosis is a prevalent type of programmed cell death (PCD) in adult tissues in which phagocytes non-autonomously eliminate viable cells. Therefore, phagoptosis can only be studied in the context of the entire tissue that includes both the phagocyte executors and the targeted cells doomed to die. Here, we describe an ex vivo live imaging protocol of Drosophila testis to study the dynamics of phagoptosis of germ cell progenitors that are spontaneously removed by neighboring cyst cells. Using this approach, we followed the pattern of exogenous fluorophores with endogenously expressed fluorescent proteins and revealed the sequence of events in germ cell phagoptosis. Although optimized for Drosophila testis, this easy-to-use protocol can be adapted to a wide variety of organisms, tissues, and probes, thus providing a reliable and simple means to study phagoptosis.
Keywords: Confocal microscopy Cyst cells Drosophila testis Germ cell progenitors LysoTracker Phagoptosis Programed cell death (PCD) Time-lapse live imaging
Background
Several types of programmed cell death (PCD) have been described, all of which function to eliminate excess or damaged cells throughout development and to maintain tissue homeostasis in adult organisms (Fuchs and Steller, 2011). Phagoptosis, a newly recognized type of PCD, is a form of cell non-autonomous cannibalism in which one cell uses the phagocytic machinery to induce death and degradation of a nearby cell that would otherwise remain alive [(Brown and Neher, 2012); Figure 1A]. Accordingly, inhibiting the phagocytic machinery within phagocytes prevents the death of targeted cells. In adults, phagoptosis is the most prevalent form of PCD, mediating the turnover of several abundant short-lived cells, including erythrocytes (Olsson and Oldenborg, 2008) and neutrophils (Jitkaew et al., 2009). This important process was only recently recognized because it can occur only in the context of entire tissues that include the two cell types, i.e., phagocytes and targeted live cells. Therefore, developing assays and finding biomarkers for this process represents a major step in characterizing the process and identifying the regulators of phagoptosis.
Figure 1. Phagoptosis of germ cell progenitors. A. The mechanism of phagoptosis. A phagocyte (pink) engulfs a live-targeted cell (green), creating a phagosome around that target, and recruits the phagocytic machinery (Rab5, Rab7, Lamp1, and lysosomes) to degrade its contents. B. Schematic representation of the apical tip of the Drosophila testis (side view). Germline (green) and cyst (pink) stem cells surround the hub cells (gray). Approximately a quarter of the spermatogonia progenitors (green) undergo spontaneous phagoptosis (red) by neighboring phagocytic cyst cells (pink). Dashed line separates progenitors from terminally differentiated spermatocytes.
In the Drosophila testis, as many as a quarter of newly emerging spermatogonia progenitors are spontaneously eliminated by germ cell death (GCD) ( Yacobi-Sharon et al., 2013; Napoletano et al., 2017). We recently showed that the underlying mechanism of GCD is phagoptosis (Zohar-Fux et al., 2022). The cyst cells that surround the germ cell progenitors rely on the phagocytic machinery to induce death and degradation of targeted germ cells (Figure 1B).
Several fluorescent markers can be used to mark the phagocytic cyst cells during the live imaging protocol, including cytoplasmic green fluorescent protein (cytGFP; Figure 2 and Video 1) or membrane-targeted CD8-GFP (Lee and Luo, 1999)]. To follow the activity of the cyst cell–derived phagocytic machinery, we used yellow fluorescent protein (YFP) tags inserted at the DNA level at the endogenous chromosomal loci of Rab5- and Rab7-containing endosomes [Video 2 (Dunst et al., 2015)]. During live imaging, we maintain the testes in a medium containing a low concentration of LysoTracker and Hoechst to visualize in situ changes in lysosomal activity and DNA integrity, respectively. Intriguingly, this technique revealed the formation of large blebs pinching off the dying germ cells that were not detected in fixed samples. While the surfaces of these blebs were positive for LysoTracker, their inner content was negative for such staining, suggesting that nutrients of the dying germ cells are transported to the cyst cells via these blebs [Video 2 (Zohar-Fux et al., 2022)]. In this protocol, we dissect whole-mount intact testes and visualize the spontaneously occurring phagoptosis process for 4–12 hours. Since the testis is a highly regenerative tissue and the germline stem and progenitor cells divide frequently, the entire tissue throbs, which may affect imaging resolution. Therefore, tight adherence of the testis to the chamber without damaging its integrity is the most critical step in obtaining a detailed live imaging movie. A previous live imaging protocol designed to study the stem cell niche of the Drosophila testis suggested the use of the isotonic Ringer's solution as the dissection medium (Greenspan and Matunis, 2017). Indeed, using Ringer's solution significantly improves the ability of the light testis to sink to the bottom of the coverslip and facilitates the adherence process.
Figure 2. Phagoptosis of germ cell progenitors. A-C. Snapshots of live-imaged testis, marked with LysoTracker (red), Hoechst (blue, nuclei), and GFP (cyst cells, c587Gal4;UAS-cytGFP). Time (hour:min) is shown on the bottom right corner. Note the rectangles and bottom images (single-channel views of the boxed regions) highlighting a phagoptosis event. White arrowheads mark DNA packed into separate nuclei (A and B) that are further involuted into one bundle (C, arrow). Asterisks mark the hub, and scale bars correspond to 10 μm.
In developing this protocol, we tested several adherence methods, including the use of 0.1% gelatin, Concanavalin A (Con A, 100 ng/mL), double layers of Con A and fetal calf serum (FCS; 10%), and two concentrations of Poly-L-Lysine (0.01% and 0.1%). We found that the dissected testes could be adhered only on Poly-L-Lysine and that the higher concentration of Poly-L-Lysine (0.1%) enabled easy adherence without applying excessive force that could damage the tissue. Coating the dish with poly-L-lysine for immobilization was shown to be detrimental to the development of early germ line cysts (Gartner et al., 2014).
Materials and Reagents
Eppendorf tubes
10 µL pipettor
Poly L-Lysine solution, 0.1% (w/v) in H2O (Sigma-Aldrich, catalog number: P8920)
Fetal bovine serum (Biological Industries, catalog number: 04-007-1A)
Penicillin/streptomycin (Biological Industries, catalog number: 03-031-1B)
Schneider’s Drosophila medium (Biological Industries, catalog number: 01-150-1A)
Hoechst stain (Invitrogen, catalog number: H3570)
LysoTracker (Thermo Fisher Scientific, catalog number: L7528)
Immersion oil (Nikon, catalog number: M210009219)
Dulbecco's phosphate buffered saline (PBS, 10×) (Biological Industries, catalog number: 02-023-5A)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G)
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911-500G)
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016-500G)
Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-100G)
Sucrose (Sigma-Aldrich, catalog number: S0389-500G)
HEPES solution (1 M, pH 6.9) (Sigma-Aldrich, catalog number: H0887-20ML)
Schneider’s medium (see Recipes)
Ringer’s solution (see Recipes)
Equipment
Standard fly husbandry equipment (e.g., vials with freshly prepared standard cornmeal molasses agar medium, 25 °C incubators, CO2 and fly pad station to anaesthetize flies, and brushes to sort and transfer the flies)
Chambered coverslip containing eight wells (ibidi, catalog number: 80826; Figure 3A)
Superfrost microscope slides (Thermo Scientific, catalog number: 631-0705). A superfrost slide is used as a dissecting dish and can be re-used after cleaning with 70% ethanol.
Dumont #5 blunter forceps (e.g., Fine Science Tools, catalog number: 11252-20)
Dumont #55 sharper forceps (e.g., Fine Science Tools, catalog number: 11255-20)
Zooming stereomicroscope with 5 × 40 magnification (e.g., Carl Zeiss, model: STEMI SV 11)
Confocal microscope. (e.g., Nikon A1R inverted laser scanning confocal microscope with a Plan Apochromat VC 60× oil objective)
Biological safety cabinet (hood) (e.g., ESCO, model: AC2-4S1)
Software
Microscope controller software (e.g., Nikon NIS Elements C v. 4.2 software, https://www.microscope.healthcare.nikon.com/products/software/nis-elements)
Cell imaging software (e.g., Imaris for Cell Biologists, Oxford Instruments, https://imaris.oxinst.com/)
ImageJ/FIJI https://imagej.net/downloads
Procedure
Schneider’s medium
In a biological safety cabinet (hood), prepare Drosophila tissue culture medium (0.5 L): 10% (v/v) fetal bovine serum and 0.5% penicillin/streptomycin in Schneider’s medium.
Prepare 600 µL aliquots of the medium in Eppendorf tubes and keep the medium bottle and aliquots at 4 °C. On the day of the experiment, transfer one aliquot to room temperature (RT).
Ringer's solution
Prepare 50 mL of Ringer's solution. The testes sink easily in the isotonic Ringer's solution, which facilitates the adherence process.
Divide the Ringer’s solution into 200 µL aliquots in Eppendorf tubes and keep at 4 °C. On the day of the experiment, transfer one aliquot to RT.
Coverslip coating
Cover the chambered coverslip containing eight wells with 300 µL per well of 0.1% Poly L-Lysine for 40 min at RT or overnight at 4 °C.
Collect the Poly L-Lysine back into an Eppendorf tube for re-use. Remove traces of Poly L-Lysine with a 10 µL pipettor and leave the coverslip to completely dry on the bench (~1 h). The coated chambered coverslip can be kept until all the wells are used.
Fly husbandry
Prepare the genotype/conditions being tested (e.g., starvation and/or aging). Obtain the appropriate transgenic line (e.g., rab5-YFP or rab7-YFP; Video 2) or establish the crosses that yield male flies expressing the appropriate fluorescent marker in the desired genetic background [e.g., to mark cyst cells with GFP (Video 1), we generated the cross c587Gal4;UAS-cytGFP].
Collect 5–6 offspring males from the desired genotype and maintain them for 2–3 days before the experiments. Phagoptosis is less abounded in 1-day-old flies and appears in all testes dissected from 2–3-day-old males or older.
Video 1. Cyst cells marked with cytGFP. A representative example of live, ex vivo young Drosophila testis is labeled with LysoTracker (red) and Hoechst (blue, nuclei). Scale bars represent 10 µm; time (hour:min) is shown on the bottom of the movie and single channels are presented in grayscale. Cyst cells are marked with cytGFP (green, c587Gal4;UAS-cytGFP). Arrow marks a phagoptosis event. Note the packed DNA in separate nuclei that becomes involuted into one bundle.
Video 2. Rab7-YFP. Live-imaged testis from Rab7-YFP (green) labeled with LysoTracker (red). Scale bars represent 10 µm; time (hour:min) is shown on the bottom of the movie and single channels are presented in grayscale. Arrow marks late endosome vesicles surrounding phagotosed germ cells and recycle blebs. Arrowhead marks a new phagoptosis event that initiates after 1 h and 20 min.
Dissection of whole-mount adult testes and mounting (see Video 3)
Video 3. Dissection of whole-mount adult testes. Forceps handling in testes dissection. The circle marks a separated pair of coiled coil testes
Prepare Ringer's solution with Hoechst stain and LysoTracker (RHL solution). First, prepare Hoechst stock solution of 10 mg/mL (dissolved in water) and a LysoTracker stock solution of 1:10 diluted in PBS (1 µM, can be kept for one week at 4 °C). Add 1 µL of the LysoTracker stock solution (0.03 µM, final concentration) and 0.5 µL of Hoechst stock solution (0.3 µM, final concentration) to the 200 µL Ringer's solution aliquot and maintain at RT. This final RHL solution can be used only on the day of the experiment.
Transfer one aliquot (600 µL) of Schneider’s medium to RT.
Clean the two forceps (#5 and #55), superfrost microscope slide, and CO2 pad with 70% ethanol before dissecting.
Anesthetize adult flies using CO2. Using the stereomicroscope, collect 5–6 males with a paintbrush and leave them on the pad to be dissected.
Under moderate magnification (10×) of the stereomicroscope, add a drop of 50 µL of RHL solution onto the superfrost microscope slide and avoid liquid dispersion. Use the blunter forceps in your non-dominant hand to gently transfer one male from the CO2 pad into the drop of RHL solution. Lay the fly on its back or side and keep holding it gently, submerged in the RHL solution. Use the sharper forceps in your dominant hand to gently tear and grab the cuticle near the bottom of the male's abdomen to release the testes into the RHL solution. Hold the posterior cuticle that contains the testes with the blunter forceps and gently pull with the sharper forceps, until you separate the pair of coiled coil testes (Video 3). Discard the rest of the fly and dissect a second male.
Add 10 μL of RHL solution to one of the eight wells of the Poly L-Lysine-coated chambered coverslip (Figure 3A). Use one prong of the forceps to transfer the dissected testes from the slide dish to the drop in the chamber (Figure 3B–3C). Use one prong of the #5 blunter forceps to gently pull each testis from its basal edge to open the coiled shape, so as to adhere the sample to the bottom of the well and expose the apical tip without damaging the tissue (Figure 3D). Damaged samples should be removed to avoid affecting the intact samples by death signals.
Figure 3. Testis mounting. A. Poly L-Lysine-coated chambered coverslip containing eight wells. B–C. Dissected testis in one well containing 10 μL of RHL solution can appear as a completely (B) or partially (C) coiled coil. D. White and black arrows mark the apical and basal edges of the testis, respectively. Pulling the testis from its basal edge with one prong of the forceps adheres the testis to the bottom of the well and exposes the apical tip for live imaging.
Repeat steps E5 and E6 to obtain a total of two wells, each containing four testes. The rest of the wells can be left empty for the next experiment.
Gently add 300 μL of Schneider’s medium to the corner of the well to avoid detachment of the samples. Cover the chambered coverslip with the lid and proceed to the confocal microscope. Live imaging is performed at RT. At the end of the experiment (after live imaging is completed), add 70% ethanol to the used wells to prevent contamination and allow subsequent use of the empty wells for the next experiment.
Time-lapse confocal imaging
Use confocal or spinning disk imaging system (the protocol can be adapted for virtually any imaging setup). We used a Nikon A1R laser scanning confocal inverted microscope equipped with four lasers (406, 492, 561, and 639 nm), a detector unit with four normal PMTs (four channels), a galvano scanner, a motorized XYZ stage, a widefield fluorescence module, a cMOS camera and NIS Elements C software, and a 60× (NA = 1.4, Plan Apo VC) oil immersion objective.
Turn on the system and place the 8-well coated chambered coverslip on the stage. Use the eye piece and widefield fluorescence to find intact testes with the low magnification objective (10×). Take widefield images using the capture function of the camera for each of the eight testes in order to later locate and examine each sample at 60× and to pick the desired one for final time-lapse imaging.
Lower the 10× objective and swap it to 60× objective and apply immersion oil on it. Find all the eight testes in captured images and direct the stage to relocate there automatically. Pick the testis that is best adhered, shows strong GFP, LysoTracker, and Hoechst signals, and is undamaged. Raise the objective back and fine tune the focus. Switch from widefield mode to confocal.
Set the imaging parameters or reuse from a previously saved image file. We use NIS Elements C v. 4.2 software, and the following parameters are implemented to image phagoptosis in live Drosophila testis.
Open the Dye and Spectral Setting (Filter and Dye) window (Figure 4).
Figure 4. Filter and Dye window
Choose settings as follows:
Setting Mode: Auto
Ch1 (excitation laser 406 nm, filter 425–475 nm) for Hoechst, Ch2 (excitation laser 492 nm, filter 500–550 nm) for GFP, and Ch3 (excitation laser 562, filter 570–620) for LysoTracker Red.
Scanner Unit: Galvano
Detector: Standard Detector (DU4)
Pseudocolor can be chosen for each channel by pressing the channel button.
Open ND Acquisition window for adjusting Z-stack range and time-lapse settings (Figure 5).
For Z-stack imaging, check the Z tab, choose left option of Define Top Bottom. Choose the Top and Bottom positions by using the focus roller while in Find Mode scanning (Figure 6; a quick scan) and pressing Top and Bottom buttons when wanted positions are reached.
Check Save to File and set the Path for automatic file storing.
Set the step to 3 μm.
For time-lapse imaging, check Time tab, check a Phase, and set the Interval (3 min) and Duration (6–12 h).
Figure 5. ND Acquisition window
Open A1plus Settings window for confocal adjustment for final imaging (Figure 6).
Figure 6. A1plus Settings window
Scan Setting:
Scan Direction: line by line
Scan Size: 1024 × 1024 pixels
Scan Speed: 0.125 (Pixel Dwell = ~6 microsecond).
Line Average/Integrate: None
Zoom: 1
Acquisition:
Set pinhole to 1.2.
Adjust the settings for each channel separately in Live mode scanning to get the appropriate image (in this mode, the scanning is performed according to the final parameters listed above).
It is recommended to minimize the laser power as much as possible to avoid photodamage and photobleaching. If the signal is good, adjusting HV (High Voltage, Gain) will be enough to reach the best image quality. Offset is used to reduce the background if needed (it should be done cautiously to avoid signal reduction).
While adjusting the channels Acquisition settings, Saturation indicator should be on to avoid oversaturated pixels.
Go back and forth within the Z-stack range, using the focus roller or clicking within the range in ND acquisition window (Z tab), to ensure the adjustment of each channel setting is balanced for all focal planes.
To start acquiring Z-stack with Time-lapse images, press “Run now” at the bottom of ND acquisition window.
Data analysis
The obtained images can be analyzed in any software of choice. Here, we first used Imaris and then ImageJ/FIJI.
Convert the microscope file to an Imaris file using Imaris File Converter 9.8.2.
Hold right mouse click to rotate the testis to point up apical edge of the testis.
Choose the specific Z-slice that best shows your immunofluorescence staining. Use: Edit → Crop 3D → Select the Z-Slice.
Zoom in to the part you are interested in.
Start recording the time series by pressing the Record function. Record different movies for each separate and merged channel.
Save the movies you have recorded as type: H.264 Movie (*.mp4).
Use the software ImageJ to generate a movie that shows a panel representing separate and merged channels.
Import the movies you have recorded by using: File → Import → Movie.
Combine all the movies together to create a panel by using: Image → Stacks → Tools → Combine.
Save the last panel movie of all channels together: frame rate: 10, format: mp4, and codec H.264.
Notes
To label cyst cells with cytGFP, we crossed females of c587Gal4 driver (on the X chromosome, T. Xie, Stowers Institute of Biomedical Research) that drives expression in early cyst cells with UAS-cytGFP males (c587Gal4;UAS-cytGFP). Rab5-YFP or Rab7-YFP (B. Lemaitre, EPFL) express YFP tags that were inserted at the DNA level at the endogenous chromosomal loci of Rab5- and Rab7-containing endosomes (Dunst et al., 2015). Flies and crosses were raised at 25 °C on freshly prepared standard cornmeal molasses agar medium. In addition, any fluorescent tagged proteins that are expressed in germ and/or cyst cells can be used to follow the GCD process.
Quantification of DNA (Hoechst) and phagosome (LysoTracker) volumes can be drawn from the movies, using Imaris (Bitplane) software with an appropriate iso-surfacing threshold.
This protocol can be adapted to other tissues that consist of both the phagocytes and the targeted cells, and that require the combination of genetic labeling of the phagocytes and the use of LysoTracker and Hoechst to establish the kinetic of events.
Recipes
Schneider's medium
0.5 L Schneider’s medium
10% (v/v) fetal bovine serum
0.5% penicillin/streptomycin in Schneider’s medium
Ringer's solution
128 mM NaCl
2 mM KCl
1.8 mM CaCl2
4 mM MgCl2
35.5 mM sucrose
5 mM HEPES, pH 6.9
Acknowledgments
This work was funded by the Israel Science Foundation (207/20). We thank A. Kolpakov for helping with the protocol setup and B. Lemaitre for the gracious gifts of Drosophila stocks. This protocol is derived from Zohar-Fux et al. (2022).
Competing interests
The authors declare that they have no competing interests.
References
Brown, G. C. and Neher, J. J. (2012). Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'. Trends Biochem Sci 37(8): 325-332.
Dunst, S., Kazimiers, T., von Zadow, F., Jambor, H., Sagner, A., Brankatschk, B., Mahmoud, A., Spannl, S., Tomancak, P., Eaton, S., et al. (2015). Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila. Dev Cell 33(3): 351-365.
Fuchs, Y. and Steller, H. (2011). Programmed cell death in animal development and disease. Cell 147(4): 742-758.
Gartner, S. M., Rathke, C., Renkawitz-Pohl, R. and Awe, S. (2014). Ex vivo culture of Drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment. J Vis Exp (91): 51868.
Greenspan, L. J. and Matunis, E. L. (2017). Live Imaging of the Drosophila Testis Stem Cell Niche. Methods Mol Biol 1463: 63-74.
Jitkaew, S., Witasp, E., Zhang, S., Kagan, V. E. and Fadeel, B. (2009). Induction of caspase- and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment. J Leukoc Biol 85(3): 427-437.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22(3): 451-461.
Napoletano, F., Gibert, B., Yacobi-Sharon, K., Vincent, S., Favrot, C., Mehlen, P., Girard, V., Teil, M., Chatelain, G., Walter, L., Arama, E. and Mollereau, B. (2017). p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis. PLoS Genet 13(9): e1007024.
Olsson, M. and Oldenborg, P. A. (2008). CD47 on experimentally senescent murine RBCs inhibits phagocytosis following Fcgamma receptor-mediated but not scavenger receptor-mediated recognition by macrophages. Blood 112(10): 4259-4267.
Yacobi-Sharon, K., Namdar, Y. and Arama, E. (2013). Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart. Dev Cell 25(1): 29-42.
Zohar-Fux, M., Ben-Hamo-Arad, A., Arad, T., Volin, M., Shklyar, B., Hakim-Mishnaevski, K., Porat-Kuperstein, L., Kurant, E. and Toledano, H. (2022). The phagocytic cyst cells in Drosophila testis eliminate germ cell progenitors via phagoptosis. Sci Adv 8(24): eabm4937.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Cell Biology > Cell imaging > Confocal microscopy
Developmental Biology > Reproduction
Cell Biology > Tissue analysis > Tissue imaging
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Peer-reviewed
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SS Sheng Shu
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Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4638 Views: 1368
Reviewed by: David PaulJose Antonio Reyes-Darias Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Nature Communications Aug 2022
Abstract
The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.
Keywords: Separating E. coli Inner membrane Outer membrane Gradient ultracentrifugation
Background
Gram-negative bacteria are characterized by three layers of cell envelope: an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM) (Silhavy et al., 2010). The IM is a phospholipid bilayer consisting of glycerophospholipids like most plasma membranes, while the OM has an asymmetric lipid structure, with phospholipids in the periplasmic leaflet and lipopolysaccharides (LPS) on the cell surface (Funahara and Nikaido, 1980). Besides the difference in lipid components, the IM and OM also have different membrane protein components (Silhavy et al., 2010). The distinct components bestow their specialized functions: the IM plays a major role in active transport, while the OM protects the cell against harmful compounds like enzymes and large antibiotics and also prevents the leaking of proteins from periplasmic space (DiRienzo et al., 1978).
Separating the IM and OM helps to decipher the location and functions of proteins and lipids in the cell (Schnaitman, 1970) and can also provide the first step in membrane protein purification using styrene-maleic acid (Shu and Mi, 2022). Sucrose gradient ultracentrifugation is the most widely used method to separate the IM and OM of Gram-negative bacteria (Miura and Mizushima, 1968; Osborn et al., 1972a and 1972b; Cian et al., 2020). Lysozyme/EDTA (ethylenediaminetetraacetic acid) is used in those methods to treat the bacterial cells, in which EDTA sequesters divalent cations, thus disrupting the lateral electrostatic bridging interactions between adjacent LPS molecules (Nikaido, 1976). However, the chelating of cations by EDTA may affect the functions or structures of some membrane proteins, which need divalent cations, such as Ca2+, Mg2+, or Zn2+, to stabilize tertiary structure or/and keep activities. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli, the extensively studied model organism of Gram-negative bacteria. In this method, lysozyme or EDTA are not used, so it is beneficial for subsequent membrane protein purification. To further test the purity of separated OM and IM, anti-OM (e.g., OmpC) and anti-IM proteins (e.g., AcrB) western blot can be used (Guzman-Flores et al., 2017).
Materials and Reagents
125 mL flask (PYREX, catalog number: 4980)
2,800 mL flask (PYREX, catalog number: 4420)
Pipette tips
Serological pipets
1 L centrifuge bottle (Thermo Scientific, NalgeneTM, catalog number: 3141-1006)
70 mL ultracentrifuge bottle (Beckman Coulter, catalog number: 355622)
37 mL ultracentrifuge tube (Beckman Coulter, catalog number: 344058)
Tissue grinder (DWK Life Sciences, catalog number: 8853030040)
Luria broth (Research Products International, catalog number: L24400-5000.0)
Terrific broth (Research Products International, catalog number: T15100-5000.0)
Agar (BD, BactoTM, catalog number: 214010)
Tris (American Bio, catalog number: AB02000-05000)
Sodium chloride (NaCl) (Dot Scientific, catalog number: DSS23020-5000)
Glycerol (J.T. Baker, catalog number: 2136-03)
Kanamycin sulfate (American Bio, catalog number: AB01100-00010)
Spectinomycin (Research Products International, catalog number: S23000-25.0)
Chloramphenicol (Sigma, catalog number: C0378-25G)
Sucrose (Sigma-Aldrich, catalog number: S0389-1 KG)
IPTG (isopropyl β-d-1-thiogalactopyranoside) (American Bio, catalog number: AB00841-00010)
PMSF (phenylmethylsulfonyl fluoride) (Dot Scientific, catalog number: DSP20270-25)
2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250-100ML)
Glycerol stock of BL21 (DE3) star plysS strain expressing the YejM/LapB complex (Shu and Mi, 2022)
Hydrochloric acid (J.T. Baker, catalog number: 9535-00)
LB medium (see Recipes)
LB agar plate (see Recipes)
TB medium (see Recipes)
1 M Tris-HCl, pH 7.8 (see Recipes)
Lysis buffer (see Recipes)
Dilution buffer (see Recipes)
73% sucrose (see Recipes)
53% sucrose (see Recipes)
20% sucrose (see Recipes)
Equipment
Pipettes (P200, P1000)
Pipette controller (BrandTech Scientific, model: accu-jet pro)
-80 °C ultra-low temperature freezer (PHCbi, model: MDF-U76VA-PA)
Microbiological incubator (VWR, model: F Air 6.3 CF)
Incubator shaker (New Brunswick, model: InnovaTM 44)
Centrifuge (Thermo Scientific Sorvall, model: BP8)
Ultracentrifuge (Beckman, model: OptimaTM LE-80K)
High-pressure microfluidizer (Avestin, model: Emulsi Flex-C3)
Fixed angle rotor (Beckman Coulter, model: Type 45Ti)
Swinging-bucket rotor (Beckman Coulter, model: SW 28)
Ultrapure water system (Millipore, model: Reference A+)
pH meter (Apera, model: pH700)
Procedure
Prepare total cell membrane
Streak the glycerol stock of BL21 (DE3) star plysS strain expressing the YejM/LapB complex on an LB agar plate containing 50 µg/mL of kanamycin sulfate, 50 µg/mL of spectinomycin, and 25 µg/mL chloramphenicol (Kan+Spe+Cam+). Incubate the plate at 37 °C on a microbiological incubator overnight.
Inoculate a single colony from the overnight culturing plate into 20 mL of LB medium (Kan+Spe+Cam+) in a 125 mL flask. Grow the cells at 37 °C with shaking at 220 rpm overnight.
Transfer 10 mL of overnight culture to 1 L of TB medium (Kan+Spe+Cam+) in 2,800 mL flasks (2 L in total). Grow the cells at 37 °C with shaking at 220 rpm to OD600 of 0.6.
Add 200 µL of 1 M IPTG to 1 L of cell culture (final concentration 0.2 mM) and incubate the cells at 30 °C with shaking at 220 rpm for another 4 h.
Centrifuge the culture at 5,000 × g for 10 min at 4 °C to harvest the cells.
Resuspend the cell pellet in 40 mL of lysis buffer (see Recipes) supplemented with 1 mM PMSF and 5 mM 2-Mercaptoethanol. At this step, resuspended cells can be flash-frozen in liquid nitrogen and stored in a -80 °C ultra-low temperature freezer, or move on directly to step A7 to break the cells.
Transfer the cells to a 40 mL tissue grinder. Homogenize the cells by pushing and pulling the grind pestle 10–20 times.
Break the cells in a high-pressure microfluidizer at 15,000 psi with two passes.
Transfer the broken cells to 50 mL centrifuge tubes and remove cell debris by centrifugation at 5,000 × g for 10 min at 4 °C.
Transfer the supernatant to a 70 mL ultracentrifuge bottle and add lysis buffer to the final volume of ~50 mL. Carefully balance the ultracentrifuge bottles.
Ultracentrifuge in a Beckman Coulter type 45Ti rotor at 40,000 rpm (185,677 × g) for 2 h at 4 °C. Keep the membrane pellet after centrifugation.
Separate inner and outer membranes
Resuspend ~3 g of membrane in 55 mL of 20% sucrose with a 40 mL tissue grinder.
Prepare six 37 mL ultracentrifuge tubes. In each tube, from the bottom to the top, layer 14 mL of 73% sucrose, 14 mL of 53% sucrose, and 9 mL of membrane resuspended in 20% sucrose.
Ultracentrifuge without braking at 23,000 rpm (65,000 × g) in a SW 28 rotor for 18 h at 4 °C.
After separation, there should be two bands: the upper band is the IM and the lower one is the OM (Figure 1).
Figure 1. Separation of IM and OM
Cut the end of a P1000 pipette tip, approximately 5 mm from the point. Collect the upper IM layer using the pipette.
Use a new pipette tip and repeat step B5 to collect the OM layer.
To remove sucrose from the membrane, transfer the IM and OM fractions into 70 mL ultracentrifuge bottles. Add dilution buffer into the bottles to a final sucrose concentration of <10%. Carefully balance the ultracentrifuge bottles.
Ultracentrifuge in a Beckman Coulter type 45Ti rotor at 40,000 rpm (185,677 × g) for 2 h at 4 °C.
Discard the supernatant and weigh the wet weight of the membrane. For 1 g of the membrane, add 10 mL of lysis buffer to resuspend using a 40 mL tissue grinder.
Flash-freeze the membrane with liquid nitrogen and store in a -80 °C ultra-low temperature freezer.
Notes
This protocol uses E. coli strain BL21 (DE3) star plysS expressing the YejM/LapB complex as an example to separate IM and OM. This protocol also applies to other E. coli strains.
All the buffers used in this protocol are kept at 4 °C.
When separating the IM and OM, for each 37 mL ultracentrifuge tube, do not add more than 0.5 g of the total membrane. Otherwise, the IM and OM may not be separated well.
To remove the sucrose in the membrane by ultracentrifuge, the membrane must be first diluted to a final sucrose concentration of less than 10%.
Recipes
LB medium
25 g Luria broth, dissolve in 1 L of deionized water. Autoclave at 121 °C for 30 min.
LB agar plate
LB medium with 1.5% w/v agar.
TB medium
47.6 g Terrific broth and 4 mL of glycerol. Dissolve in 1 L of deionized water.
Autoclave at 121 °C for 30 min.
1 M Tris-HCl, pH 7.8
Dissolve 121.14 g of Tris in 800 mL of Milli-Q water. Adjust the pH to 7.8 using hydrochloric acid. Then add Milli-Q water to a final volume of 1 L.
Lysis buffer
50 mM Tris-HCl, pH 7.8
300 mM NaCl
10% glycerol (v/v)
Dilution buffer
50 mM Tris-HCl, pH 7.8
300 mM NaCl
73% sucrose
73% w/v sucrose
20 mM Tris-HCl, pH 7.8
53% sucrose
53% w/v sucrose
20 mM Tris-HCl, pH 7.8
20% sucrose
20% w/v sucrose
20 mM Tris-HCl, pH 7.8
Acknowledgments
Research reported in this publication was supported by NIAID and NIGMS of the National Institutes of Health under award numbers R21AI156595 and R01GM137068. This protocol was previously used in our reported results (Shu and Mi, 2022).
Competing interests
S. S and W. M declare no conflicts of interest.
References
Cian, M. B., Giordano, N. P., Mettlach, J. A., Minor, K. E. and Dalebroux, Z. D. (2020). Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii. J Vis Exp (158). doi: 10.3791/60517.
DiRienzo, J. M., Nakamura, K. and Inouye, M. (1978). The outer membrane proteins of Gram-negative bacteria: biosynthesis, assembly, and functions. Annu Rev Biochem 47: 481-532.
Funahara, Y. and Nikaido, H. (1980). Asymmetric localization of lipopolysaccharides on the outer membrane of Salmonella typhimurium. J Bacteriol 141(3): 1463-1465.
Guzman-Flores, J. E., Alvarez, A. F., Poggio, S., Gavilanes-Ruiz, M. and Georgellis, D. (2017). Isolation of detergent-resistant membranes (DRMs) from Escherichia coli. Anal Biochem 518: 1-8.
Miura, T. and Mizushima, S. (1968). Separation by density gradient centrifugation of two types of membranes from spheroplast membrane of Escherichia coli K12. Biochim Biophys Acta 150(1): 159-161.
Nikaido, H. (1976). Outer Membrane of Salmonella-typhimurium Transmembrane Diffusion of Some Hydrophobic Substances. Biochimica Et Biophysica Acta 433(1): 118-132. 1
Osborn, M. J., Gander, J. E. and Parisi, E. (1972a). Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem 247(12): 3973-3986.
Osborn, M. J., Gander, J. E., Parisi, E. and Carson, J. (1972b). Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247(12): 3962-3972.
Schnaitman, C. A. (1970). Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol 104(2): 890-901.
Shu, S. and Mi, W. (2022). Regulatory mechanisms of lipopolysaccharide synthesis in Escherichia coli. Nat Commun 13(1): 4576.
Silhavy, T. J., Kahne, D. and Walker, S. (2010). The bacterial cell envelope. Cold Spring Harb Perspect Biol 2(5): a000414.
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Microbiology > Microbial cell biology > Organelle isolation
Biochemistry > Lipid > Membrane lipid
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Determining Bone-forming Ability and Frequency of Skeletal Stem Cells by Kidney Capsule Transplantation and Limiting Dilution Assay
HU Hitoshi Uchida
TM Takamitsu Maruyama
WH Wei Hsu
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4639 Views: 372
Reviewed by: Olga Kopach Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Translational Medicine Mar 2021
Abstract
Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.
Keywords: Skeletal Regeneration Calvaria Cell-based Therapy Craniofacial Suture Stem Cell Renal Capsule Transplantation Skeletogenic Mesenchyme Mesenchymal Stem Cell Skeletogenesis
Background
Conventional methods have shown the skeletogenic abilities of mesenchymal stromal cells (MSCs) isolated from bone marrow and other tissues in a Petri dish. However, in vivo transplantation studies indicate that the majority of MSCs lack engraftment, survival, and differentiation abilities (Caplan and Correa, 2011; Zeitouni et al., 2012). Only a small portion of MSCs are genuine skeletal stem cells (SSCs) (Robey et al., 2014; Sacchetti et al., 2007). In addition, in vitro experiments cannot examine certain features that are essential for stem cell stemness, thus lacking critical characteristics to fit into the rigorous criteria for the modern definition of SSCs. Animal models of ectopic bone formation have clear advantages over orthotopic transplantation because of the environments lacking cytokine interference and interactions with endogenous cell types, e.g., bone-forming cells. Three ectopic locations— subcutaneous, intramuscular, and kidney capsule—have been mainly used for transplantation studies (Scott et al., 2012). Subcutaneous implantation is the simplest method, but pertinent concerns arise because of several caveats related to the paucity of bone formation (Yang et al., 1996). Intramuscular implantation implies close contact with native skeletal muscle progenitors, raising concerns about the cellular origin of the ectopic bone that could be converted from host cells by osteogenic stimulus or traumatic injury (Takaoka et al., 1988; Yu et al., 2008). However, no endogenous cell interferes with the analysis of the transplant, which can be easily identified through kidney capsule transplantation. Although technically more demanding, the environment provides the most nutrient-rich resource for robust bone formation from significantly fewer cell numbers (Maruyama et al., 2021). Using kidney capsule transplantation, we have demonstrated ectopic bone generation at a single-cell level (Maruyama et al., 2016). This method also enables the determination of stem cells’ switch from an osteogenic to a chondrogenic fate for cartilage generation (Maruyama et al., 2016; Maruyama et al., 2022a). Furthermore, because kidney capsule transplantation permits in vivo clonal expansion analysis, it is sensitive enough to determine stem cell frequency by limiting dilution analysis (Maruyama et al., 2016; Maruyama et al., 2021). These analyses utilizing kidney capsule transplantation are powerful tools with clear advantages to advance skeletal stem cell research.
Materials and Reagents
50 mL conical polypropylene centrifuge tube (Fisher Scientific, catalog number: 12-565-271)
1.5 mL microtube (Fisher Scientific, catalog number: 05-408-129)
Insulin syringes with Ultra-FineTM needle (BD Biosciences, catalog number: 08290-3284-38)
Artificial tears ointment (Rugby Laboratories, catalog number: 0536-6550-91)
Cotton swab, puritan 6” sterile (Puritan, catalog number: 25-806 1WC)
Povidone-Iodine cotton swab stick, individual packet 10% strength (Medicine, catalog number: MDS093901)
Alcohol pads (PDI/Professional Disposables, catalog number: B60307)
Nonidet P-40 substitute (Sigma-Aldrich, catalog number: 98379)
Capillary tube (Drummond Scientific, catalog number: 1-000-800)
POLYSYN violet braided coated polyglycolic acid absorbable suture (catalog number: G391NV, TruStitch)
Latex gloves (VWR, catalog number: 56617-180), 5 years shelf life at room temperature
SCID mouse (NOD.CB17-Prkdcscid/NCrCrl, Charles River) older than two months of age
Dulbecco’s phosphate-buffered saline without calcium & magnesium (Gibco, catalog number: 10010-023), two years shelf life at room temperature
Dulbecco’s phosphate-buffered saline with calcium & magnesium (Gibco, catalog number: 14040-133), two years shelf life at room temperature
Matrigel (BD Biosciences, catalog number: 354234), two years of shelf life at -20 °C
Ketamine HCl (10 mg/mL) (COVETRUS, catalog number: 071069), two years shelf life at room temperature
Xylazine (1 mg/mL) (COVETRUS, catalog number: 061035), two years shelf life at room temperature
NYLON blue monofilament suture (TruStitch, catalog number: A662NV)
Ethiqa XR (Fidelis Pharmaceuticals LLC, catalog number: 86084-100-30), one year shelf life at room temperature
Paraformaldehyde (J.T. Baker, catalog number: S898-0), seven years of shelf life at room temperature
Silver nitrate (Sigma-Aldrich, catalog number: 10220-100G), >5 years shelf life at room temperature
4% PFA (see Recipes)
2% Nonidet P-40 (see Recipes)
0.02% NP40, 2% PFA (see Recipes)
5% sodium thiosulfate (see Recipes)
Anesthesia (see Recipes)
Equipment
Centrifuge (Thermo Scientific, Sorvall ST 16R Centrifuge)
Incubator (Thermo Forma, Series II Water Jacketed CO2 Incubator)
Procedure/Dissection Board (Aims Lab Products, GPM2)
Fiber-Optic Illuminator with dual goosenecks (Nikon)
Scissors (Iris scissors, Fine Science Tools)
Forceps (Dumont #5SF, Ring, and Graefe forceps, Fine Science Tools)
Needle holder: Halsted-Mosquito Hemostats (Fine Science Tools, catalog number: 91308-12)
Reflex Wound Clips (VWR, catalog number: 203-1000101326-476)
Stapler: Reflex Wound Clips (VWR, catalog number: 203-1000101326-476)
Electrocautery: Cautery Loop Tip High-Temperature 2200 °F (Bovie Medical Corporation)
Heat pad, microwavable (K&H Pet Products, catalog number: 69553)
Electric shaver (OSTER, catalog number: 078996-101-001)
Nikon stereoscope (NIKON, catalog number: SMZ1500)
Spot imaging system (Insight CMOS Digital Camera, SPOT)
Procedure
Preparation of primary suture cells for transplantation (102 to 105 cells)
Count cell numbers after suture cell isolation from mouse or human tissues.
Hint: Details for suture cell isolation can be found in the published protocol (Maruyama et al., 2022b).
Attention: Keep cells on ice.
Melt frozen Matrigel on ice.
Attention: Handle Matrigel on ice to prevent gelation before filling in a syringe.
Transfer the cell suspension from a 50 mL conical tube to a 1.5 mL microtube using a pipette.
Spin the cells down by centrifugation: 400 × g (400 rcf) for 7 min at 4 °C.
Carefully remove the supernatant from the cell pellet.
Hint: Gently tap the microtube to mix the cell pellet with the residual solution.
Resuspend the cells in 5 µL of Matrigel.
Fill the cell-embedded Matrigel (CEM) into an insulin syringe (Figure 1).
Figure 1. Filling the cell-embedded Matrigel into an insulin syringe
Incubate the cell-filled syringe at 37°C for (at least) 5 min.
Hint: The CEM undergoes gelation at temperatures in the range of 22–35 °C.
Making the fire-polished glass capillary tube
Hold a glass capillary on both sides and apply heat at the center using a gas burner (Figure 2A).
Immediately after starting to feel the softness of the tube, gently pull the capillary tube toward both ends, forming a tip of approximately 0.2 mm in diameter.
Fire-polish the pulling side to smooth the tip (Figure 2B).
Figure 2. Preparation of the fire-polished capillary tube. (A) Pulling the capillary tube. (B) Fire polishing the pulling side.
Setup of the surgery field
Clean and sterilize the procedure table using 70% ethanol.
Assemble an area for surgery: a stage, light, an electrocautery device, a needle holder, a heating pad, and surgical tools, e.g., scissors, forceps, and staplers (Figure 3).
Attention: Surgical instruments are sterilized by autoclave.
Figure 3. Preparation of the operation field. The image shows an example of the surgery field.
Anesthesia and disinfection
Weigh the recipient SCID mouse to determine the dose of anesthetics: Ketamine and Xylazine.
Hint: Ketamine: 90–100 mg/kg (body weight) and Xylazine: 9–10 mg/kg (body weight).
Administer the anesthetic cocktail via intraperitoneal injection.
Transfer the animal to a sterile preoperative preparation bench.
Apply lubricating eye ointment to prevent dryness of the eyes during surgery (Figure 4A).
Shave back furs using the electric shaver and wipe the area with an alcohol pad (Figure 4B).
Disinfect the shaved area by gentle swabbing with an iodine solution (Figure 4C).
Wait a couple of minutes for disinfection (Figure 4D).
Remove the iodine by gentle swabbing with an alcohol pad (Figure 4E).
Figure 4. Preparation of animals for surgical operation. (A) Applying eye ointment. (B) Shaving the back furs. (C) Swabbing iodine. (D) Two minutes waiting. (E) Swabbing the iodine with an alcohol pad.
Transplantation
Lift the skin with forceps and use a pair of scissors to make a small incision (~1 cm) below the ribs and 0.5 cm off-center but parallel to the spine (Figure 5A).
Continue to cut through the peritoneum (Figure 5B).
Gently squeeze the abdomen under the incision to expose the kidney (Figure 5C).
Attention: Keep the kidney in a moist condition by applying PBS (Figure 5D).
Figure 5. Preparation of animals for kidney transplantation. Incision of the skin (A), and the peritoneum (B). (C) Exposure of the kidney. (D) Apply moisture to the kidney.
Carefully scratch the surface of the kidney with the needle to make a small opening at the injection site.
Gently insert the fire-polished tip of the capillary tube into the space between the kidney and outer membrane to make some room.
Hint: The idea is to see the opening to enable the syringe to easily penetrate the kidney capsule.
Hint: Do not damage the kidney; especially avoid significant bleeding, which would reduce transplantation efficacy.
Gently insert the insulin syringe containing CEM through the surface opening (Figure 6A).
Attention: The needle should go in a direction right underneath the outer membrane (kidney capsule region), but not into the kidney (Figure 6A’).
Inject the CEM underneath the kidney capsule (Figure 6B).
Hint: The CEM is visible and stable in the kidney capsule (Figure 6B’).
Close the small opening using electrocautery (Figure 6C).
Figure 6. Implantation of cell-embedded Matrigel to the kidney capsule. (A, A’) Insertion of the syringe into the kidney capsule region. (B, B’) Injection of cell-embedded Matrigel. (C) Closure of the small opening.
Gently move the kidney back to the abdominal cavity.
Close the peritoneum using absorbable polystyrene sutures (Figure 7A).
Close the skin using silk or nylon sutures (Figure 7B).
Hint: Alternatively, clip the sutured area using a stapler (Figure 7C).
Administer analgesics (e.g., Ethiqa) subcutaneously to mitigate the pain.
Transfer the animal to a clean heating pad to recover from anesthesia.
Monitor the animal continuously to observe any signs of labored breathing.
Once the animal starts to show signs of being responsive, transfer the animal to a clean cage, allowing free access to food and water.
Hint: There is no need to administer analgesic post-surgery because Ethiqa is effective up to 72 h.
Monitor the mouse daily for at least four consecutive days after surgery.
Retrieve and analyze the transplanted kidney at 2–4 weeks post-operation.
Figure 7. Post-surgical operation procedures. Closure of the peritoneum (A) and skin (B) and staple the sutured area (C).
Whole-mount von Kossa staining
Euthanize animals in a CO2 chamber.
Dissect the transplanted kidney and the contralateral kidney (as experimental control) and soak them in PBS on ice.
Photograph the transplant and control under the Nikon stereoscope using the SPOT image system (Figure 8A).
Prefix the transplant and control with 0.02% NP40/2% PFA in PBS at room temperature for 3 h.
Wash with distilled water at room temperature three times for 10 min each.
Hint: The washing process is critical to reduce background staining.
Place the sample in 1% aqueous silver nitrate at room temperature.
Expose the transplant and control under UV light for 20–60 min at room temperature.
Hint: Monitor the tissue every 10 min, until mineral deposition occurs and turns brown, to terminate the process.
Wash in distilled water at room temperature three times for 10 min each.
Place the stained kidney in 5% sodium thiosulfate at room temperature for 5 min.
Wash in distilled water at room temperature for 10 min.
Evaluate and photograph the stained transplant and control under the Nikon stereoscope using the SPOT imaging system (Figure 8B).
The sample can be post-fixed and decalcified for histological and immunological evaluations.
Figure 8. Analysis of transplanted kidney by whole mount von Kossa staining. Transplanted (right) and non-transplanted (left) kidneys before (A) and after (B) von Kossa staining. Arrows indicate the formation of ectopic bones at the kidney capsule. Enlargements of the inset in A and B are shown in a and b. Scale bars, 5 mm (A, B); 1 mm (a, b).
Data analysis
Stem cell frequency
Evaluation of bone formation after kidney capsule transplantation
Evaluate bone formation by whole-mount von Kossa staining of the transplanted kidney capsule using the Nikon stereoscope with the SPOT imaging system (Figures 4, 5, 6, 7, 8, Supplemental Figures S9, S10 of Maruyama et al., 2021).
Input data in a table format including dose (number of cells), tested (number of transplantations), and response (number of positive transplantations) to the ELDA software on the webpage (http://bioinf.wehi.edu.au/software/elda/).
Enter a confidence interval (95% as default) and select additional checkboxes if necessary.
Click on Run to determine stem cell frequency, based on the successful generation of bone in the transplants.
The “Estimate” indicates the stem cell frequency in the control and experimental groups.
Validate the likelihood ratio test for a single-hit model, to obtain the p-value for the determination of statistical significance (Figure 4 of Maruyama et al., 2021).
Recipes
4% PFA
40 g of paraformaldehyde in 1 L of PBS
2% NP 40
2 g of Nonidet P 40 in 100 mL of distilled water
0.02% NP40, 2% PFA
20 mL of 4% PFA
400 µL of 2% NP 40
19.6 mL of PBS
5% sodium thiosulfate
5 g of sodium thiosulfate in 100 mL of distilled water
Anesthesia
8.5 mL of DPBS (Dulbecco’s Phosphate-buffered Saline without calcium & magnesium)
1 mL of Ketamine (10 mg/mL)
0.5 mL of Xylazine (1 mg/mL)
Acknowledgments
The authors thank former and current lab members for their technical and intellectual support. This work is supported by the National Institutes of Health (DE15654, DE269369) and NYSTEM (C029558) to W.H. This protocol is derived from the original research papers published in Science Translational Medicine (Maruyama et al., 2021) and Science Advances (Maruyama et al., 2022a).
Competing interests
The authors declare no competing financial interests.
Ethics
The care and use of experimental animals described in this work comply with the guidelines and policies of IACUC at the Forsyth Institute and the University Committee on Animal Resources at the University of Rochester.
References
Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
Maruyama, T., Hasegawa, D., Valenta, T., Haigh, J., Bouchard, M., Basler, K. and Hsu, W. (2022a). GATA3 mediates nonclassical beta-catenin signaling in skeletal cell fate determination and ectopic chondrogenesis. Sci Adv 8(48): eadd6172.
Maruyama, T., Jeong, J., Sheu, T. J. and Hsu, W. (2016). Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. Nature Commun 7: 10526.
Maruyama, T., Stevens, R., Boka, A., DiRienzo, L., Chang, C., Yu, H. I., Nishimori, K., Morrison, C. and Hsu, W. (2021). BMPR1A maintains skeletal stem cell properties in craniofacial development and craniosynostosis. Sci Transl Med 13(583).
Maruyama, T., Yu, H. I. and Hsu, W. (2022b). Skeletal Stem Cell Isolation from Cranial Suture Mesenchyme and Maintenance of Stemness in Culture. Bio Protoc 12(5): e4339.
Robey, P. G., Kuznetsov, S. A., Riminucci, M. and Bianco, P. (2014). Bone marrow stromal cell assays: in vitro and in vivo. Methods Mol Biol 1130: 279-293.
Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, P. G., Riminucci, M., et al. (2007). Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131(2): 324-336.
Scott, M. A., Levi, B., Askarinam, A., Nguyen, A., Rackohn, T., Ting, K., Soo, C. and James, A. W. (2012). Brief review of models of ectopic bone formation.Stem Cells Dev 21(5): 655-667.
Takaoka, K., Nakahara, H., Yoshikawa, H., Masuhara, K., Tsuda, T. and Ono, K. (1988). Ectopic bone induction on and in porous hydroxyapatite combined with collagen and bone morphogenetic protein. Clin Orthop Relat Res (234): 250-254.
Yang, Z., Yuan, H., Tong, W., Zou, P., Chen, W. and Zhang, X. (1996). Osteogenesis in extraskeletally implanted porous calcium phosphate ceramics: variability among different kinds of animals. Biomaterials 17(22): 2131-2137.
Yu, P. B., Deng, D. Y., Lai, C. S., Hong, C. C., Cuny, G. D., Bouxsein, M. L., Hong, D. W., McManus, P. M., Katagiri, T., Sachidanandan, C., et al. (2008). BMP type I receptor inhibition reduces heterotopic [corrected] ossification.Nat Med 14(12): 1363-1369.
Zeitouni, S., Krause, U., Clough, B. H., Halderman, H., Falster, A., Blalock, D. T., Chaput, C. D., Sampson, H. W. and Gregory, C. A. (2012). Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration.Sci Transl Med 4(132): 132ra155.
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© 2023 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
Stem Cell > Adult stem cell > Cell transplantation
Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Cell Transplantation > Xenograft
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464 | https://bio-protocol.org/en/bpdetail?id=464&type=0 | # Bio-Protocol Content
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Northern Blot of tRNA in Yeast
Yuehua Wei
Published: Vol 3, Iss 7, Apr 5, 2013
DOI: 10.21769/BioProtoc.464 Views: 14690
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Original Research Article:
The authors used this protocol in The EMBO Journal Aug 2009
Abstract
tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by northern blot.
Keywords: TRNA Northern Blot Yeast Urea P TRNA Northern Blot Yeast Urea PAGE Small RNA
Materials and Reagents
RapidGel (500 ml) (Affymetrix, catalog number: 75848 )
Urea (CO(NH2)2) (Sigma-Aldrich, catalog number: U6504 )
Tetramethylethylenediamine (TEMED, C6H16N2) (Thermo Fisher Scientific, catalog number: 110-18-9 )
APS/ Ammonium persulfate ((NH4)2S2O8) (Sigma-Aldrich, catalog number: A3678 )
Formamide (CH3NO) (Thermo Fisher Scientific, catalog number: 75-12-7 )
DEPC/ Diethylpyrocarbonate (O(COOC2H5)2) (Sigma-Aldrich, catalog number: D5758 )
Bromophenol Blue (C19H10Br4O5S) (Sigma-Aldrich, catalog number: B0126 )
Xylene Cyanol FF (C25H27N2NaO6S2) (Sigma-Aldrich, catalog number: X4126 )
Probe DNA oligo
[γ-32P]-ATP, 10 Ci/mmol 2 mCi/ml, 250 μCi (PerkinElmer, catalog number: BLU002250UC )
Whatman 3 M paper (Whatman, catalog number: 3 MM Chr )
Microspin G-25 column (GE Healthcare, catalog number: 27-5325-01 )
Hybond N+ membrane (Amersham, catalog number: RPN303B )
Ficoll (Sigma-Aldrich, catalog number: F4375 )
Sodium citrate
BSA
Polyvinlypyrrolidone (Sigma-Aldrich, catalog number: PVP360 )
T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201S )
10x PNK buffer (New England Biolabs, catalog number: B0201S )
Tris base (Thermo Fisher Scientific, catalog number: 77-86-1 )
Boric acid (Thermo Fisher Scientific, catalog number: 10043-35-3 )
EDTA (Sigma-Aldrich, catalog number: EDS-1KG )
Methylene blue (Sigma-Aldrich, catalog number: 28514-100G )
Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
Salmon sperm DNA (Life Technologies, InvitrogenTM, catalog number: 15632-011 )
Equipment
Bench top centrifuge
UV cross linker (Strata linker,model: 1800 )
Power supply with constant voltage > 450 V
Exposure cassette / intensifier screen (Sigma-Aldrich, catalog number: C5479-1EA )
Hybridization bottle
Hybridization oven
Large gel cassette (around 20 x 40 cm)
Film developer
Procedure
Part I: Separate tRNAs by denaturing gel
Prepare total RNA by either Trizol RNA extraction kit or by hot phenol method described in Wei (2012). Good quality RNA will have an OD260/280 ratio of 1.8 to 2 and an OD260/230 of 1.8 or greater.
Prepare 10% Acrylamide-6 M Urea denaturing gel in a large gel cassette (around 20 x 30 cm. Mini gel did not work well in my experiment).
Pre-run the gel in 1x TBE buffer with constant 450 V until the gel is heated to 50 °C (about 1 h). Note: I found this to be critical. One reason could be that pre-running the gel to this temperature could help get rid of excessive Urea in the gel, making RNA possible to go through. I usually attached a thermometer to ensure that the temperature reach 50 °C.
RNA samples are mixed with 2x with Formamide loading dye and heated to 70 °C for 2 min.
Turn off power. Rinse out the wells with 1x TBE using a syringe and needle, make sure that urea is rinsed out.
Load the RNA samples (25 μg) and run the gel at constant 450 V for about 2 h (Bromophenol Blue runs around 12 nt and cyanol around 55 nt).
Let the gel cool down to room temperature and disassemble the gel set.
Part II: transfer tRNAs to membrane
Measure gel and cut a piece of Hybond N+ membrane with clean gloves and scissors (washed with DEPC water and autoclaved). Soak the membrane in 1x TBE, attach to the gel. Soak 2 pieces of autoclaved Whatman 3 M paper in 1x TBE and sandwich the gel-membrane.
Transfer gel/membrane/whaman paper sandwich to semi-dry transfer apparatus. Use a sterile transfer pipet to squeeze out bubbles between the membrane and the gel.
Wipe off excess liquid around the edges of gel and transfer at constant 10 V for 2.5 h.
Disassemble the gel and membrane, soak membrane in DEPC water for 1 min. Put the membrane on autoclaved filter paper for 10 min to dry the membrane.
Crosslink the RNAs to the membrane with UV cross linker (Strata Linker 1800) at 1,200 x 100 m Joles. It takes roughly 2 min.
Stain with methylene blue RNA staining solution for 1 min. De-stain by washing with DEPC water several times for 30 sec. This is to evaluate the efficiency of RNA transfer. If transfer is not good, I suggest repeating the above procedures since radioactive material is involved in later steps.
Save the membrane in DEPC water for Northern blotting.
Part III: 5' End-Labeling of Probe
To examine the newly synthesized tRNA, design DNA oligo annealing specifically to the pre-tRNA rather than mature tRNA, because mature RNA is very abundant. Pre-tRNAs will be processed to mature tRNA. During this process, certain DNA sequence will be cleaved. Primers will be designed annealing to the cleaved DNA sequence For example, pre-tRNA Leu3 probe is 5'-CCAAACAACCACTTATTTGTTGA-3'.
Prepare 5' end labeling reaction buffer and label the probe with radioactive ATP at 37 °C for 1 h (see Recipes for detail).
Prepare the microspin G-25 column by vortexing the resin in the column.
Snap off bottom and loosen the cap, place in 1.5 ml Eppendorf tubes and spin the column for 1 min at 700 x g. The gentle centrifugation is critical for the column to form a smooth angled surface.
Place the column in new Eppendorf tube and slowly apply the labeling reaction to center of angled surface of resin bed. Do not disturb the resin.
Spin column for 2 min at 700 x g. Discard column in radioactive waste.
Take out 1 μl of labeled probe in 0.5 ml eppendorf tube, put tube in scintillation vial and count the cpm.
Part IV: Hybridization
Mix hybridization buffer and take 20 ml into 50 ml conical tube.
Heat the buffer in 65 °C water bath until clear. It should be clear within 10 min.
Transfer 10 ml to hybridization bottle with membrane (note transferred side is the proved side) and rotate gently at 37 °C in a hybridization oven for 60 min.
Put the remaining 10 ml hybridization at 37 °C for 30 min.
Heat 100 μl salmon sperm DNA at 90 °C for 1 min and add to the remaining 10 ml hybridization buffer.
Add 20 μl labeled probe into the 10 ml hybridization buffer and shake vigorously.
Decant the buffer in the hybridization bottle and add the new hybridization buffer with labeled probe. Rotate gently at 37 °C in a hybridization oven overnight.
Pre-warm wash buffer to 37 °C in a water bath.
Transfer hybridization buffer with probes to 50 ml conical tube and store it at -20 °C. The probes can be re-used within 2 weeks for 2-3 times, but the auto-radioactive signal will be decreasing.
Wash the membrane with 50 ml wash buffer by rotating at 42 °C for 30 min in the hybridization oven.
Repeat washing for 2 more times.
Take out membrane and wrap with Saran-wrap.
Place the membrane in exposure cassette with intensifier screen and film, put at -80 °C refrigerator.
Develop the film after 3 days. If signal is weak, expose for 1 week.
Recipes
DEPC water
Add 1 ml DEPC to 1 L ddH2O2, mix and put at room temperature overnight. Autoclave.
10x TBE in DEPC water
In 800 ml DEPC water, add 108 g Tris base, 55 g boric acid, 40 ml of 0.5 M EDTA (pH 8.0).
Mix to dissolve and add DEPC water to 1L.
Autoclave.
10% Acrylamide-6 M Urea denaturing gel
Mix 2.5 ml 10x TBE, 6.25 ml Rapid Gel (40%) and 15 g Urea, heat to 50 °C and mix to dissolve.
Add DEPC water to 25 ml then filter through 0.45 μM filter syringe.
Add 25 μl TEMED and 50 μl 25% APS, mix vigorously and transfer to large gel set with appropriate comb.
2x Formamide loading dye
95% (v/v) formamide in DEPC water, add tiny amount of Bromophenol Blue (0.01~0.1%) and Xylene Cyanol FF (0.01~0.1%) , vortex to mix.
Methylene blue RNA staining solution
Dissolve sodium acetate in water to 0.3 M and adjust to pH 5.5 with glacial acetic acid and NaOH. Dissolve 0.02 g methylene blue in 100 ml of above solution and filtered.
5' end labeling reaction buffer
2 μl
10x PNK buffer (fresh from NEB)
30 pmol
probe DNA oligo
3.3 μl
[γ-32P] ATP
1 μl
T4 Polynucleotide Kinase 10 U/ml
Add DEPC H2O to 20 μl
Incubate at 37 °C at least 1 h.
20X SSC (1 L)
NaCl 175 g
Sodium citrate 88 g
Add DEPC water to 1 L and autoclave
50x Denhardt's Solution (100 ml)
Ficoll
1 g
Polyvinlypyrrolidone
1 g
BSA
1 g
Add DEPC water to 100 ml to dissolve, filter solution through 0.45 μm filter syringe.
Hybridization buffer (1 L)
250 ml
20x SSC
100 ml
Denhart's reagent (50x)
10 ml
10% SDS (autoclaved)
Add DEPC water to 1 L, store in 4 °C
Wash buffer (1 L)
100 ml 20x SSC
10 ml 10% SDS (autoclaved)
Add 890 ml DEPC water and store at room temperature.
Acknowledgments
This protocol is derived from the following papers, Wei et al. (2009a) and Wei et al. (2009b), and the relevant references therein. The work was supported by NIH grants R01-CA099004 and R01-CA123391 to Dr. X.F. Steven Zheng at Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey.
References
Wei, Y., Tsang, C. K. and Zheng, X. F. (2009a). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.
Wei, Y. and Zheng, X. F. (2009b). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
Wei, Y. (2012). A simple preparation of RNA from yeast by hot phenol for northern blot. Bio-protocol 2(9): e209.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Molecular Biology > RNA > RNA detection
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4,640 | https://bio-protocol.org/en/bpdetail?id=4640&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Protein Pull-down Assay Using HiBiT-tag-dependent Luciferase Activity Measurement
MA Masashi Arakawa
EM Eiji Morita
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4640 Views: 1094
Reviewed by: Gal HaimovichKeisuke Tabata Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Biological Chemistry Mar 2022
Abstract
Co-immunoprecipitation or pull-down assays are frequently used to analyze protein–protein interactions. In these experiments, western blotting is commonly used to detect prey proteins. However, sensitivity and quantification problems remain in this detection system. Recently, the HiBiT-tag-dependent NanoLuc luciferase system was developed as a highly sensitive detection system for small amounts of proteins. In this report, we introduce the method of using HiBiT technology for the detection of prey protein in a pull-down assay. Using this protocol, we demonstrate the formation of a ternary complex consisting of Japanese encephalitis virus NS4B and two host factors, namely valosin-containing protein, and nuclear protein localization protein 4, which is a critical biological event during flavivirus replication in cells.
Keywords: Pull-down assay HiBiT tag Split nano luciferase Flavivirus Japanese encephalitis virus
Background
Most proteins interact with their partner proteins to carry out their biological activity. To understand the role of proteins in cells, co-immunoprecipitation (co-IP) or pull-down assays are frequently used to characterize protein–protein interactions. Western blotting (WB) is commonly used to detect prey proteins in co-IP or pull-down assays. However, its sensitivity is low or dependent on the primary antibody, and it gives inaccurate quantification.
High Bit peptide of NanoLuc Binary Technology (NanoBiT) (HiBiT-tag, amino acid sequence: VSGWRLFKKIS) is a part of split NanoLuc luciferase that can reconstitute intact NanoLuc luciferase when another part of split NanoLuc luciferase, Large Bit peptide of NanoBiT (LgBiT), is present. Therefore, the addition of recombinant LgBiT protein and NanoLuc luciferase substrate and measurement of luminescence facilitates the detection of HiBiT-tag (Dixon et al., 2016). Since HiBiT-tag is a short peptide tag with a length of 11 amino acids, it has a minimum effect on the function of fused proteins. Furthermore, a HiBiT-tag-dependent NanoLuc luciferase detection system is useful for quantifying small quantities of protein, because the signal-to-noise ratio is significantly high in NanoLuc luciferase–dependent luminescence, which allows for small-scale experiments. The procedure is simple and therefore suitable for high-throughput assays. In this report, we introduce the method of using the HiBiT-tag-dependent NanoLuc luciferase system for the detection of prey protein in a pull-down assay. Generally, proteins form an oligomeric complex with various partners. In these cases, the depletion of key subunits significantly affects the formation of the entire complex. In this report, we demonstrate that depletion of a mediator protein affects ternary complex formation.
Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus, is a human pathogenic flavivirus. In JEV-infected cells, endoplasmic reticulum membrane–derived large compartments (also called viral replication organelles) are observed. Viruses have been considered to efficiently replicate in these compartments, which may be a target for the development of antiviral reagents (Arakawa and Morita, 2019). Previously, our group reported that the host factor valosin-containing protein (VCP) is recruited to the viral replication organelle and helps in viral genome replication (Tabata et al., 2021). However, no direct interaction between VCP and viral proteins were detected. Our previous study revealed a mediator that bridges the interaction between them. We found that the interaction of nuclear protein localization protein 4 (NPL4), a VCP-associating co-factor, and NS4B, a nonstructural viral protein that localizes on the viral replication site, is important for the recruitment of VCP to the viral replication organelle. We have shown that depletion of NPL4 via siRNA knockdown significantly reduces the affinity between VCP and NS4B, through pull-down assay utilizing HiBiT-tag-dependent luciferase activity (HiBiT activity) measurement (Arakawa et al., 2022) (Figure 1A). HiBiT-tagged NS4B and One-Strep-FLAG (OSF)-tagged VCP were co-expressed in 293T cells and the VCP were affinity-purified using Strep-Tactin beads (Figure 1B). The amount of NS4B in the VCP-bound fraction was then determined in the presence of NPL4 and compared with that in the absence of NPL4 by measuring HiBiT activity (Figure 2C). This protocol is not only useful for studying virus–host interactions, but also has broader applications in the investigation of general protein–protein interactions.
Materials and Reagents
1.5 mL tubes (WATSON, catalog number: 8064131815C)
0.22 μm filter (AS ONE, catalog number: 033022SO-SFCA)
6 cm cell culture dish (Thermo Scientific, catalog number: 150462)
Cell scraper (VIOLAMO, catalog number: 1-2249-01)
White 384-well immuno plates (Thermo Scientific, catalog number: 460372)
293T cells (ATCC: CRL-3216)
Plasmid pCAG-NS4B-FLAG-HiBiT, which encodes HiBiT-tagged JEV NS4B protein (Arakawa et al., 2022)
Plasmid pCAG-OSF-VCP, which encodes One Strep–tagged VCP proteins or is empty (Arakawa et al., 2022)
siRNA-luciferase, which targets firefly luciferase (sense sequence: 3′-CGUACGCGGAAUACUUCGAtt-5′)
siRNA-NPL4, which targets NPL4 (sense sequence: 3′-CUGAAGUGGUCGAUGAAAUtt-5’)
Nano Glo HiBiT lytic detection system (Promega, catalog number: N3040)
Dulbecco’s modified Eagle’s medium (Nacalai Tesque Inc., catalog number: 08458-16)
Fetal bovine serum (FBS) (Thermo Scientific, catalog number: 10270-106)
Phosphate-buffered saline (PBS) without calcium and magnesium (Nacalai Tesque Inc., catalog number: 14249-24)
Benzylpenicillin potassium (Fujifilm Wako Pure Chemical Corporation, catalog number: 021-07732)
Streptomycin sulfate (Tokyo Chemical Industry, catalog number: S0585)
Strep-Tactin Sepharose 50% suspension (IBA Lifesciences GmbH, catalog number: 2-121-010)
Strep-tag elution (10× buffer E) (IBA Lifesciences GmbH, catalog number: 2-1000-025)
cOmplete, EDTA-free, protease inhibitor cocktail (Roche, catalog number: 11873580001)
LipofectamineTM 3000 transfection reagent (Thermo Scientific, catalog number: L3000015)
LipofectamineTM RNAiMAX transfection reagent (Thermo Scientific, catalog number: 13778150)
Opti-MEM (Thermo Scientific, catalog number: 31985062)
Tris (Tris[hydroxymethyl]aminomethane) (Nacalai Tesque Inc., catalog number:35406-91)
NaCl (Nacalai Tesque Inc., catalog number: 31320-05)
Triton X-100 (Nacalai Tesque Inc., catalog number: 35501-15)
100× penicillin G + streptomycin stock solution (see Recipes)
Culture medium (see Recipes)
Pull-down washing buffer (see Recipes)
100× concentrated cOmplete stock solution (see Recipes)
Lysis buffer (see Recipes)
HiBiT reagent (see Recipes)
1× Strep-tag elution buffer (see Recipes)
Equipment
Humidified incubator (37 °C, 5% CO2)
Microplate luminometer (Thermo Scientific, Varioskan LUX Multimode Microplate Reader)
Vortex mixer (Scientific Industries, model: Vortex-Genie 2)
Centrifuge machine for microtube (Thermo Scientific, model: SorvallTM LegendTM Micro 21R)
Rotator (BIO CRAFT, model: BC-710I)
Procedure
Add 60 pmol siRNA-NPL4 (or siRNA-luciferase for negative control) to 250 µL of Opti-MEM. Mix well using a vortex mixer (Figure 1).
Note: Steps 1–8 pertain specifically to the siRNA transfection protocol, while step 9 marks the beginning of the pull-down protocol. To perform siRNA transfection, please follow the "Reverse transfection protocol" of LipofectamineTM RNAiMAX transfection reagent.
Figure 1. Overview of the protein pull-down HiBiT assay. A. Schematic illustration of the protein pull-down HiBiT assay. B. Flow diagram of the protein pull-down HiBiT assay.
Add 6 µL of LipofectamineTM RNAiMAX transfection reagent to 250 µL of Opti-MEM. Mix well using a vortex mixer.
Add the mixture from step 1 to that from step 2 and mix well using a vortex mixer.
Incubate the mixture for 5 min at room temperature.
Seed 293T cells in a 6 cm dish at a density of 750,000 cells/well in 4.5 mL of culture medium.
Gently add approximately 500 µL of the siRNA-Lipofectamine mixture to the cell-containing dish.
Place the dish in a CO2 incubator for 24 h.
Replenish culture medium at 24 h post transfection.
Note: Slowly add medium to the side of the well to avoid cell detachment.
Add 1.5 µg pCAG-NS4B-FLAG-HiBiT, 1.5 µg pCAG-OSF-VCP, and 120 pmol siRNA-NPL4 (or siRNA-luciferase for negative control) to 250 µL of Opti-MEM. For siRNA transfection, repeat the initial transfection using the same siRNA. Mix well using a vortex mixer.
(Optional) Set control experiments using an empty vector (pCAG-OSF) in place of pCAG-OSF-VCP.
Add 6 µL of LipofectamineTM 3000 transfection reagent to 250 µL of Opti-MEM. Mix well using a vortex mixer.
Add the mixture from step 9 to that from step 10 and mix well using a vortex mixer.
Incubate the mixture for 10 min at room temperature.
Apply the mixture from step 12 to the cell-containing dish (~500 µL).
Place the dish in a CO2 incubator for 48 h.
Aspirate the culture medium and add 1 mL of ice-cold PBS to each dish. Collect the cells using a cell scraper and transfer them to a 1.5 mL tube.
Centrifuge the 1.5 mL tubes at 500 × g for 10 min at 4 °C.
Aspirate PBS. Add 1 mL of ice-cold PBS to each tube and suspend by pipetting.
Centrifuge the 1.5 mL tubes at 500 × g for 10 min at 4 °C.
Aspirate PBS. Add 500 µL of ice-cold lysis buffer to each tube and mix by pipetting.
Centrifuge the 1.5 mL tubes at 20,000 × g for 10 min at 4 °C.
Transfer 5 µL of the supernatant to 1.5 mL tubes for input and add 45 µL of lysis buffer. Mix well using a vortex mixer.
Transfer 10 μL of diluted lysate in each well of a 384-well plate.
Note: To ensure accurate measurements, it is recommended to use more than three wells per sample. If the obtained values are outside the reading range, a dilution series should be carried out.
Add 10 μL of HiBiT reagent to each well.
Incubate for 10 min at room temperature.
Measure the luminescence for 1,000 ms measurement time/well using a microplate reader.
Transfer 480 µL of the clear lysate to fresh 1.5 mL tubes.
Add 15 µL of Strep-Tactin Sepharose 50% suspension to the lysate and rotate at 4 °C for 1 h.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Aspirate the supernatant. Add 1 mL of cold pull-down washing buffer to each tube and mix via inversion.
Repeat steps 28 and 29 thrice.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Aspirate the supernatant. Add 450 µL of 1× Strep-tag elution and rotate at 4 °C for 1 h.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Transfer 400 µL of elution buffer to fresh 1.5 mL tubes.
Transfer 10 μL of elution in each well of a 384-well plate.
Note: To ensure accurate measurements, it is recommended to use more than three wells per sample. If the obtained values are outside the reading range, a dilution series should be carried out.
Add 10 μL of HiBiT reagent from Nano Glo HiBiT lytic detection system to each well.
Incubate for 10 min at room temperature.
Measure the luminescence for 1,000 ms measurement time/well using a microplate reader (Figure 2).
Figure 2. HiBiT activity of the Strep-Tactin-purified fraction and the input fraction. A. HiBiT activity of the Strep-Tactin-purified fraction. B. HiBiT activity of the input fraction. C. The compensated HiBiT value of the Strep-Tactin purified fraction. The HiBiT activity of the Strep-Tactin purified fraction was calculated by dividing its value by that of the input fraction. The means between two groups were compared using a Student’s t-test. Differences were considered significant at **P < 0.01.
Notes
In the NanoLuc/HiBiT system, strong luminescence emission may be obtained and causes leaks to adjacent wells in a multi-well plate. Therefore, we recommend loading the samples in every other well.
The materials used in this study can be obtained from the corresponding author, Eiji Morita, upon reasonable request.
Recipes
100× penicillin G + streptomycin stock solution
PBS 100 mL
Benzylpenicillin potassium 0.626 g
Streptomycin sulfate 1 g
Sterilize using a 0.22 μm filter and store in a refrigerator.
Culture medium
Dulbecco’s modified Eagle’s medium 500 mL
FBS, heat-inactivated via incubation at 56 °C for 45 min 50 mL
100× penicillin G + streptomycin stock solution 5 mL
Pull-down washing buffer
1 M Tris pH 7.5 20 mL
5 M NaCl 30 mL
Triton X-100 1.07 g
ddH2O up to 1 L
100×-concentrated cOmplete stock solution
cOmplete, EDTA-free, protease inhibitor cocktail 1 tablet
ddH2O 500 µL
Store at -20
Lysis buffer
1 M Tris pH 7.5 20 mL
5 M NaCl 30 mL
Triton X-100 10.7 g
ddH2O up to 1 L
Add 100× concentrated cOmplete stock solution just before use.
HiBiT reagent from Nano Glo HiBiT lytic detection system
Nano-Glo HiBiT lytic buffer 500 µl
Nano-Glo HiBiT lytic substrate 10 µl
LgBiT protein 5 µl
1× Strep-tag elution buffer
Strep-tag elution (10× buffer E) 2 mL
ddH2O 18 mL
Acknowledgments
This work was supported by JSPS KAKENHI (Grant Number 22H00553, 22H02873, 22K18378, 20H05305, 20K21874), JST CREST, Japan (grant number JPMJCR17H4). The protocol for HiBiT detection was adapted from previous work (Goto et al., 2020). This protocol is derived from the original research paper (Arakawa et al., 2022; DOI: 10.1016/j.jbc.2022.101597).
Competing interests
The authors have no competing interests directly relevant to the content of this article.
References
Arakawa, M. and Morita, E. (2019). Flavivirus Replication Organelle Biogenesis in the Endoplasmic Reticulum: Comparison with Other Single-Stranded Positive-Sense RNA Viruses. Int J Mol Sci 20(9).
Arakawa, M., Tabata, K., Ishida, K., Kobayashi, M., Arai, A., Ishikawa, T., Suzuki, R., Takeuchi, H., Tripathi, L. P., Mizuguchi, K., et al. (2022). Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication. J Biol Chem 298(3): 101597.
Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T., Kirkland, T. A., et al. (2016). NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11(2): 400-408.
Tabata, K., Arakawa, M., Ishida, K., Kobayashi, M., Nara, A., Sugimoto, T., Okada, T., Mori, K. and Morita, E. (2021). Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation. J Virol 95(15): e0223420.
Goto, S., Ishida, K., Suzuki, R. and Morita, E. (2020). Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus. Bio Protoc 10(9): e3606.
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Microbiology > Microbe-host interactions > Virus
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4,641 | https://bio-protocol.org/en/bpdetail?id=4641&type=0 | # Bio-Protocol Content
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The Assessment of Beta Cell Mass during Gestational Life in the Mouse
YK Yury Kryvalap
JC Jan Czyzyk
Published: Vol 13, Iss 6, Mar 20, 2023
DOI: 10.21769/BioProtoc.4641 Views: 447
Reviewed by: RAMESH KUDIRA Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Translational Medicine Apr 2021
Abstract
Successful advancement in the treatment of diabetes mellitus is not possible without well-established methodology for beta cell mass calculation. Here, we offer the protocol to assess beta cell mass during embryonic development in the mouse. The described protocol has detailed steps on how to process extremely small embryonic pancreatic tissue, cut it on the cryostat, and stain tissue slides for microscopic analysis. The method does not require usage of confocal microscopy and takes advantage of enhanced automated image analysis with proprietary as well as open-source software packages.
Keywords: Beta cell mass Insulin area Embryonic pancreas Timed pregnancy Frozen sections
Background
This protocol offers state-of-art detailed steps to assess beta cell mass on immunofluorescent (IF)-stained sections of pancreatic tissue. Quite frequently, beta cell area and mass estimation are heavily biased due to examination of too few (often just 2–3) sections of the pancreatic tissue. Here, we offer an unbiased novel approach to cut through the pancreatic tissue and make representative layers as well as backup slides for later staining. Using our approach, the number of sections and tissue levels that are necessary to accurately estimate the size of beta cell mass is dependent only on the pancreas developmental stage. Moreover, this protocol yields consistent estimations of beta cell mass among individual samples per single treatment group.
Materials and Reagents
Pair of straight sharp non-serrated forceps (Fisher Scientific, catalog number: 12000127)
Pair of 1 cc syringes (McKesson, catalog number: 16-ST1C) with 30.5 G needles (BD, catalog number: 305106)
Petri dishes (Corning, catalog number: 351029)
500 mL Stericup Quick Release Millipore Express PLUS 0.22 μm filter unit (Millipore, catalog number: S2GPU05RE)
250 mL Stericup Quick Release Millipore Express PLUS 0.22 μm filter unit (Millipore, catalog number: S2GPU02RE)
Super Pap-pen, small (Electron Microscopy Sciences, catalog number: 71312)
Invisible tape (Staples, catalog number: 487908)
Adhesive glass slide 75 × 25 × 1 mm (Matsunami, catalog number: SUMGP12)
Immunofluorescence (IF) staining tray (Simport Scientific, catalog number: M9202)
Neo Micro cover glass (Matsunami, catalog number: 24x50)
Nail polish (Electron Microscopy Sciences, catalog number: 72180)
Paper towel (Scott Kimberly-Clark Professional, catalog number: 09-24-589-0-06)
15 mL conical tubes (Thermo Fisher Scientific, catalog number: 339651)
50 mL conical tubes (Thermo Fisher Scientific, catalog number: 339653)
Weigh boats (Electron Microscopy Sciences, catalog number: 70042)
BALB/cJ male mice (The Jackson Laboratory, catalog number: 000651)
Prechilled PBS pH 7.4 (Gibco, catalog number: 10010-023)
Paraformaldehyde (PFA) 8% aqueous solution, EM grade (Electron Microscopy Sciences, catalog number: 157-8)
Sucrose (Macron Fine Chemicals, catalog number: 8360-06)
OCT compound (Scigen, catalog number: 4586)
Biopsy cryomold (Sakura Tissue-Tek, catalog number: 4565)
Dry ice pellets
100% ethanol
Fisherfinest chemically resistant marker (Fisher Scientific, catalog number: 22-026-700)
Bovine serum albumin (BSA) (fraction V) (Fisher Bioreagents, catalog number: BP1600-100)
Sodium azide (Sigma, catalog number: S-8032)
Anti-insulin mouse Alexa-647 (BD, catalog number: 565689)
Anti-glucagon mouse Alexa-594 (SantaCruz, catalog number: sc-514592 AF594)
Anti-somatostatin mouse Alexa-488 (BD, catalog number: 566032)
Shandon Immu-Mount (Thermo Fisher Scientific, catalog number: 9990402)
DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, catalog number: D1306)
30% sucrose (see Recipes)
5% BSA with 0.1% sodium azide (see Recipes)
DAPI in slide mounting media (see Recipes)
Equipment
Scissors (Fisher Scientific, catalog number: 13820002)
Dissecting microscope with mirror and light below (Olympus, model: SZX7)
Cryostat (Microm, model: HM 525)
pH meter (Mettler Toledo, model: S220)
IF-capable microscope or IF Virtual Slide scanner (Olympus VS 120, Zeiss AxioScan Z1)
Analytical weigh scale (Mettler Toledo, model: XS105)
Software
Visiopharm 2021.07 or later
Alternatively:
ImageJ with Trainable Weka Segmentation plugin (Arganda-Carreras et al., 2017).
Procedure
Setting up timed pregnancy in mice
Place each male mouse into an individual cage, twice the number needed for each night of breeding. For example, if 5 male mice will be used for breeding at a given night, then 10 male mice should be set up. Let the animals rest for one night.
At the end of the day (approximately between 4:00 and 6:00 PM), place one female mouse with each individual male and leave the remaining half of single-housed male mice alone.
Next morning, check each female mouse for the presence of a vaginal plug (Figure 1), which suggests successful breeding. Tag the plugged female mice, measure their body weight, and place them in a new cage. This day is referred to as E0.5. Make appropriate notes in your notebook or on the computer, including the mouse number and the day when the vaginal plug was confirmed, as well as the body weight of the mouse at the day of the vaginal plugging.
Figure 1. Vaginal plug (indicated by an orange arrow) during morning assessment after overnight breeding of Balb/c female mouse
Repeat breeding with the second half of males during following night. Let the first half of males, which were used during the first night of breeding, to rest. Continue this mating strategy until all desired females are plugged. In order to facilitate execution of experimental design for specific embryonic days and correctly predict experimental needs utilizing a timed-pregnancy model, we created a dedicated MS Excel program (YKTPPaL.xsls).
At 10 days after E0.5, check the body weight of female mice. An increase by more than 2 g strongly suggests pregnancy (accordingly, the age of embryos is considered E10.5 at this stage) (Heyne et al., 2015). Apply any experimental and control treatment, as originally designed. Alternatively, the treatments can be initiated at E0.5, although this approach requires additional mice as not all plugged female mice become pregnant.
Tissue processing and freezing
Euthanize pregnant mice according to the IACUC approved protocol.
Prepare ice bucket with the following Petri dishes (Figure 2):
One dish with PBS for initial placement of embryos
One dish with PBS for individually separated embryos
Two dishes with PBS for pancreatic tissue isolation from each embryo being processed
One or more Petri dish lids for tissue processing (Figure 3)
Figure 2. Preparation for embryonic pancreas isolation
Figure 3. The processing steps of embryonic pancreatic tissue, E12.5 (indicated by orange arrows)
Harvest pancreatic tissue as desired (e.g., at E12.5, E14.5, and E16.5) (Video 1). If experimental design and conditions permit, it is better to euthanize only one pregnant mouse at a time and process its litter. Proceed then with euthanasia of the next pregnant mouse and processing of its litter. With good skills, each pancreatic sample can be consistently obtained within 2–4 min. Specifically, we were successful with up to two litters at the same time.
Video 1. Dissecting technique of developing pancreatic tissue during mouse embryogenesis
Outline with Pap-pen three circles per pancreatic rudiment as needed on Petri dish lids and fill them up with PBS, 2% PFA, and 30% sucrose, respectively. Gently, place each isolated pancreatic tissue in a circle with PBS (Figure 3).
This step can be simplified by pulling pancreatic rudiments from the same experimental group into one processing circle. For example, pancreatic rudiments at E12.5 treated with control/vehicle can be placed into one processing circle, as long as it is the only parameter being assessed during the study.
If each pancreatic rudiment has its own unique features, e.g., genotype, type of treatment, gender, etc., then they should be placed into individual processing circles.
With the help of dissecting microscope and 1 cc syringe with 30 G needle, transfer isolated samples into the PAP-pen circles containing 2% PFA for 2–4 h at room temperature (RT). Next, transfer samples into the last circle with 30% sucrose until they sink down to the bottom of solution (Video 2).
Video 2. Embryonic pancreatic tissue explant processing technique for cryopreservation
Prepare Tissue-Tek biopsy cryomolds: label molds with chemically resistant marker and tape with clear tape over marker writing. Fill up the molds with OCT compound. Using the syringe with 30 G angled or straight needle, transfer samples from sucrose circle into the mold with OCT under the dissecting microscope. Position the sample in the middle depth of the mold. Repeat it for all samples.
Crush dry ice with mortar and pestle into very small pieces and transfer it into a borosilicate container filled up with 100% ethanol, to make freezing solution.
Quickly, under the dissecting microscope and using syringe with 30 G needle, push pancreatic sample to the bottom of the mold, making sure the tissue is oriented in the middle of the most bottom area (Figure 4). Place the mold on top of the dry ice/ethanol bath for freezing. Repeat freezing procedure for all samples as quickly as possible.
Figure 4. Placement of embryonic pancreatic tissue E16.5 in the cryomold
Tissue cutting
Since embryonic pancreatic rudiments (especially at E12.5–E14.5) are very tiny, it is important to freeze them in the middle of the most bottom area of cryomold. This approach will make tissue visible as a brown-to-yellow dot and facilitate its finding for cutting on cryostat. For a backup, each slide should be cut in duplicate. All tissue levels obtained from a single pancreatic rudiment should be positioned in sequential order on 1–2 slides. The thickness of sections should be 5 μm.
First tissue section is referred to as level one (e.g., level 1 section 1) and should be placed on the first slide in the top left corner, allowing a reasonable distance to the edge of the slide. The very next pancreatic section is referred to as a duplicate section of the first layer (e.g., level 1 section 2) and should be placed in the same spot of duplicate slide. Repeat this cutting technic until all pancreatic tissue is cut through.
Using the above-described approach, E12.5 pancreatic section levels are spaced approximately 15 μm apart. For level separation of larger embryonic pancreatic tissue, make an additional seven (E14.5) or ten (E16.5) cuts (5 μm each) into waste to space adjacent levels to 35 and 50 μm apart, respectively. The total level output may vary and be dependent on treatment and genotype. On average, expect 10–14 tissue levels per single pancreatic bud (Table 1 and Figure 5).
Table 1. Recommendation of pancreatic tissue cutting in relation to its size
Pancreas size Main slide section per level Duplicate slide section per level 5 µm cuts to waste (skip) Distance between levels, µm Levels per pancreas
E12.5 1 1 0 15 10–12
E14.5 1 1 7 35 10–14
E16.5 1 1 10 50 10–14
E18.5 1 1 10 50 10–14
0P 1 1 12 60 10–14
Figure 5. E14.5 Embryonic pancreas sections (outlined with orange rectangles) from level 1 through 13 (left to right, top to bottom) on a slide
Note that depending on experimental needs, it is possible to prepare additional backup slides of pancreatic tissue starting at E14.5. Accordingly, the number of 5 μm cuts to be skipped into waste should be decreased (Table 2).
Table 2. Example of pancreatic tissue cutting recommendation with three backup slides
Pancreas size Main slide section per level Duplicate slide section per level 5 µm cuts to waste (skip) Distance between levels, µm Levels per pancreas
E16.5 1 3 8 50 10–14
Store slides in a -80 °C freezer in slide boxes. Slides can be stored for up to four years.
IF staining
De-freeze and dry slides at RT. Keep duplicate slides frozen as backup. Outline a big circle around all tissue sections on each slide with PAP-pen.
Add 5% BSA dropwise on a slide to cover tissue sections. Incubate 30–60 min at RT in moisturized IF staining tray.
Prepare working dilution of mouse anti-insulin Alexa-647 antibody in 5% BSA (the dilution factor will depend on the microscope and lot of the antibody). If needed, you can combine this antibody with anti-glucagon Alexa-594 and anti-somatostatin Alexa-488 antibodies.
For example, we successfully used a combination of anti-insulin Alexa-647 (1:500), anti-glucagon Alexa-594 (1:1,000), and anti-somatostatin Alexa-488 (1:200) Abs together in a single-step staining procedure (Figure 6).
Figure 6. 23P pancreas section stained simultaneously with antibodies to insulin (Alexa-647, red), glucagon (Alexa-594, purple), and somatostatin (Alexa-488, green). 20× magnification. Scale bar, 50 μm.
Remove excess BSA solution from slide by blotting the edge of the slide against paper towel and add 100 μL of Abs working solution on each slide. Cover moisturized staining tray with a lid and incubate in dark at 4 °C overnight.
The following morning, remove excess of staining solution from the slide by blotting the edge of the slide against paper towel, and place the slide in a Coplin jar with PBS for 5 min. Remove excess of PBS solution and repeat washing step one more time.
Place two coverslips on a paper towel and add one bubble-free drop of mounting media with DAPI in the center of the coverslip.
Take the slide out from washing Coplin jar, blot its edge against paper towel, quickly swing it in the air to remove residual PBS and, with tissue sections facing down, put it on a coverslip with DAPI mounting media. Repeat it for a second slide. The weight of the slide will help to evenly spread out DAPI mounting media across the tissue sections. Repeat this step for all slides in the staining batch.
Air dry slides in dark for 60–120 min and apply nail polish around the edge of coverslip to seal the slide for long-term storage. Slides are ready for microscopy, but if time permits, cure slides at 4 °C overnight for best autofocusing results.
Microscopy
Take pictures of each tissue section with an IF camera–equipped microscope by focusing on a DAPI channel. For small tissue samples (e.g., E12.5), at most instances a single field of view at 20× will fit the entire tissue section. For bigger sections (e.g., E13.5 and older) it will be necessary to take overlapping pictures of different fields of view and stitching them together in a photo-editing software. Alternatively, a virtual microscope slide scanner (e.g., Zeiss Axioscan Z1, Olympus V120) can be effectively used to automatically photograph, using DAPI channel as an autofocusing channel, and stich tissue sections together for further analysis (Figure 7). As a general rule of better autofocusing, it is recommended to set up a shorter exposure time (faster DAPI focusing), and DAPI signal intensity should not exceed 30%–50% of its maximum histogram value.
Figure 7. E16.5 pancreas section stained with anti-insulin Alexa-647 Ab with multiple fields of view stitched together. 2× magnification. Scale bar, 500 µm.
Image analysis
Use software (e.g., Visiopharm software with Author module) for multicolored IF imaging (Figure 8A):
Uncheck all unnecessary staining channels (e.g., amylase, CK19, and DAPI, as shown in Figure 8A) and draw small regions corresponding to at least three classes for segmentation purposes: ins+ (staining positive for insulin), the tissue (tissue area stained negatively for insulin), and background (slide area without tissue) (Figure 8B and 8G).
If necessary, apply preprocessing step to better prepare image for segmentation (in example described in Figure 8, we used a mean filter on insulin-staining channel, which helps to negate and equalize individual pixel variations in the background and specific staining patterns) and train a software, using Bayesian algorithm (or any other one suited for a given task). Make sure that the software can consistently recognize these areas (Figure 8C and 8G).
Using postprocessing steps, remove unnecessary labels and small artifacts. In the example shown in Figure 8D and 8G, the label “background” was removed completely as it was not used in further calculations. The smallest ins+ spots measuring less than 12 μm in diameter and “tissue” labels measuring less than 20 μm in diameter also were removed as they usually represent artifacts.
Perform segmentation of the entire image (Figure 8E) and, if necessary, check its consistency with other stains (Figure 8F).
Figure 8. E16.5 pancreas, stained for insulin (green), amylase (purple), cytokeratin 19 (red), and nuclei (blue) was preprocessed, segmented, and fine-tuned (A-F) by using Visiopharm software Author module (G). 14× magnification. Scale bar, 100 µm.
Comparable results can be obtained using open-sources software, including ImageJ/FiJi with Trainable Weka Segmentation package (Heyne et al., 2015).
For each pancreatic sample, calculate the following parameters:
Total area occupied by insulin-producing cells
Total tissue section area
Calculate the percentage of area occupied by insulin-producing cells, according to the following formulas:
Area occupied by insulin-producing cells/(tissue area + insulin-positive area) × 100% (if total section area value does not include insulin-positive area) or:
Area occupied by insulin-producing cells/(tissue area) × 100% (if total section area value includes insulin-positive area already)
Calculate beta cell mass: % of area occupied by insulin-producing cells × pancreas weight expressed in mg (optional if weighting scale permits such a small, consistent reading).
For example, E16.5 healthy Balbc/cJ pups, on average, have 0.3857% ± 0.02327 of insulin-positive beta cell area (Kryvalap et al., 2021).
Recipes
30% sucrose
Dissolve 300 g of sucrose in 500 mL of PBS on a magnetic stirrer until solution becomes clear.
Add distilled water up to 1,000 mL.
Sterilize solution with two units of Stericup Quick Release Millipore Express PLUS 0.22 μm filters, aliquot into 50 mL conical tubes, and store at 4 °C.
5% BSA with 0.1% sodium azide
Prepare 10% stock solution of sodium azide: dissolve 10 g of sodium azide in 50 mL of distilled water on a magnetic stirrer until solution becomes clear. Add distilled water up to 100 mL. Store at room temperature.
Prepare 5% BSA in PBS: dissolve 12.5 g of BSA (fraction V) in 150 mL of PBS pH 7.4 on a magnetic stirrer until solution becomes clear. Add PBS up to 250 mL.
Add 2.5 mL of 10% sodium azide to the 5% BSA solution. Mix well.
(optional) Pass through a 250 mL Stericup Quick Release Millipore Express PLUS 0.22 μm filter unit to clear up solution. Store at 4 °C.
DAPI in slide mounting media
Make 14.3 mM DAPI stock: to the vial with 10 mg of DAPI, add 2 mL of distilled water. Vortex well. Aliquot and store at -20 °C.
Add 1 μL of 14.3 mM DAPI stock solution into the bottle with 20 mL of mounting media. Cover with foil and rotate it to mix on a shaker for 1 h at RT. Store at 4 °C and protect from the light.
Acknowledgments
Some of the work described in the manuscript was supported by grants from the Juvenile Diabetes Research Foundation (grant 17-2012-428) and the American Diabetes Association (grant 1-17-ICTS-083). This protocol is derived from the original research paper (Kryvalap et al., 2021; DOI: 10.1126/scitranslmed.abf1587).
Competing interests
The authors declare no duality of interest associated with this manuscript.
References
Arganda-Carreras, I., Kaynig, V., Rueden, C., Eliceiri, K. W., Schindelin, J., Cardona, A. and Sebastian Seung, H. (2017). Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33(15): 2424-2426.
Heyne, G. W., Plisch, E. H., Melberg, C. G., Sandgren, E. P., Peter, J. A. and Lipinski, R. J. (2015). A Simple and Reliable Method for Early Pregnancy Detection in Inbred Mice. J Am Assoc Lab Anim Sci 54(4): 368-371.
Kryvalap, Y., Jiang, M. L., Kryvalap, N., Hendrickson, C. and Czyzyk, J. (2021). SerpinB13 antibodies promote β cell development and resistance to type 1 diabetes. Sci Transl Med 13(588): eabf1587.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Developmental Biology > Morphogenesis > Cell structure
Cell Biology > Cell imaging > Cryosection
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4,642 | https://bio-protocol.org/en/bpdetail?id=4642&type=0 | # Bio-Protocol Content
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Peer-reviewed
Determination of Paraquat in Arabidopsis Tissues and Protoplasts by UHPLC-MS/MS
MZ Mingming Zhao *
QW Qi Wang *
MS Muyu Shi
ZS Ziyan Sun
HT Huiru Tang
XG Xiaochun Ge
(*contributed equally to this work)
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4642 Views: 580
Reviewed by: Demosthenis Chronis Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Plant Sep 2021
Abstract
Paraquat is a cost-effective herbicide, widely used in many countries, that can induce severe oxidative stress in photosynthetic tissues. Studying plant herbicide resistance or antioxidant stress mechanisms requires determining the cellular paraquat level when plants are treated by paraquat. The traditional isotopic labeling method has the potential risk to cause problems to both human health and the environment. For radioisotope manipulation, special operation spaces and strict environmental inspection are also required. In addition, the radiolabeled paraquat is increasingly hard to buy due to the extended production cycle. Here, we describe a nonradioactive method to determine the paraquat level in a small number of Arabidopsis tissues or protoplasts, using a high resolution ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry (MS)/MS method. This method is highly selective and sensitive, and more environmentally compatible and technically feasible than the isotope detection method.
Keywords: Paraquat Arabidopsis Oxidative stress UHPLC-MS/MS
Background
Paraquat (PQ, 1,1-dimethyl-4,4-bipyridinium dichloride, or commonly called methyl viologen) is a nonselective herbicide that has been widely used in many countries for over half a century (Farrington et al., 1973; Fussell et al., 2011). It acts on chloroplasts and kills plants in several hours. PQ accepts electrons from photosystem I and transfers them to molecular oxygen, thus generating highly toxic reactive oxygen species (ROS) and causing severe oxidative stress in green tissues (Farrington et al., 1973; Fussell et al., 2011;Hawkes, 2014). In the past decades, a lot of PQ-resistant weeds have been found in the field. Studying plant resistance mechanisms to PQ not only helps to understand how weeds evolve herbicide resistance, but also promotes the understanding of plant antioxidant stress mechanisms. In Arabidopsis, a number of PQ-resistant mutants have been reported, including rmv1 (resistant of methyl viologen 1) (Fujita et al., 2012), par1 (paraquat-resistant 1) (Li et al., 2013), atpdr11 (pleiotropic drug resistance 11) (Xi et al., 2012), rcd1 (radical-induced cell death 1) (Fujibe et al., 2004), pst1 (photoautotrophic salt tolerance 1) (Tsugane et al., 1999), par2 (paraquat-resistant 2) (Chen et al., 2009), pqt3 (paraquat tolerance 3) (Luo et al., 2016), dtx6 (Lv et al., 2021; Xia et al., 2021), and atpqt11 (paraquat tolerance 11) (Huang et al., 2022), and the mutant genes responsible for this resistance have been identified. The resistance mechanisms revealed so far have been classified into three types: impaired uptake and transport of PQ, enhanced sequestration of PQ, and enhanced ROS scavenging ability (Nazish et al., 2022).
We identified a new PQ-resistant mutant rtp1 (resistant to paraquat 1), in which a MATE (multidrug and toxic compound extrusion) family protein DTX6 is disrupted (Lv et al., 2021). MATE family proteins are a type of transporters with distinct substrate specificity. To elucidate the resistance mechanism of DTX6, it is important to measure the PQ level in cells after treatment. The isotopic labeling method has been used to track exogenous molecules in plants; however, radiolabeled PQ is becoming increasingly difficult to obtain, and the manipulation of radioisotopes needs very complex approval and inspection procedures. Therefore, we established a nonradioactive method to determine PQ content in cells by combining ultra-high-performance liquid chromatography (UHPLC) and mass spectrometry (MS) systems. The UHPLC-MS/MS can determine PQ by using the multiple reaction monitoring mode of triple-quadrupole mass spectrometry. It has high selectivity and sensitivity and can achieve a resolution of 10-15 mol/L on column. Compared with traditional radiolabeling, the UHPLC-MS/MS method is more environmentally compatible and easier to implement.
Materials and Reagents
50 mL conical centrifuge tube (PROMETHE, catalog number: PCT010500, sterile)
15 mL conical centrifuge tube (PROMETHE, catalog number: PCT010150, sterile)
2 mL centrifuge tube (Shanghai Jing Xin, catalog number: JX-LG0100, nonsterile)
2.0 mL amber screw vial (Thermo Fisher, catalog number: C5000-2W)
150 μL liner pipe (Thermo Fisher, catalog number: C4012-530)
Two-week-old Arabidopsis seedlings grown on 1/2 MS plates (mutant seeds are available via email request to the corresponding author, and wildtype seeds are available from TAIR website https://www.arabidopsis.org)
Four-week-old Arabidopsis plants grown in soil
Murashige & Skoog basal medium with vitamins (PhytoTech, catalog number: M519)
KCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7447-40-7)
CaCl2 (Solarbio, CAS: 10043-52-4)
NaCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7647-14-5)
HCl (Sinopharm Chemical Reagent Co., Ltd, CAS: 7647-01-0)
MES (BBI, CAS: 145224-94-8)
NH4OAC (Sinopharm Chemical Reagent Co., Ltd, CAS: 631-61-8)
Paraquat (Aladdin, CAS: 1910-42-5)
Water (LC-MS-grade) (Merck, Milli-Q IQ7000)
Methanol (LC-MS-grade) (Merck, CAS: 67-56-1)
Acetonitrile (LC-MS-grade) (Sigma-Aldrich, CAS: 75-05-8)
Formic acid (LC-MS-grade) (Sigma-Aldrich, CAS: 64-18-6)
Liquid nitrogen
W5 buffer (see Recipes)
Paraquat treatment buffer (see Recipes)
Wash buffer (see Recipes)
Paraquat extraction solution (see Recipes)
Redissolving solution (see Recipes)
LC mobile phase (see Recipes)
Equipment
Automatic sample fast grinder (Shanghai Jing Xin, model: JXFSTPRP-CL)
Ultrasonic cleaners (Supmile, model: KQ-200VDE)
Nitrogen blowing concentrator (Beijing Jiayuan Industrial Technology Co., Ltd., model: MD200-2)
High-speed refrigerated centrifuge (KUBOTA, model: 6500)
Table high-speed centrifuge (Hettich, model: MIKRO 220R)
Light microscopy (Leica, model: Stereo microscope S8 APO)
-80 freezer
Water bath (HEDE Laboratory, model: DK-420BS)
Hemocytometer
UHPLC system (Shimadzu Nexera UHPLC LC30A system, including sample manager, solvent manager, and column oven)
Mass spectrometer system (SCIEX QTRAP 6500 plus mass spectrometer equipped with an electrospray ionization)
UHPLC column (ACQUITY UPLC BEH HILIC Column, 130Å, 1.7 µm particle size, 2.1 mm × 100 mm, Waters)
Procedure
Treat Arabidopsis seedlings with paraquat
Surface-sterilize the Arabidopsis seeds with 70% ethanol and then grow them on vertical 1/2 Murashige & Skoog square plates (1% agar) for 10 days under standard long daylight growth conditions (16:8 h light/dark cycle) (Weigel and Glazebrook, 2002).
Lay the plates horizontally and then leave them in darkness for two days to allow the hypocotyl to grow upward quickly. After that, return the plates to long daylight conditions again to grow for another two days (Figure 1).
Figure 1. Brief diagram of seedling growth and treatment process
Note: On each plate, mutants and the Col-0 wildtype control were grown side-by-side. The growth procedure illustrated in Figure 1 will facilitate the subsequent paraquat treatment and root harvest. Roots mainly grow on the surface of the media and can easily be treated and harvested, while leaves do not touch the media surface.
Use a pipette to add 10 mL of 10 μM paraquat slowly into the plates and let the paraquat distribute evenly on the media surface. Keep the plates horizontally in darkness for 6 h.
Note: Use the same quantity of paraquat to treat each plate. The volume of paraquat should be enough to submerge the media surface but not enough to touch the leaves.
Harvest roots and leaves separately. Rinse the materials shortly three times with distilled water, blot them dry with paper towels, and then weigh. For mutants and controls, it is better to use a similar weight of materials for further experiments.
Freeze the materials in liquid nitrogen and store them at -80 °C for paraquat determination.
Treat Arabidopsis protoplasts with paraquat
Prepare protoplasts from the leaves of Arabidopsis grown under short daylight conditions (8:16 h light/dark cycle) for four weeks based on the method described previously (Yoo et al., 2007). Suspend the protoplasts in cold W5 buffer.
Count the protoplasts using a hemocytometer. Remove the W5 buffer and adjust the protoplasts to a final concentration of 2 × 106 protoplasts/mL with paraquat treatment solution. Use at least 10 mL protoplasts for one assay.
Incubate the protoplasts in darkness at room temperature for 3 h. Gently shake the protoplasts by hand every 20 min.
Note: This step is to load the paraquat into the protoplasts.
Centrifuge at 100 × g for 2 min at 4 °C and remove the supernatant carefully.
Add 10 mL of wash buffer into the tube and resuspend the protoplasts gently.
Repeat steps B4–B5 two more times.
Note: The above steps can be used for paraquat uptake assay. The following steps are carried out for paraquat efflux assay.
After the final wash, suspend the protoplasts in wash buffer again and aliquot them equally into several tubes. Each tube corresponds to one time point. Set at least three replicates for each time point. For this assay, we use 0, 10, 30, and 60 min time points. Keep the tubes in darkness at room temperature and start timing using a timer.
At each time point, centrifuge the tubes as in step B4. Collect the supernatants and protoplasts, respectively.
Freeze the samples in liquid nitrogen and then store them at -80 °C for paraquat determination.
Paraquat extraction
Use 1 × 106–2 × 106 protoplasts or 20–30 mg of plant tissues to determine paraquat content in a 2 mL centrifuge tube. Conduct three freeze-thaw cycles (freeze the protoplasts in liquid nitrogen for 1 min, then thaw them in a water bath at room temperature for 2 min) to rupture the protoplasts. For plant tissues, grind the materials into powder in liquid nitrogen.
Add 800 µL of paraquat extraction solution into each tube.
Sonicate each sample for 20 min (1 min on, 1 min off) in total on ice.
Put the samples in a water bath at 60 °C for 30 min and then cool down to room temperature.
Centrifuge at 13,500 × g for 10 min at 4 °C and collect the supernatant.
Repeat steps C2–C5 and combine the supernatants together.
Evaporate the organic solvents with a nitrogen blowing concentrator and store the dried samples at -80 °C until determination.
Paraquat determination
Take the samples from -80 °C and add 100 μL of the room-temperature redissolving solution into the 2 mL centrifuge tubes to dissolve the samples.
Lightly flick the tubes several times until the samples are completely dissolved. Centrifuge at 14,000 × g for 10 min at 4 °C. Pipette 80 μL of the supernatant into a 2 mL amber screw vial with a 150 μL liner pipe.
Inject 1 μL of the sample into the system with a Waters BEH HILIC column (1.7 μm particle size, 2.1 mm × 100 mm).
Perform a linear gradient elution program using LC mobile phase consisting of solvent A and solvent B. Set the flow rate of the mobile phase at 0.4 mL/min to conduct a linear gradient elution program (7 min): 90% B hold for 1 min, 90% B decreased to 20% B in 1 min, 20% B hold for 2.3 min, 20% B increased to 90% B in 0.2 min, and 90% B hold for 2.5 min. (The procedure was 0 min, 90% B; 1 min, 90% B; 2 min, 20% B; 4.3 min, 20% B; 4.5 min, 90% B; and 7 min, 90% B.)
Detect the samples in positive mode (ESI+) with multiple reaction monitoring mode on the LC-MS/MS system.
Set the gas parameters in ion source, including ion source gas 1, ion source gas 2, and the curtain gas to 55, 55, and 35 psi, respectively. Set the temperature to 500 °C and the ion spray voltage to 5,500 V. Monitor two multiple reaction transitions of each analyte for quantification and confirmation of paraquat (186.0–171.0 for quantification and 93.2–171.3 for confirmation). The voltages of declustering potential and collision energy are compound-specific parameters. For paraquat, they are 100, 60, 25, and 17, respectively. Set other parameters to default values as recommended by the manufacturer.
Integrate the chromatographic peak of the target analyte. Integration parameters were set to 9 and 1,000 points for the minimum peak width and height, and S/N integration threshold and baseline subtraction window were set to 3 and 2 min, respectively.
Data analysis
Calculate the chromatographic peak area of paraquat directly by UHPLC-MS/MS. The peak area of paraquat represents the amount of analyte on the column. Since the injection volume is 1 µL and the total volume of the sample is 100 µL, we multiply the chromatographic peak area by 100 to obtain the total quantity of analyte in the sample.
Paraquat efflux assay results are presented in Figure 2A. The same number of protoplasts was sampled at each time point for paraquat determination. Paraquat content in protoplasts was indicated by its chromatographic peak area. The paraquat level at time point 0 was set as 100% and the relative levels of paraquat at other time points were calculated according to the formula in Figure 2B.
Root uptake assay results are presented in Figure 2C. The normalized paraquat level in tissues was obtained by dividing the chromatographic peak area of paraquat by the quantity of materials, that is, the weight of Arabidopsis materials (such as roots and leaves). The paraquat content in different samples was compared.
Figure 2. Paraquat determination in protoplasts and plant tissues by UHPLC-MS/MS. (A) Paraquat efflux assay using Arabidopsis leaf protoplasts. The paraquat level in protoplasts was indicated by its chromatographic peak area. At least three replicates for each time point were determined. The paraquat level at time point 0 was set as 100%, and the relative levels of paraquat at other time points were calculated using the formula given in Figure 2(B). The samples shown are Col-0 (wildtype control), DTX6m-11 (DTX6m-overexpression line), and dm-5 (dtx5 dtx6 double mutant) (Lv et al., 2021). Values are mean ± SD (n = 3). Significance of the differences between Col-0 and the other materials is analyzed by Student’s t-test. PQ, paraquat. ** P ≤ 0.01; * P ≤ 0.05. (B) Calculation formula of relative paraquat level at different time points in Arabidopsis protoplasts. PAt: peak area of paraquat at each time point; PA0: peak area of paraquat at time point 0. (C) Paraquat uptake assay using Arabidopsis roots. Arabidopsis were treated with 10 μM paraquat in darkness for 6 h and the paraquat level in tissues was defined as the chromatographic peak area of paraquat divided by the fresh weight of the tissues. Values are mean ± SD (n = 3). Two-way ANOVA is performed to show the significance of difference.
Recipes
W5 buffer (for protoplast experiment)
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES (pH 5.7)
Note: Prepare mother solutions for each reagent, then filter and store them at 4 °C. Make W5 buffer freshly using mother solutions.
Paraquat treatment solution (for protoplast experiment)
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES (pH 5.7)
5 mM paraquat
Note: Make this solution freshly using the stored mother solutions of each component. The mother solution of paraquat should be stored at -20 °C in the dark.
Wash buffer (for protoplast experiment)
154 mM NaCl
125 mM CaCl2
5 mM KCl
2 mM MES (pH 5.7)
Note: Make wash buffer freshly using the stored mother solutions.
Paraquat extraction solution
60% methanol
40% 0.5 M HCl
Note: This solution can be stored at -20 °C for three months.
Redissolving solution
50% acetonitrile
50% Milli-Q water
Note: Make this solution freshly.
LC mobile phase
Solvent A (pH = 3.95):
100 mM NH4OAC
0.5% formic acid in water
Solvent B:
Acetonitrile
Note: Make solvent A freshly.
Acknowledgments
This protocol was developed based on the methods described previously (Pizzutti et al., 2016; Zou et al., 2015). This work was supported by the National Natural Science Foundation of China (Grants No. 31970343 and 31770274)
Competing interests
No conflict of interest declared.
References
Chen, R., Sun, S., Wang, C., Li, Y., Liang, Y., An, F., Li, C., Dong, H., Yang, X., Zhang, J. and Zuo, J. (2009). The Arabidopsis PARAQUAT RESISTANT2 gene encodes an S-nitrosoglutathione reductase that is a key regulator of cell death. Cell Res 19(12): 1377-1387.
Farrington, J. A., Ebert, M., Land, E. J. and Fletcher, K. (1973). Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides. Biochim Biophys Acta 314(3): 372-381.
Fujibe, T., Saji, H., Arakawa, K., Yabe, N., Takeuchi, Y. and Yamamoto, K. T. (2004). A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation. Plant Physiol 134(1): 275-285.
Fujita, M., Fujita, Y., Iuchi, S., Yamada, K., Kobayashi, Y., Urano, K., Kobayashi, M., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2012). Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis. Proc Natl Acad Sci U S A 109(16): 6343-6347.
Fussell, K. C., Udasin, R. G., Gray, J. P., Mishin, V., Smith, P. J., Heck, D. E. and Laskin, J. D. (2011). Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase. Free Radic Biol Med 50(7): 874-882.
Hawkes, T. R. (2014). Mechanisms of resistance to paraquat in plants. Pest Manag Sci 70(9): 1316-1323.
Huang, Y. J., Huang, Y. P., Xia, J. Q., Fu, Z. P., Chen, Y. F., Huang, Y. P., Ma, A., Hou, W. T., Chen, Y. X., Qi, X., et al. (2022). AtPQT11, a P450 enzyme, detoxifies paraquat via N-demethylation. J Genet Genomics S1673-8527(22)00127-8. doi: 10.1016/j.jgg.2022.04.007.
Li, J., Mu, J., Bai, J., Fu, F., Zou, T., An, F., Zhang, J., Jing, H., Wang, Q., Li, Z., et al. (2013). Paraquat Resistant1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat. Plant Physiol 162(1): 470-483.
Luo, C., Cai, X. T., Du, J., Zhao, T. L., Wang, P. F., Zhao, P. X., Liu, R., Xie, Q., Cao, X. F. and Xiang, C. B. (2016). PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b. PLoS Genet 12: e1006332.
Lv, Z., Zhao, M., Wang, W., Wang, Q., Huang, M., Li, C., Lian, Q., Xia, J., Qi, J., Xiang, C., et al. (2021). Changing Gly311 to an acidic amino acid in the MATE family protein DTX6 enhances Arabidopsis resistance to the dihydropyridine herbicides. Mol Plant 14(12): 2115-2125.
Nazish, T., Huang, Y. J., Zhang, J., Xia, J. Q., Alfatih, A., Luo, C., Cai, X. T., Xi, J., Xu, P. and Xiang, C. B. (2022). Understanding paraquat resistance mechanisms in Arabidopsis thaliana to facilitate the development of paraquat-resistant crops. Plant Commun 3(3): 100321.
Pizzutti, I. R., Vela, G. M., de Kok, A., Scholten, J. M., Dias, J. V., Cardoso, C. D., Concenco, G. and Vivian, R. (2016). Determination of paraquat and diquat: LC-MS method optimization and validation. Food Chem 209: 248-255.
Tsugane, K., Kobayashi, K., Niwa, Y., Ohba, Y., Wada, K. and Kobayashi, H. (1999). A recessive Arabidopsis mutant that grows photoautotrophically under salt stress shows enhanced active oxygen detoxification. Plant Cell 11(7): 1195-1206.
Xi, J., Xu, P. and Xiang, C. B. (2012). Loss of AtPDR11, a plasma membrane-localized ABC transporter, confers paraquat tolerance in Arabidopsis thaliana. Plant J 69(5): 782-791.
Xia, J. Q., Nazish, T., Javaid, A., Ali, M., Liu, Q. Q., Wang, L., Zhang, Z. Y., Zhang, Z. S., Huang, Y. J., Wu, J., et al. (2021). A gain-of-function mutation of the MATE family transporter DTX6 confers paraquat resistance in Arabidopsis. Mol Plant 14(12): 2126-2133.
Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.
Zou, T., He, P., Cao, J. and Li, Z. (2015). Determination of paraquat in vegetables using HPLC-MS-MS. J Chromatogr Sci 53(2): 204-209.
Weigel, D. and Glazebrook, J. (2002). Arabidopsis: A laboratory manual. Cold Spring Harbor Laboratory Press.
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A Protocol for Mitotic Metaphase Chromosome Count Using Shoot Meristematic Tissues of Mulberry Tree Species
Raju Mondal *
SA Sreya Antony *
BG Belaghihalli N. Gnanesh
GT G. Thanavendan
GR G. Ravikumar
BS B. T. Sreenivasa
SD S. Gandhi Doss
KV K. Vijayan
(*contributed equally to this work)
Published: Vol 13, Iss 17, Sep 5, 2023
DOI: 10.21769/BioProtoc.4643 Views: 779
Reviewed by: Wenrong HeAnuradha Singh Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS ONE Aug 2023
Abstract
Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant physiology/performance. Despite its significance, the classical cytogenetic study is now frequently avoided because of its tedious job. In general, root meristems are used to study the mitotic chromosome number, even though the use of root tips was restricted because of sample availability, processing, and lack of standard protocols. Moreover, to date, a protocol using shoot tips to estimate chromosome number has not yet been achieved for tree species’ germplasm with a large number of accessions, like mulberry (Morus spp.). Here, we provide a step-by-step, economically feasible protocol for the pretreatment, fixation, enzymatic treatment, staining, and squashing of meristematic shoot tips. The protocol is validated with worldwide collections of 200 core set accessions with a higher level of ploidy variation, namely diploid (2n = 2x = 28), triploid (2n = 3x = 42), tetraploid (2n = 4x = 56), hexaploid (2n = 6x = 84), and decosaploid (2n = 22x = 308) belonging to nine species of Morus spp. Furthermore, accession from each ploidy group was subjected to flow cytometry (FCM) analysis for confirmation. The present protocol will help to optimize metaphase plate preparation and estimation of chromosome number using meristematic shoot tips of tree species regardless of their sex, location, and/or resources.
Keywords: Chromosome number Flow cytometry Morus spp. Polyploidy Shoot meristem
Background
The plant genome is organized into chromosomes, preserving hereditary information and facilitating its replication, transcription, and transfer. One of the fundamental aspects of plant evolutionary biology is to understand the genome organization and its functional aspects that directly or indirectly act in concert with plant adaptability. Chromosomal studies imply a historical impression of natural consequences, for example, the patterns of chromosomal evolution influenced by factors like natural/artificial selection pressure or crop domestication (Tang et al., 2010). The evolution of chromosome size, structure, and number and the change in DNA composition suggest high plasticity of nuclear genomes at the chromosomal level (Guerra, 2008). In the recent past, integrated cytogenetics, chromosome-level sequencing, and comparative analysis were implemented to grasp the essential component of the evolutionary mechanisms of plant genomes. There are approximately 450,000 species of green plants, but only approximately 300 genome assemblies at the chromosome-scale, corresponding to 812 species (Arita et al., 2021; Kress et al., 2022). Additionally, polyploidy (or whole-genome duplication, WGD) is considered an evolutionary and ecological force in times of stress adaptation (Van de Peer et al., 2021). Hence, chromosomal information has significant importance in plant breeding, genetic, and biotechnological studies including chromosome-scale genome assembly, which can complement molecular phylogeny towards the understanding of complex evolutionary consequences.
Despite its significance, the estimation of chromosome numbers using the squash technique has been restricted (Goldblatt, 2007). For the majority of monocots, root tips are widely used to study the mitotic chromosome number, as the use of shoot tips is laborious, and squashing reduces the spread quality (Anamthawat-Jónsson, 2003). However, mitotic metaphase plate preparation using root tips or floral tissues present considerable disadvantages, specifically for tree plants: (a) living material with actively growing tissues, like meristems, are prerequisite (Guerra, 2008), and the collation of meristematic root tips from the mature tree plant is not easy (Sinha et al., 2016), (b) seed-derived root tips do not represent the same chromosome complements as their mother trees, because of introgressive hybridization (Anamthawat-Jónsson, 2003), (c) grafted plants do not comprise a true root system; hence, chromosomal study is difficult, and (d) the availability of floral tissues depends on the favorable season (Anamthawat-Jónsson, 2003). On the other hand, metaphase plate preparation using shoot meristematic tips present major advantages compared to root tips or floral tissues: (a) ease of collecting intact healthy explants from plants; (b) shoot tips tend to have less condensed chromosomes, and longer or extended chromosomes are desirable as the mapping resolution will be better (Anamthawat-Jónsson, 2003); further, for a highly heterozygous crop like mulberry, the chromosome size and number show huge variations (Datta et al., 1954), and shoot tips could be precious explants to estimate the chromosome number of higher ploidy plants; and (c) for recalcitrant seeds as well as endangered tree species, shoot tips of original mother plants may serve the purpose.
So far, published articles/protocols using shoot tips are very limited; cytogenetics cannot be applied in population studies unless samples are obtained from actual plants in the field, because chromosome number varies among the progeny (Anamthawat-Jónsson, 2003). Recently, Chang et al. (2018) suggested that the small size of chromosomes of mulberry also limits distinguishing euploid/aneuploid, and karyotypic analysis could help identifying different ploidy. It is, therefore, necessary to develop a standard, economically feasible base protocol for the screening and characterization of large-scale germplasm accessions. In addition, high-cost flow cytometry (FCM) analysis for large-scale accessions of germplasm may not be affordable for all researchers (Windham et al., 2020). This barrier is exacerbated by a lack of sufficient details on critical aspects of the protocol like tissue choice, maceration, and squashing (Windham et al., 2020).
Mulberry (Morus spp.) has been commercially exploited as the host of the monophagous pest silkworm (Bombyx mori L.). It belongs to the Moraceae family, which comprises 37 genera, with more than 1,100 species (Clement and Weiblen, 2009). The genus Morus has over 10 species with more than 1,000 cultivated varieties spanning Asia, Europe, Africa, and the United States (He et al., 2013). Efforts were made to classify Morus species; however, to date, taxonomic nomenclature remains doubtful. Besides, genetics of inheritance are also complicated in Morus species due to the higher level of heterozygosity as well as WGD (Jain et al., 2022). Mulberry has a wide range of polyploidy; for example, M. notabilis was reported as a haploid, having a chromosome complement of 2n = x = 14 (He et al., 2013), while M. alba, M. atropurpurea, M. bombycis, M. indica, M. latifolia, and M. rotundiloba were considered diploids, having 2n = 2x = 28 (Datta, 1954). The majority of triploids (2n = 3x = 42) and tetraploids (2n = 4x = 56) have been identified in M. laevigata (Das, 1961). Hexaploid species (2n = 6x = 84), such as thick leaf M. serrata (Basavaiah et al., 1989) and M. tiliaefolia (Seki, 1952), are also recognized; the ploidy can extend up to decosaploid (2n = 22x = 308), as in M. nigra (Basavaiah et al., 1990).
The generation of chromosomal/ploidy-related information of non-model tree plants like mulberry, where the occurrence of polyploidization is common, can be a logistic strategy to create a foundation for future molecular cytogenetics and next-generation sequencing–based work, toward potential crop development and conservation. In this context, the metaphase chromosome number of 200 germplasm accessions of different accessible Morus spp. was counted using shoot meristematic tissue. Accessions were obtained from the Central Sericultural Germplasm Resources Centre (CSGRC), Hosur, India. Hopefully, the present protocol will help to optimize metaphase plate preparation and chromosome number estimation using meristematic shoot tips of tree species regardless of sex, location, and/or resources.
Materials and reagents
Consumables
Eppendorf tubes, 1.5 mL (Tarsons, India)
Reagent bottles, amber 25 mL (Borosil, catalog number: 1519009)
Glass slides, 76 mm × 26 mm (Borosil, Product code 9100P02)
Coverslip, rectangular, 24 mm × 60 mm (Blue Star, India)
Aluminum foil, 72 m (Century, India)
Dissecting needle (Labkafe, India Product code: LKBI 008/1)
Plant Material
Tree plants approximately 15 years old, maintained at CSGRC’s field gene bank, of different cytotypes as diploids (MI-0014 and MI-0308), triploids (MI-0173 and MI-0799), tetraploids (K2-4X), hexaploids (ME-0126, MI-0426, and MI-0571), and decosaploid (ME-0241) were selected for this study. In the present protocol, for metaphase chromosome count, apical shoot meristematic tips were collected after 20 days of pruning.
Reagents
Sterile, double-distilled water (ddH2O)
100% glacial acetic acid (Rankem, catalog number: A0031)
0.5 M ethylenediaminetetraacetic acid (EDTA) (GeNei, catalog number: FC43)
Ethanol (HiMedia, catalog number: MB106)
Potassium chloride (KCl) (HiMedia, catalog number: PCT0012)
2.5 (w/v) pectinase (HiMedia, catalog number: PCT1519)
2.5 (w/v) pectolyase (HiMedia, catalog number: PCT1520)
1:1 (v/v) cellulase (HiMedia, catalog number: RM3331)
1% acetocarmine (HiMedia, catalog number: PCT1304)
1% Orcein (HiMedia, catalog number: RM277)
Saturated para-dichlorobenzene (PDB) (HiMedia, catalog number: GRM6907) (see Recipes)
0.002 M 8-hydroxyquinoline (HQ) (HiMedia, catalog number: GRM7135) (see Recipes)
45% GAA (glacial acetic acid) (see Recipes)
70% ethanol (see Recipes)
3EtOH:1GAA (see Recipes)
75 mM KCl (see Recipes)
Enzyme cocktail (see Recipes)
1% aceto-orcein (see Recipes)
Equipment
Surgical blade, size 22 (Surgeon, India, REF 10122)
Personal protection equipment (Oriley, model: ORPPE6), including gloves (model: KSN30) and safety glasses (Augen, model: safety glass-SG-03)
Ice flaker (PAREX, PSW-130)
Squeeze bottle (Borosil, model: 0166024)
Highly absorbent blotting paper (Swastik, India)
Forceps, pointed, 5" (Borosil, model: LAFP8888005)
Minicooler (Tarson, model: 525060)
Incubator (ESCO, model: CCL-050B-8)
Micropipette (Eppendorf)
Freezer (Whirlpool, model: ICEMAGIC FF-350)
Stereo zoom microscope (Leica, model: Wild M8-308700)
Portable digital microscope (Medprime, model: BT-E2020) with iPad (AppleInc.)
iMac 27’’ M1 chip-macOS Monterey (Apple Inc.)
Sankalp immersion oil (Oil LV, model: 1017)
Spirit lamp (HiMedia, model: LA275)
Sealing wax (Alpha Chemika, model: AL2934)
Software
Cilika (Version 1.30), Medprime Technology Pvt. Ltd. (www.medprimetech.com), image capture software
Microsoft Excel, Microsoft
Keynote presentation software (Apple Inc. Version 10)
Floreada.io (https://floreada.io/flow-cytometry-software)
Procedure
Collections of samples and pre-treatment
Select 3–5 young healthy shoots and use needle and forceps to dissect fresh apical shoot meristematic tips, approximately from 0.5 to 1.0 cm, between 9:00 and 10:00 am. Immediately transfer the collected samples to the pre-fixative solution, i.e., 1 mL of PDB with 20 µL of HQ in a 1.5 mL Eppendorf tube (Figure 1).
Note: For pretreatment of shoot apical meristematic tips, a minimum of three (for large size tetraploid accession) and a maximum of five (small size apical tips specifically for diploid accession) samples per Eppendorf tube can be used. The sample should be transferred immediately to the pre-fixative solution, to enhance the metaphase arrest stage. PDB and HQ stock solutions should be kept separately in amber reagent bottles at room temperature (RT) and mixed properly by gently inverting five times before collection of the sample.
Figure 1. Detailed steps and daily activity for mitotic metaphase plate preparation using shoot meristematic tissues of Morus spp.
Transfer the pretreated samples to a mini cooler at 0 °C for 5 min, followed by 4 °C for 4 h.
Note: Place the mini cooler in the ice bucket before conducting sample collection for the maintenance of the mini cooler’s temperature.
Discard the pre-fixative (PDB+HQ) solution and remove young leaf primordia using pointed forceps and a needle under a stereo zoom microscope. Transfer the trimmed apical shoot tips to a strainer and wash thoroughly under running tap water for 5 min, followed by 5 mL of ddH2O for 10 min in a watch glass (Figure 2).
Note: Removal of the young leaf primordia (usually 3–4 numbers) is useful to enhance enzymatic treatment (Step C9). Thorough washing is necessary to remove PDB+HQ residues.
Figure 2. Step-by-step flowchart of sample collection, pretreatment, fixation, staining, and squash for microscopic observation
Fixation of meristems
Transfer the washed samples to 1 mL of ice-cold 3EtOH:1GAA in a 1.5 mL Eppendorf tube and incubate for 1 h at RT (24 ± 2 °C).
Note: Prepare 3EtOH:1GAA fresh before fixation.
Discard the 3EtOH:1GAA solution and replace with freshly prepared ice-cold 3EtOH:1GAA solution. Incubate the samples at 4 °C for a minimum of two days. Replace with fresh 3EtOH:1GAA solution every 12 h.
Discard the 3EtOH:1GAA solution and wash thoroughly in 5 mL of ddH2O for 10 min; then, store the samples in 1 mL of 70% ethanol in a 1.5 mL Eppendorf tube at 4 °C for further use.
Wash the samples in 5 mL of ddH2O twice in a watch glass for 10 min each. Transfer the samples to 1 mL of 45% GAA in a 1.5 mL Eppendorf tube and incubate for 1 h at RT.
Wash thoroughly in 5 mL of ddH2O for 10 min in a watch glass and remove water from the sample with a blotting paper.
Enzymatic digestion
Treat the samples with an enzyme cocktail comprised of cellulase (2%), pectinase (2.5%), and pectolyase (1%) for 4 h at 37 °C in the dark using an incubator.
Staining
Transfer enzyme-treated samples to 500 μL of 1% aceto-orcein in a 1.5 mL Eppendorf tube covered with aluminum foil (to maintain dark conditions) and incubate the sample-containing tubes for 12–14 h at RT.
Squashing (see Video 1)
Video 1. Squash technique for chromosome study of Morus spp.
Transfer one of the processed (stained) samples from the aceto-orcein stain to a glass slide.
Note: Remove the excess stain with the help of blotting paper if required.
Add two drops of 45% GAA to the sample and gently dissolve the tissue with the back side of the needle.
Gently mix the sample with the addition of one drop of acetocarmine and three drops of aceto-orcein stain; subsequently, remove the debris with a dissecting needle.
Place a coverslip over the slide. Keep the slide inside a folded blotting paper and gently press with your thumb to remove the excess stain and to spread uniformly.
Gently tap over the coverslip using the backside of the needle to obtain an optimum spread of cells as well as chromosomes.
Note: To enhance the spreading quality, continuous tapping is required until the clump of cells spread over the slide as a thin layer of cells. Ensure that no air bubbles remain.
Apply flame heat (using spirit lamp) on the bottom side of the mounted slide for 2–3 s and gently tap for precise chromosome spreading.
Seal the mounted slide with wax (optional).
Place the slide and visualize the chromosome under the microscope (Portable digital microscope, Medprime, model: BT-E2020). Any compound microscope with 40× or 100× objectives with oil immersion can be used. Microscopic images of representative accessions from each ploidy group are represented in Figure 3.
Figure 3. Metaphase plates and estimated chromosome number of different cytotypes of Morus spp. (A) diploid V1 (2n = 2x = 28), (B) triploid AR12 (2n = 3x = 42), (C) tetraploid M. laevigata L. (2n = 4x = 28), (D) hexaploid M. Serrata Roxb. (2n = 6x = 84), and (E) decosaploid M. nigra L. (2n = 22x = 308). Scale bar = 5 µm.
Flow cytometry (FCM) analysis
To confirm the ploidy level cytotypes, genome size was estimated by FCM of selected ploidy (2x, 3x, 4x, 6x, and 22x) accessions, which were identified through chromosome number count (Figure 4). A dual laser FACSCaliburTM (BD Biosciences, United States) was used to estimate genome size with some modifications to the protocol described by Galbraith et al. (1983). In brief, young mulberry leaves (5–6 days old) of approximately 0.5 cm2 were collected between 8:30 and 9:00 am. With a razor blade, the leaf sample was chopped in 2 mL of nuclear isolation buffer [hypotonic propidium iodide (50 μg/mL), trisodium citrate dihydride (3 g/L), 0.05% (v/v) Nonidet P-40, and RNase A (2 mg/mL)]; filtered (30 μm nylon mesh) nucleus suspensions were collected in tubes. The tubes were capped and kept at 37 °C for 30 min. Then, the samples were subjected to FCM analysis. Pisum sativum was used as the standard reference and measurements were made in triplicates.
Figure 4. Confirmation of different mulberry cytotypes using flow cytometry analysis. Histogram of propidium-iodine-A (PI-A) fluorescence intensity of (A) reference of Pisum sativum, (B–C) diploid, (D–E) triploid, and (F) tetraploid. (G) List of accessions studied, genome size [in mega base pairs (Mbp) and pictogram (pg)], and predicted ploidy level. (H) Histogram of PI fluorescence intensity (count vs. PI-A) of diploid (2x), triploid (3x), tetraploid (4x), hexaploid (6x), and decosaploid (22x) with reference to Pisum sativum. (I) Scatterplot (PI-W vs. PI-A) of diploid (2x), triploid (3x), tetraploid (4x), hexaploid (6x), and decosaploid (22x) nuclei, showing they are evenly spaced in respect to fluorescence and represent a well-defined series of areas that correspond to 2C, 3C, 4C, 6C, and 22C nuclei.
Data analysis
Metaphase plate images were captured (with an automated measuring scale bar) and a presentation (karyomorphological drawing) was prepared in Keynote, Apple Inc. (Version 10).
Genome size (Mbp) was calculated according to the formulae by Lysak and Dolezel (1998) with the conversion of 1 pg equal to 980 Mbp (Dolezel et al., 2003). Finally, the genome size of studied accessions was calculated using the standard reference of Pisum sativum. DNA content of the mulberry accessions ranged from 0.85 (diploid) to 8.67 pg (decosaploid) and the coefficient of variation was 3.21 (<5%; Figure 4H). The floreada.io (https://floreada.io/flow-cytometry-software) online tool was used to generate histograms and scatterplots for FCM analysis.
Notes
In step B5, change 3EtOH:1GAA solution once every 30 min for bleaching of chlorophyll.
In step B6, incubation for a minimum of two days and resuspending the sample in freshly prepared ice-cold 3EtOH:1GAA solution at 12-h-intervals is essential.
In step C9, enzymatic treatment (pectinase, cellulase, and pectolyase) for 4 h at 37 °C in the dark is recommended for karyotype analysis of higher ploidy level (2n = 3x, 4x, 6x, and 22x). For general cytological analysis and chromosome study, treating only with pectinase for 6 h at 37 °C is optimal.
Recipes
PDB
Reagent Final concentration Amount
PDB Saturated 10 g
H2O n/a 95 mL
Total n/a 100 mL
70% ethanol
Reagent Final concentration Amount
Ethanol (absolute) 70% 70 mL
H2O n/a 30 mL
Total n/a 100 mL
0.002 M HQ
Reagent Final concentration Amount
HQ 0.002 M 0.073 g
ddH2O n/a 250 mL
Total n/a 250 mL
45% GAA
Reagent Final concentration Amount
GAA 45% 45 mL
ddH2O n/a 55 mL
Total n/a 100mL
Potassium chloride (KCl)
Reagent Final concentration Amount
KCl 1 M 7.5 g
ddH2O n/a 100 mL
Total n/a 100 mL
EtOH (3):GAA (1)
Reagent Final concentration Amount
Ethanol 3 parts 75 mL
GAA 1 part 25 mL
Total n/a 100 mL
Enzyme cocktail
Reagent Final concentration Amount
KCl 75 mM 7.5 mL
Cellulase 2% 2.0 g
Pectinase 2.5% 2.5 g
Pectolyase 1% 1.0 g
EDTA (0.5 M) 7.5 mM 1.5 mL
ddH2O n/a 91 mL
Total n/a 100 mL
1% Aceto-orcein
Reagent Final concentration Amount
GAA 45% 45 mL
Orcein 1% 1.0 g
ddH2O n/a 55 mL
Total n/a 100 mL
Acknowledgments
The work was supported by a grant (PIG 06004 SI) from Central Silk Board (CSB), Ministry of Textiles, GoI, Bengaluru, India. Ms. Sreya Antony thanks CSB for the Junior Research Fellowship. We are grateful to Dr. Chandish R. Ballal, Former Director, ICAR-National Bureau of Agricultural Insect Resources, Bengaluru, India for continuous encouragement and support. We thank Mr. Shreyas M. Burji, Auxochromofours Solutions Pvt. Ltd., and the team of the National Centre for Biological Sciences (NCBS), Bengaluru, India for the flow cytometry facility, and Mr. C. Ventakeshappa, CSGRC-Hosur for technical support. We would like to thank Subhankar Biswas, Department of Botany, ISc, Banaras Hindu University, Varanasi, India for constructive suggestions for video editing. We are also thankful to all three expert reviewers for their constructive suggestions/comments.
This protocol is based on the research paper (Gnanesh et al. 2023).
Competing interests
The authors declare that they have no competing interests.
References
Anamthawat-Jónsson, K. (2003). Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization. Methods Cell Sci 25(3-4): 91-95.
Arita, M., Karsch-Mizrachi, I. and Cochrane, G. (2021). The international nucleotide sequence database collaboration. Nucleic Acids Res 49(D1): D121-D124.
Basavaiah, Dandin, S. B. and Rajan, M. V. (1989). Microsporogenesis in hexaploid Morus serrata Roxb. Cytologia 54(4): 747-751.
Basavaiah, Dandin, S. B., Dhar, A. and Sengupta, K. (1990).Meiosis in natural decosaploid (22x) Morus nigra L.Cytologia55(3): 505-509.
Chang, L.Y., Li, K.T., Yang, W.J., Chung, M.C., Chang, J.C. and Chang, M.W. (2018). Ploidy level and their relationship with vegetative traits of mulberry (Morus spp.) species in Taiwan. Sci Hortic 235: 78-85.
Clement, W. L. and Weiblen, G. D. (2009). Morphological evolution in the mulberry family (Moraceae). Systematic Botany 34(3): 530-552.
Das, B. C. (1961). Cytological studies on Morus indica L. and Morus laevigata Wall. Caryologia14(1): 159-162.
Datta, M. (1954). Cytogenetical studies on two species of Morus. Cytologia19(1): 86-95.
Dolezel, J., Bartos, J., Voglmayr, H. and Greilhuber, J. (2003). Nuclear DNA content and genome size of trout and human. Cytometry A 51(2): 127-128; author reply 129.
Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M., Sharma, D. P. and Firoozabady, E. (1983). Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220(4601): 1049-1051.
Gnanesh, B. N., Mondal, R., Arunakumar, G. S., Manojkumar, H. B., Singh, P., Bhavya, M. R., Sowbhagya, P., Burji, S. M., Mogili, T., Sivaprasad V., et al. (2023). Genome size, genetic diversity, and phenotypic variability imply the effect of genetic variation instead of ploidy on trait plasticity in the cross-pollinated tree species of mulberry. PLoS One 18(8): e0289766.
Goldblatt, P. (2007). The index to plant chromosome numbers: past and future. Taxon56(4): 984-986.
Guerra, M. (2008). Chromosome numbers in plant cytotaxonomy: concepts and implications. Cytogenet Genome Res 120(3-4): 339-350.
He, N., Zhang, C., Qi, X., Zhao, S., Tao, Y., Yang, G., Lee, T. H., Wang, X., Cai, Q., Li, D., et al. (2013). Draft genome sequence of the mulberry tree Morus notabilis. Nat Commun 4: 2445.
Jain, M., Bansal, J., Rajkumar, M. S., Sharma, N., Khurana, J. P. and Khurana, P. (2022). Draft genome sequence of Indian mulberry (Morus indica) provides a resource for functional and translational genomics. Genomics 114(3): 110346.
Kress, W. J., Soltis, D. E., Kersey, P. J., Wegrzyn, J. L., Leebens-Mack, J. H., Gostel, M. R., Liu, X. and Soltis, P. S. (2022). Green plant genomes: What we know in an era of rapidly expanding opportunities. Proc Natl Acad Sci U S A 119(4): e2115640118.
Lysak, M. A. and Dolezel, J. (1998). Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 51(2): 123-132.
Seki, H. (1952). Cytological studies of Moraceae plants (V) On the chromosome number of Morus tiliaefolia Makino. J Fac Text Seric Shinshu Univ 2: 13-17.
Sinha, S., Karmakar, K., Devani, R., Banerjee, J., Sinha, R. and Banerjee, A. (2016). Preparation of Mitotic and Meiotic Metaphase Chromosomes from Young Leaves and Flower Buds of Cocciniagrandis. Bio-protocol 6(7): e1771.
Tang, H., Sezen, U. and Paterson, A. H. (2010). Domestication and plant genomes. CurrOpin Plant Biol 13(2): 160-166.
Van de Peer, Y., Ashman, T. L., Soltis, P. S. and Soltis, D. E. (2021). Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell 33(1): 11-26.
Windham, M. D., Pryer, K. M., Poindexter, D. B., Li, F. W., Rothfels, C. J. and Beck, J. B. (2020). A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited. Appl Plant Sci 8(4): e11342.
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Plant Science > Plant molecular biology > Chromatin
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4,644 | https://bio-protocol.org/en/bpdetail?id=4644&type=0 | # Bio-Protocol Content
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In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method
FN Fayrouz Naim *
RH Rie Hasebe *
SH Shintaro Hojyo *
YS Yukatsu Shichibu *
AI Asuka Ishii
YT Yuki Tanaka
KT Kazuki Tainaka
SK Shimpei I. Kubota
KK Katsuaki Konishi
MM Masaaki Murakami
(*contributed equally to this work)
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4644 Views: 969
Reviewed by: Meenal SinhaJulie WeidnerMarieta Ruseva
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Original Research Article:
The authors used this protocol in Cell Feb 2012
Abstract
Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.
Keywords: Experimental autoimmune encephalomyelitis (EAE) CD4+ T cells Myelin oligodendrocyte glycoprotein (MOG) Gateway reflex Microinflammation CUBIC Au13 nanoclusters
Background
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), mediated by myelin-specific autoreactive CD4+ T cells (International Multiple Sclerosis Genetics et al., 2011). Active experimental autoimmune encephalomyelitis (aEAE) is a well-established animal model of MS that is induced by immunizing mice with myelin oligodendrocyte protein–derived peptide (MOG) and complete Freund's adjuvant (CFA) together with pertussis toxin (PTx). However, where and how pathogenic CD4+ T cells enter the CNS from peripheral blood and cause the disease had been unclear, since it was believed that the CNS is protected from immune cells by the blood–brain barrier (Liu et al., 2012). Using a passive transfer model of EAE, in which pathogenic CD4+ T cells including autoreactive CD4+ T cells from the secondary lymphoid tissues of aEAE mice are transferred to naïve mice, we demonstrated that sensory-sympathetic crosstalk, triggered by gravity stimulation via the soleus muscle, leads to the accumulation of pathogenic immune cells at the dorsal vessels of the fifth lumbar cord (L5) locally, which in turn causes microinflammation, a chronic inflammation that enhances the permeability of specific blood vessel sites by elevated local inflammatory mediators, such as IL-6 and TNF-α (Arima et al., 2012). Importantly, this permeability enables autoreactive CD4+ T cells to enter the CNS. We named this specific neuro-immune interaction the gateway reflex and have since demonstrated it can be activated by six types of environmental stimuli: gravity, electricity, pain, stress, light, and inflammation (Arima et al., 2012, 2015 and 2017; Hasebe et al., 2022; Murakami et al., 2019; Stofkova et al., 2019).
Coordinated gold nanoclusters (AuNCs) have an atomically precise molecular structure, ultrasmall size, facile surface modification, and characteristic optical properties (Jin et al., 2016). Some AuNCs have been reported to exhibit unique photoluminescence emission with long lifetimes and large Stokes shift from the visible to near-infrared wavelengths (Chen et al., 2015; Yu et al., 2019), as well as high compatibility and photostability. Thus, they are good candidates as luminophores for long-term imaging, high-sensitivity detection, and target-specific treatment (Luo and Liu, 2022; Palmal and Jana, 2014; Zhang et al., 2022). The usefulness of AuNCs as fluorescent probes has already been shown in imaging, detection, and therapy. Phosphine-coordinated AuNCs represent a luminescent gold cluster family (Konishi et al., 2018; Sugiuchi et al., 2017). Among them, diphosphine-coordinated Au13 nanoclusters with an icosahedral gold core motif are interesting, because of their easy accessibility, robustness, and moderately intense photoluminescence in the near-infrared region (Shichibu and Konishi, 2010; Sugiuchi et al., 2015).
The development of tissue-clearing reagents and protocols, efficient fluorescent labeling, and rapid volumetric imaging by light sheet microscopy has enabled new tissue-clearing methods for the 3D imaging of intact tissues and even entire organisms (Hama et al., 2015; Ueda et al., 2020). Despite tissue-clearing approaches enabling high tissue transparency within a few days using organic chemicals (e.g., benzyl alcohol and benzyl benzoate), concerns about the quenching of fluorescent proteins and safety have remained. In response, Tainaka et al. (2014) developed the hydrophilic unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method, which efficiently removes lipids, pigments, and calcium phosphate to retain the fluorescent signals and enable whole-body imaging with single-cell resolution in mice. Combining light sheet microscopy and CUBIC can visualize rare cells and complex structures in various tissues at the whole organ level.
Chronic microinflammation is a hallmark of many inflammatory diseases, including autoimmune, neurodegenerative, and metabolic syndrome–driven diseases. In such inflammatory diseases with stochastic and proliferative processes, single-cell events ultimately affect the health status of the entire organism. Therefore, a detection system that enables quick identification of the development and progression of microinflammation is desired. However, conventional protocols for identifying inflammation sites are mainly based on 2D histology using inflammatory mediator-specific reporter mice or antibodies, which makes it difficult to accurately identify the sites depending on the tissue sections.
In this protocol, we show a rapid 3D-imaging method to detect microinflammation in aEAE mice using Au13 nanoclusters and CUBIC. Au13 signals leaked out from the blood vessels around the L5, indicating enhanced vascular permeability caused by microinflammation, supporting our previous findings of a vascular gate formed for pathogenic immune cells to enter the CNS in this region (Arima et al., 2012). Thus, our method is useful for studying the stepwise development of microinflammation at the whole organ level in various inflammatory diseases and will also provide future perspectives of Au13 nanocluster-based imaging techniques to study biological events, which are induced by the enhancement of vascular permeability at specific vessel sites.
Materials and Reagents
C57BL/6NCrSlc mice (6–8-week-old male; Japan SLC)
50 mL polypropylene conical tube (Corning, Falcon®, catalog number: 352070)
15 mL polypropylene conical tube (Corning, Falcon®, catalog number: 352096)
1 mL syringe for tuberculin slip tip (TERUMO, catalog number: SS-01T)
Injection needle 25G × 1 (TERUMO, catalog number: NN-2525R)
Insulin syringe (with 27G × 1/2 needle) (TERUMO, catalog number: SS-10M2713)
Normal winged needle for vein D type (25G × 5/8 needle) (TERUMO, catalog number: SV-25DLK)
Three-way stopcock (TERUMO, catalog number: TS-TL1K)
30 mL syringe lock type 30 (TERUMO, catalog number: SS-30LZ)
5 mL Costar Stripette (Corning, catalog number: 4487)
10 mL Costar Stripette (Corning, catalog number: 4488)
25 mL Costar Stripette (Corning, catalog number: 4489)
MOG35-55 (MEVGWYRSPFSRVVHLYRNGK, Sigma-Aldrich)
Incomplete Freunds’ adjuvant (IFA) (Sigma-Aldrich, catalog number: F5506)
Mycobacterium tuberculosis H37RA (DIFCO, catalog number: 231141)
PTx from Bordetella pertussis (Sigma-Aldrich, catalog number: P7208-50UG)
Saline (Otsuka Pharmaceutical, catalog number: K6E85)
Isoflurane (Pfizer)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625)
Potassium chloride (KCl) (Kanto Chemical, catalog number: 32326-00)
Disodium hydrogen phosphate (Na2HPO4) (Nacalai Tesque, catalog number: 31801-05)
Potassium dihydrogen phosphate (KH2PO4) (Kishida Chemical, catalog number: 000-63955)
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034)
Sodium hydroxide (NaOH) (FUJIFILM Wako Chemicals, catalog number: 198-13765)
4% paraformaldehyde phosphate buffer solution (PFA) (Nacalai Tesque, catalog number: 09154-85)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Sodium azide (FUJIFILM Wako Chemicals, catalog number: 197-11091)
Casein from bovine milk (Sigma-Aldrich, catalog number: C3400)
1,2-Hexanediol (Tokyo Chemical Industry, catalog number: H0688)
1-Methylimidazole (Tokyo Chemical Industry, catalog number: M0508)
2,3-Dimethyl-1-phenyl-5-pyrazolone (Tokyo Chemical Industry, catalog number: D1876)
Nicotinamide (Tokyo Chemical Industry, catalog number: N0078)
Silicone oil (Shin-Etsu Silicone, catalog number: HIVAC-F4)
Mineral oil (Sigma-Aldrich, catalog number: M8410-1L)
Anti-actin, α-smooth muscle (α-SMA)–FITC antibody, mouse monoclonal (clone: 1A4) (Sigma-Aldrich, catalog number: F3777)
Purified rat anti-mouse CD31 (clone: MEC 13.3) (BD Biosciences, catalog number: 550274)
Goat anti-rat IgG (H+L) cross-absorbed secondary antibody, Alexa FluorTM 555 (Thermo Fisher Scientific, catalog number: A-21434)
Dimethyl sulfoxide (DMSO) for spectrochemical analysis (FUJIFILM Wako Chemicals, catalog number: 045-28335)
Sodium borohydride (NaBH4) (Tokyo Chemical Industry, catalog number: S0480)
Ethanol (Kanto Chemical, catalog number: 14033-00)
[1,2-Bis(diphenylphosphino)ethane]dichlorodigold(I) (Au2(dppe)Cl2) (Sigma-Aldrich, catalog number: 717363)
Dichloromethane (Kanto Chemical, catalog number: 10158-81)
Hydrochloric acid (FUJIFILM Wako Chemicals, catalog number: 080-01061)
Acetone (Kanto Chemical, catalog number: 01026-81)
Methanol (Kanto Chemical, catalog number: 25183-81)
[Au13(dppe)5Cl2]Cl3 (Au13) [synthesized according to the literature (Shichibu and Konishi, 2010), see Recipes]
1 M HEPES buffer, pH 7.2 (see Recipes)
Phosphate buffered saline (PBS) (see Recipes)
Delipidation/decoloration buffer (see Recipes)
Staining buffer (see Recipes)
CUBIC-R (see Recipes)
Observation oil (see Recipes)
Equipment
Micro-dissecting scissors (Hammacher, catalog number: HSB014-11)
Micro scissors 105 mm straight sword (AS ONE, catalog number: YS-7105)
Angled serrated tip forceps (Hammacher, catalog number: HSC187-11)
KFI tweezers GG (Kowa Forceps Industry, catalog number: K-3 GG)
PIPETMAN P (Gilson, model: P20, catalog number: F123600)
PIPETMAN P (Gilson, model: P200, catalog number: F123601)
PIPETMAN P (Gilson, model: P1000, catalog number: F123602)
Pipet-aid (FALCON/catalog number: 357590)
Light sheet microscopy (LaVision BioTec/UltraMicroscope II)
Shake-LR (TAITEC, catalog number: 0054809-000)
Wave-SI (TAITEC, catalog number: 0054334-000)
Double shaker NR-30 (TAITEC, catalog number: 0000205-000)
Neo shaker NS-LR (AS ONE, catalog number: 2-7827-01)
Stereomicroscope (OLYMPUS, model: SZ61)
LED illuminator stand (OLYMPUS, model: SZ2-ILST)
Refractive index measurement equipment (ATAGO, catalog number: PAL-RI)
Software
Imaris version 9.0.0 (Bitplane, https://imaris.oxinst.com)
Procedure
Figure 1 shows an overview of the protocol.
Figure 1. Overview of procedures for microinflammation detection using Au13 nanoclusters and the CUBIC tissue-clearing method. Mice were immunized with MOG35-55/CFA (IFA containing M. tuberculosis H37RA) and underwent PTx injection. On days 16–20, when experimental autoimmune encephalomyelitis (EAE) develops with clinical scores of 2–3, the EAE mice were injected with Au13 nanoclusters, anesthetized after 2 h, and transcardially perfused with PBS and then PFA. The spine with the ribs was taken out and further subjected to PFA fixation for one day. Then, L1–L6 were excised carefully and immersed in delipidation/decoloration reagent (hexanediol/methylimidazole) for four days (days 1–5). After delipidation and decoloration, L1–L6 were stained with anti-SMA or anti-CD31 for seven days (days 5–12) and subjected to refractive index adjustment in CUBIC-R (days 12–14). The Au signal was visualized by light sheet microscopy and analyzed by Imaris software.
Induction of EAE
Mix 100 μL (400 μg) of 4 mg/mL of MOG35-55 in PBS and 100 μL of CFA [10 mL of IFA plus two vials (20 mg) of M. tuberculosis H37RA] in two syringes using a three-way stopcock. Emulsify by pressing the syringe back and forth, transferring the contents from one syringe to the other for 5–10 min until a stable white emulsion is produced (for details, see Tanaka et al., 2017).
Immunize C57BL/6 mice with 200 μL of MOG35-55/CFA subcutaneously (s.c.) at the tail base using a 1 mL syringe with a 25G needle on day 0.
Inject i.v. 400 ng of PTx in 200 μL of saline into the tail vein of mice using an insulin syringe on days 0, 2, and 7.
Assess clinical scores of EAE daily with a 0–5 scoring system (0, no signs; 1, flaccid tail; 2, impaired righting reflex and/or gait; 2.5, paralysis of one hind limb; 3, paralysis of both hind limbs; 4, hind limb paralysis with partial fore limb paralysis; 5, moribund or dead).
Preparation of Au13 nanoclusters
Synthesize Au13 nanoclusters ([Au13(dppe)5Cl2]Cl3; see Recipe 1) and isolate as crystalline solids according to the procedures described in Shichibu and Konishi (2010).
Store 160 μM Au13 nanoclusters in DMSO.
Fixation of spines from Au13 nanoclusters–treated EAE mice
Prior to injecting into mice, Au13 nanoclusters are diluted 5-fold with 10 mM HEPES (pH 7.2, see Recipe 2) or saline to 32 μM.
Inject i.v. 200 μL of 32 μM Au13 nanoclusters into the tail vein of aEAE mice with clinical score 2–3 using the insulin syringe.
After 2 h, anesthetize the mice with 4% isoflurane.
Perfuse the mice with PBS (see Recipe 3) and PFA using the winged needle and three-way stopcock locked by syringes.
Make an incision in the right atrial appendage of the heart.
Perfuse with 25 mL of cold PBS via the left ventricle of the heart using the syringe with the 25G winged needle.
After the PBS perfusion, change the solution to 4% PFA using the three-way stopcock.
Perfuse with 20 mL of cold 4% PFA.
Take out the spine with the ribs (Video 1) and immerse in 40 mL of 4% PFA in a 50 mL tube at 4 °C for 24 h with shaking at 60 rpm in an orbital motion on a TAITEC Shake-LR.
Video 1. Procedures for the L1-L6 preparation. After transcardial perfusion with PBS followed by PFA, the spine with the ribs was immediately taken out and further fixed with PFA. Then, L1–L6 were excised by carefully cutting the intervertebral discs between T13-L1 and L6-S1 with a razor, with the T13 rib as a position marker under stereomicroscopy installed with an LED illuminator.
Delipidation/decoloration
Wash the spine with the ribs with 40 mL of PBS at room temperature for 30 min three times while shaking at 30 rpm in a seesaw motion on a TAITEC Wave-SI.
Excise L1–L6 carefully under stereomicroscopy installed with the LED illuminator (Video 1).
Immerse L1–L6 in 40 mL of delipidation/decoloration buffer (see Recipe 4) in a 50 mL tube at 37 °C for 4 days with shaking at 80 rpm in a reciprocating motion on an AS ONE Neo shaker NS-LR.
Antibody staining
Wash L1–L6 with 40 mL of PBS at room temperature for 1 h three times with shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
Dilute FITC-conjugated α-SMA antibody and purified rat anti-mouse CD31, which recognize smooth muscle actin on the artery to platelet endothelial cell adhesion molecule-1 on whole blood vessels, respectively, to 1:50 in staining buffer (see Recipe 5).
Immerse L1–L6 in 1.5 mL of the diluted antibody solution in a 5 mL polypropylene tube at room temperature for seven days with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
Wash L1–L6 with 40 mL of PBS in a 50 mL tube at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
For anti-CD31 staining:
Further immerse L1–L6 in 1.5 mL of Alexa Fluor 555-conjugated goat anti-rat IgG diluted to 1:200 in staining buffer at room temperature for two days with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
Wash L1–L6 with 40 mL of PBS in a 50 mL tube at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
Postfix L1–L6 with 10 mL of 1% PFA in PBS at room temperature for 5 h with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
Wash L1–L6 with 40 mL of PBS at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
Refractive index adjustment
Living organisms are composed of molecules with different refractive indices, such as water, proteins, and lipids. In order to observe mammalian tissues in 3D by light sheet microscopy, it is important to suppress light scattering by making the refractive index uniform.
Immerse L1–L6 in 10 mL of 1/2 CUBIC-R [diluted CUBIC-R (see Recipe 6) to 1:1 in dH2O] at room temperature for 5 h with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
Discard 1/2 CUBIC-R and immerse L1–L6 in 10 mL of CUBIC-R at room temperature overnight with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
Replace with fresh CUBIC-R and further immerse L1–L6 at room temperature for 4 h with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30 (Figure 2).
Figure 2. Cleared lumbar cords. Brightfield image of the lumbar cords before and after tissue clearing.
Microscopy and image analysis
Wash L1–L6 with 10 mL of observation oil (see Recipe 7).
Place L1–L6 into a glass container filled with observation oil.
Acquire 3D images using a light sheet microscope (UltraMicroscope II) as reported previously (Dodt et al., 2007; Susaki et al., 2014).
Collect all raw image data in lossless 16-bit TIFF format.
Visualize, capture, and analyze 3D-rendered images using Imaris software, version 9.0.0 (Figures 3 and 4, Videos 2 and 3).
Figure 3. Detection of microinflammation at L5 by Au13 nanoclusters and staining of the artery with anti-SMA antibody. (A) aEAE mice with clinical score 2.5 and naïve mice were injected i.v. with Au13 nanoclusters or solvent. Figures show cleared L3–L6 from each mouse stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. The signal of Au13 nanoclusters was visualized as a punctate signal with a diameter of 50–200 nm in the dorsal blood vessels centered on L5 and in the parenchyma of the spinal cords in aEAE mice. Intravascular Au signals were also observed in aEAE and naïve mice injected with Au13 nanoclusters. Scale bars, 1,500 μm. Magenta: Au13 nanocluster; green: SMA (artery). (B) Magnified images of L5 from the aEAE and naïve mice in (A). Scale bars, 1,000 μm. Magenta: Au13 nanocluster; green: SMA (artery).
Figure 4. Detection of microinflammation at L5 by Au13 nanoclusters and staining of whole blood vessels with anti-CD31 antibody. aEAE mice with clinical score 3 were injected i.v. with Au13 nanoclusters. Cleared L5 stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels, is shown. Scale bars, 400 μm. Green: Au13 nanocluster; red: CD31 (blood vessel).
Video 2. The detection of microinflammation at L5 by Au13 nanoclusters and staining of the artery with anti-SMA antibody. A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 2.5 after injecting i.v. with Au13 nanoclusters. The cleared lumbar spinal cords were stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. Magenta: Au13 nanocluster; green: SMA (artery).
Video 3. The detection of microinflammation at L5 by Au13 nanoclusters and staining of whole blood vessels with anti-CD31 antibody.A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 3 after injecting i.v. with Au13 nanoclusters. The cleared lumbar spinal cords were stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels. Green: Au13 nanocluster; red: CD31 (blood vessel).
Notes
Sodium azide–containing staining buffer is classified as a particularly hazardous substance due to high acute toxicity, especially to dermis.
CUBIC-R and other reagents are safe reagents, so there is no need to prepare them under a chemical fume hood. Further, they are highly stable and not difficult to prepare.
The injection of 200 μL of PTx or Au13 nanoclusters i.v. should be performed by slowly pressing the insulin syringe and injecting the full volume of solution for approximately five seconds.
In our aEAE induction protocol, the disease usually develops with clinical scores of 2–3 on days 16–20 after the immunization. However, the timing of the disease onset and when the clinical score reaches 2–3 may depend on the manufacturing lot of the MOG peptides, CFA, and PTx as well as the mouse age and sex.
Mice with clinical score 3–4 were humanely euthanized according to ethical regulations.
When Au13 nanoclusters were injected for 4 h, the Au signal was not detected. Therefore, we set the time to 2 h.
Mice were anesthetized with 4% isoflurane and PBS was then perfused while the mice were alive. Since the end of the transcardial perfusion uses 25 mL of PBS (more than 10 times the total blood volume), the mice were dead before the PFA perfusion.
In the fixation, washing, and refractive index adjustment steps, the entire tissue sample may be transferred from a decanter to a clean container and then transferred again into a tube filled with fresh buffer or solution using tweezers without a hook or spoon to minimize damage.
In step D2, L1–L6 should be excised from the spine carefully to minimize damage using micro-scissors and angled serrated tip forceps under the stereomicroscope with illuminator (Video 1).
The compounds used for delipidation/decolorization are surfactants and may affect antigenicity.
The lumbar spinal cords are regularly immersed in delipidation/decoloration buffer only once.
From step E3 onward, the samples should be protected from light.
For step E3, a 1.5 mL Eppendorf tube may be a bit small. For the spinal cord, a 2 mL tube is fine for antibody staining.
For light sheet microscopy, pictures are taken so that all tissues are included. It is recommended to acquire data in Z-stacks every 5 μm.
In our protocol, the spots were not counted. However, it would be possible to count visually or calculate the volume of each spot with a software such as Imaris.
Recipes
[Au13(dppe)5Cl2]Cl3 (Au13)
Synthesize and isolate as crystalline solids according to the method of Shichibu and Konishi (2010).
Add 10 mL of ethanol containing NaBH4 (66.2 mg, 1.75 mmol) to 240 mL of dichloromethane containing Au2(dppe)Cl2 (302 mg, 0.35 mmol).
Stir the mixture at room temperature for 3 h.
Remove the solvent, suspend the residue in dichloromethane, and filter.
Evaporate the filtrate to dryness until forming a dark-brown powder (283.6 mg).
Dissolve the powder in ethanol (49 mL) and then treat with 1 mL aqueous hydrochloric acid (12 mmol).
Stir the mixture at room temperature for 24 h.
Evaporate the volatiles.
Wash the resulting residue with acetone.
Suspend the resulting residue in methanol and filter.
Evaporate the filtrate to dryness until forming [Au13(dppe)5CI2]CI3 (Au13) as crystalline red solids (75.9 mg).
Weigh the crystalline solids of the Au13 cluster (~3 mg).
Dissolve the crystalline solids in dimethyl sulfoxide (final concentration: 160 μM).
Store the nanoclusters at room temperature for up to one month with light shielding.
1 M HEPES buffer, pH 7.2 (1,000 mL)
Add 238.3 g of HEPES to 800 mL of dH2O.
Adjust the buffer to pH 7.2 with 10 N NaOH.
Add dH2O up to 1,000 mL (final concentration: 1 M).
To prepare 10 mM HEPES buffer, 1 M HEPES buffer is diluted 100-fold with dH2O.
Store the buffer at room temperature for up to one month.
PBS (1,000 mL)
Add the following substances to 800 mL of dH2O:
8 g of NaCl (final concentration: 137 mM)
0.2 g of KCl (final concentration: 2.68 mM)
1.15 g of Na2HPO4 (final concentration: 8.1 mM)
0.2 g of KH2PO4 (final concentration: 1.47 mM)
Add dH2O up to 1,000 mL.
Store PBS at room temperature for up to one month.
Delipidation/decoloration buffer (500 g)
Add the following substances to 440 g of PBS:
50 g of 1,2-Hexanediol [final concentration: 10% (w/w)]
10 g of 1-Methylimidazole [final concentration: 2% (w/w)]
Store the buffer at room temperature for up to one month.
Staining buffer (100 mL)
Mix the following substances and add dH2O up to 100 mL:
5 mL of 10% Triton X-100 in dH2O (final concentration: 0.5%)
0.1 mL of 10% sodium azide in dH2O (final concentration: 0.01%)
5 mL of 20× PBS (final concentration: 1×)
50 mL of 1% casein in PBS (final concentration: 0.5%; should be resolved to make 1% solution in PBS by warming at 60 °C)
Store the buffer at 4 °C for up to one month.
CUBIC-R (500 g)
Add the following substances to 125 mL dH2O:
225 g of 2,3-Dimethyl-1-phenyl-5-pyrazolone [final concentration: 45% (w/w)]
150 g Nicotinamide [final concentration: 30% (w/w)]
Store CUBIC-R at room temperature for up to one month with light shielding.
Observation oil (100 g; refractive index = 1.52, 25 °C)
Add the following substances to a 100 mL tube and shake gently:
60.2 g of silicone oil (HIVAC-F4) (Refractive index = 1.555, 25 °C)
39.8 g of mineral oil (Refractive index = 1.467, 25 °C)
Measure the refractive index using a refractometer and adjust by adding either oil to 1.52.
Store the oil at room temperature for up to one month.
Acknowledgments
We appreciate the excellent technical assistance and secretarial assistance provided by Ms. C. Nakayama, Ms. S. Takano, Ms. S. Fukumoto, Ms. N. Yamamoto, Ms. S. Morita, and Ms. M. Ohsawa. We also thank Dr. Peter Karagiannis for proofreading the text. This work was supported by KAKENHI (MM, HR, SH, SIK), Japan Science and Technology Agency (JPMXS0120330644 (Q-Leap)), Japan Agency for Medical Research and Development (AMED) (MM; JP21zf0127004 and JP223fa627005), and the Takeda Science Foundation (MM, SH). This study was also supported partly by the Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University, by the Photo-Excitonix Project, Hokkaido University, and by the Promotion Project for Young Investigators at Hokkaido University (MM). This protocol is based on our previous experimental procedures for EAE induction (Arima et al., 2012; Ogura et al., 2008; Tanaka et al., 2017), Au13 nanocluster preparation (Shichibu and Konishi, 2010), and CUBIC tissue-clearing protocol (Tainaka et al., 2014).
Competing interests
The authors declare no competing interests.
Ethics
All animal experiments described herein were approved by the Institutional Animal Care and Use Committees of the Institute for Genetic Medicine, Hokkaido University, and were performed according to institutional guidelines and regulations.
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Neuroscience > Nervous system disorders > Animal model
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Rapid and Reliable Quantification of Glycerol-3-phosphate Using Gas Chromatography–coupled Mass Spectrometry
HL Huazhen Liu
KY Keshun Yu
PK Pradeep Kachroo
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4645 Views: 616
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Original Research Article:
The authors used this protocol in Science Advances Jun 2022
Abstract
Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature.
Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography–coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.
Keywords: Glycerol-3-phosphate GC-MS Arabidopsis thaliana Plant defense N-Methyl-N-(trimethylsilyl)trifluoroacetamide
Background
Glycerol-3-phosphate (G3P) is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis (Lim et al., 2017). For glycerolipid biosynthesis, G3P is first acylated with the fatty acid (FA) oleic acid (18:1), to form lyso-phosphatidic acid (lyso-PA); this reaction is catalyzed by the chloroplastic enzyme G3P acyltransferase encoded by the ACT1 gene in Arabidopsis. Lyso-PA is converted to phosphatidic acid (PA) via lysophosphatidic acid acyltransferase–catalyzed transfer of 16:0 FA to the sn-2 position of lyso-PA. PA is in turn converted to phosphatidylglycerol and diacylglycerol (DAG). The DAG pools act as precursors for the synthesis of other major plastidal membrane lipids, including monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulpholipids. This pathway of glycerolipid synthesis operates in the chloroplast and is commonly referred to as the prokaryotic pathway.
G3P levels are regulated by enzymes involved in its biosynthesis, as well as those involved in G3P catabolism (Venugopal et al., 2009). G3P is synthesized via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or the G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP) (Mandal et al., 2011; Lim et al., 2017). DHAP is derived from glycolysis via triosephosphate isomerase activity on glyceraldehyde-3-phosphate. In Arabidopsis, the total G3P pool is derived from the activities of five G3Pdh isoforms and one GK isoform. The G3Pdh enzymes are present in cytosol, mitochondria, and chloroplast, and only a mutation in the plastid-localized G3Pdh isoforms, designated GLY1, impairs glycerolipid biosynthesis (Shen et al., 2003 and 2006). Unlike GLY1, neither the second chloroplastic nor the two cytosolic isoforms of G3Pdh contribute to plastidal and/or extraplastidal lipid biosynthesis (Chanda et al., 2011). This suggests that a specific sub-pool of G3P in the chloroplast is required for lipid biosynthesis.
The cellular pool of G3P also regulates the plastidal 18:1 level, which in turn governs nitric oxide levels and thereby chloroplast-nucleus retrograde signaling associated with defense. G3P levels are also important for basal defense against the hemibiotrophic fungus, Colletotrichum higginsianum (Chanda et al., 2008). Genetic mutations affecting G3P synthesis in Arabidopsis enhance susceptibility to C. higginsianum; conversely, plants accumulating increased G3P show enhanced resistance. G3P also contributes to R-mediated defense leading to systemic acquired resistance (SAR) in Arabidopsis, soybean, and wheat (Chanda et al., 2011Yang et al., 2013; Yu et al., 2013; Gao et al., 2014; Wang et al., 2014 and 2018; Shine et al., 2019). G3P also plays an important role in root-shoot-root signaling that regulates incompatible response against rhizobia in soybean (Shine et al., 2019). Compromised SAR in G3P-deficient mutants is restored by exogenous application of G3P. G3P is systemically mobile and is transported to the distal tissues via the plasmodesmata (PD) (Lim et al., 2016). This symplastic transport of G3P involves specific isoforms of PD-localizing proteins, which regulate PD gating. Thus, quantification of G3P levels in the plant tissues is an integral part of research on plant defense. Earlier methods to quantify G3P levels from plant tissues was based on high-performance liquid chromatography–based profiling of extracts prepared from ~0.5–1 g of tissue weight. The G3P peaks in chromatograms were assigned using electrochemical detection of analytes corresponding to the G3P standard (Chanda et al., 2008 and 2011). This method was unable to confirm the precise identity of G3P; moreover, it required excessive tissue weight for reliable extraction of G3P.
In the present method, we use gas chromatography (GC)-mass spectrometry (MS)–based selective ion monitoring (SIM) mode to quantify G3P from tissues or petiole exudates. Ribitol was used as an internal standard and both ribitol and G3P were derivatized using N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Figure 1). This method can reliably quantify G3P from ~10–100 mg of fresh tissue, is cost effective, and can be used to process 100–200 samples in a day.
Figure 1. Derivatization reactions for analyte G3P (upper panel) and the internal standard ribitol (lower panel) with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). MSTFA reacts with hydroxyl groups and the derivatized products of G3P and ribitol contain four or five trimethylsilyl groups (TMS), respectively.
Materials and Reagents
BDTM tuberculin syringe, 1 mL (Becton Dickinson, catalog number: 309659)
Kimwipes, 1-ply (Fisher, catalog number: 06-666)
Autoclavable microfuge tubes, 1.5 mL (Sarstedt, catalog number: 72.690.300)
Centrifuge tubes, 15 mL (Sarstedt, catalog number: 50.809.220)
Disposable test tubes, 13 × 100 mm (VWR, catalog number: 47729-572)
GC vials, 2 mL (Agilent, catalog number: 5190-9062)
Screw caps (Agilent, catalog number: 5182-0717)
Pasteur pipets (Fisher, catalog numbers: 13-678-20A and 13-678-20C)
Micro capillary pipets (Kimble, catalog number: 71900-20)
Arabidopsis thaliana (can be obtained from Arabidopsis biological resource center, www.arabidopsis.org)
Pseudomonas syringae expressing avrRpt2 (a gift from Dr. Barbara Kunkel, Washington University in St. Louis)
N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Soltec Bio Science, catalog number: GC113)
Liquid nitrogen (American Welding & Gas, 22 Psi)
Magnesium chloride hexahydrate (Fisher, catalog number: BP214-500)
Acetonitrile (ACROS, catalog number: 26827-0040)
Acetone (Thermo Scientific, Catalog number: A949-1)
Ribitol (Adonitol; Alfa Aesar, catalog number: L03253)
Glycerol-3-phosphate (Sigma, catalog number: G7886)
Monosodium phosphate (Alfa Aesar, catalog number: 33385)
Sodium orthovanadate (MP Biomedicals, catalog number: 159664)
Sodium pyrophosphate (Fisher Scientific, catalog number: BP330)
10 mM magnesium chloride (see Recipes)
Extraction buffer (see Recipes)
Internal standard (see Recipes)
Phosphatase inhibitors (see Recipes)
Equipment
Milli-Q Advantage A10 Water purification system (EMD Millipore, model: Z00Q0V0WW)
Lyophilizer, VirTis Freezemobile 25ES freeze dryer
Autoclave (Consolidated Stills & Sterilizers, model: SSR3APB)
Electric multi-cooker/streamer (Presto Big Kettle; Walmart.com, catalog number: 06008)
Scissors (Harbor Freight Tools, catalog number: 62458)
Aluminum foil (Thermo Scientific, catalog number: 01-213-102)
Explosion proof clips (M2 Sci, catalog number: C1005-T5-lock)
Balance (METTLER TOLEDO, model: AT261)
Benchtop Dewar flask (Thermo Scientific, catalog number: 10-194-100B)
Tissue grinder (Axygen Scientific, catalog number: PES15BSI)
Vortexer with a Styroform platform (Labnet, model: S0100)
Dewar flask (Thermo Scientific, catalog number: 10-194-100A/B)
Tabletop centrifuge (Eppendorf, model: 5417C), which is used for centrifuging microfuge tubes
Parker Source TriGas Generator (Parker Hannifin, model: LCMS-5000NA, compressor model: SF120872PH)
Table-top swing-arm centrifuge (Clay Adams, model: Dynac 0101), used for centrifuging disposable test tubes
Latex bulbs, 2 mL (VWR, catalog number: 82024-554)
Gas chromatograph-mass spectrometer (GC-MS) (Agilent, model: 7890A-5977)
GC column, HP-5MS (Agilent, catalog number: 19091J-413)
Ultra-high purity helium (AWG gases, Lexington, KY, catalog number: UN1046)
Computer for GC-MS (Dell, model: 3430)
Syringe for manual GC injection, 5 µL (Hamilton, catalog number: 87993)
Software
MassHunter Workstation for qualitative and quantitative analysis (Agilent, software version: 10.0)
Enhanced MassHunter Workstation for data acquisition (Agilent, software version: 10.0)
MS spectral library (NIST; Agilent Technologies)
Procedure
Equipment setup
GC setup
Chromatography: Perform chromatography with a 30 m capillary HP-5MS GC column, using ultra-high pure helium as the carrier gas set at a constant pressure of 1.2 mL/min. Set the injection port temperature to 240 °C and the transfer line temperature to 280 °C. Run samples in splitless mode. Use the temperature program shown in Table 1 for chromatography.
Table 1. GC temperature used for profiling ribitol and G3P
Rate Temperature Hold time
0 80 °C 1 min
15 °C/min 200 °C 1 min
10 °C/min 280 °C 2 min
MS setup
Auto-tune the instrument according to the manufacturer’s instructions.
Sample preparation and analysis
For pathogen assays, infiltrate the largest three leaves of ~4-week-old plants with either 10 mM MgCl2 or the pathogen Pseudomonas syringae avrRpt2 using a needleless syringe from the abaxial side of the leaves. After leaf infiltration, remove excess droplets of 10 mM MgCl2 or pathogen suspension with Kimwipes.
Approximately 16 h later, collect ~10–100 mg infiltrated leaf tissue. Sample the entire leaf using a pair of scissors.
Weigh the tissue using a balance that is accurate to the 1 mg digit. Record the weight of each sample. ∆CRITICAL STEP
Lyophilize the leaf tissue overnight in aluminum foil. Weigh and place approximately 1–10 mg of dry leaf tissue in an autoclaved microfuge tube.
Drop the microfuge tubes containing lyophilized tissue into a Dewar flask containing liquid nitrogen. Store frozen tissues at -80 °C until further analysis. •PAUSE POINT
Grind the tissue to a fine powder with a plastic pestle and immediately add 1,000 µL of extraction buffer before the tissue is thawed. ∆CRITICAL STEP
Add 2 µg of internal standard to the extract.
Cap the tubes with explosion proof clips and boil immediately in water for 5 min using an electric multi-cooker. ∆CRITICAL STEP
Centrifuge the tubes at 18,000 × g for 15 min.
Transfer the supernatant to a 13 × 100 mm glass test tube.
Dry the supernatant with a stream of nitrogen gas. Dried extract can be stored at -20 °C indefinitely. •PAUSE POINT
Add 250 µL of water to the dried extract and vortex the tube for 30 s.
Centrifuge the tubes at ~80% speed (approximately 1,250 × g) for 1 min in a tabletop swing-arm centrifuge.
Transfer the supernatant to a fresh microfuge tube followed by incubation at -80 °C for 30 min (this step is for sample cleanup). Thaw the samples at room temperature and then centrifuge at 18,000 × g for 6 min.
Transfer the supernatant to a GC vial and dry with a stream of nitrogen gas.
Wash the vial with 500 µL of acetone to remove residual water in the sample and then dry again with a stream of nitrogen gas.
Use microcapillary pipets to add 50–75 µL of acetonitrile to the dried samples, vortex the vials for 10 s, and add 50–75 µL of MSTFA containing 1% trimethylchlorosilane (TCMS). Incubate the samples at 65 °C for 1 h and transfer the contents to a GC insert with a Pasteur pipet.
Inject 0.5–1 µL into GC/MS for analysis using selective ion monitoring mode (SIM) using quantifier ions 299 (G3P) and 217 (ribitol) and qualifier ions 357 and 445 for G3P, and 147 and 307 for ribitol.
GC-MS data from standard compound analysis
Figure 2 shows the total ion chromatogram (top panel) and corresponding MS of ribitol (middle panel) and G3P (bottom panel) standards run in scan mode.
Figure 2. Total ion chromatogram and corresponding mass spectrometry of ribitol and glycerol-3-phosphate (G3P) standards run in scan mode. GC-MS chromatograph of equal weight (2 µg each in 100 µL) mixture of ribitol and G3P derivatized with MSTFA-TCMS as described in Procedure B using scan mode (Top panel) Ribitol-TMS (at 9.77 min) and G3P-TMS (at 10.07 min) are the two major peaks. (Middle panel) Mass spectra of ribitol-TMS derivative. (Bottom Panel) Mass spectra of G3P-TMS derivative.
Figure 3 shows the GC-MS data from standard compound analysis using SIM mode. Based on the mass spectra shown in Figure 2, three major ions were used to monitor G3P (299, 357, 445) and ribitol (217, 147, 307) in SIM mode.
Figure 3. Total ion chromatogram and corresponding MS of ribitol and G3P standards run in selective ion monitoring (SIM) mode. GC-MS chromatograph of equal weight (2 µg each in 100 µL) mixture of ribitol and G3P derivatized with MSTFA-TCMS as described in Procedure B using SIM mode (Top panel) Ribitol- (at 9.77 min) and G3P-TMS (at 10.07 min) derivatives are the two major peaks (indicated by 1/2). (Middle panel) Mass spectra of ribitol-TMS derivative in SIM mode selected for the major ions 217, 147, 307. (Bottom panel) Mass spectra of G3P-TMS derivative in SIM mode selected for the major ions 299, 357, 445. The dwell time for each ion was set at 25 ms. The relative abundance of major and qualifier ions monitored in SIM and scan modes was similar.
G3P levels in Arabidopsis leaf tissue
GC-MS data for G3P analysis from Arabidopsis thaliana leaf tissue samples using scan (Figure 4) and SIM (Figure 5) modes.
Figure 4. Total ion chromatographs of wild-type Columbia-0 leaf tissues (upper panel). Ribitol (4 µg) was used as an internal standard as described in Procedure B. The middle and bottom panels show MS spectra of ribitol- and G3P-TMS derivatives, respectively. The vertical line in the top panel corresponds to the region that shows G3P specific spectra.
Figure 5. Chromatographs of wild-type Columbia-0 leaf tissues. Ribitol (4 µg) was used as an internal standard as described in Procedure B. (Middle panel) Mass spectra of ribitol-TMS derivative in SIM mode selected for the major ions 217, 147 and 307. (Bottom panel) Mass spectra for G3P-TMS derivative in SIM mode selected for the major ions 299, 357, and 445.
Relative G3P levels in Col-0 plants after pathogen infection
Figure 6 shows the relative G3P levels in Col-0 plants inoculated with mock or pathogen. The pathogen-inoculated wild-type plants accumulated ~2–4-fold higher G3P levels compared to mock-inoculated plants.
Figure 6. Total ion chromatographs showing ribitol and G3P peaks in mock (10 mM MgCl2)- and pathogen (P. syringae expressing avrRpt2)-inoculated wild-type Columbia-0 leaf tissues at 16 h post inoculation (upper panel). The bottom panel shows quantified G3P levels in mock- and pathogen-inoculated plants. Plants were inoculated with 105 CFU mL-1 in 10 mM MgCl2. Error bars indicate standard deviation (n = 4), and asterisks indicate statistical significance (P > 0.001).
Data analysis
MassHunter Workstation 10.0 was used for qualitative and quantitative data analysis. The ion ratios between quantitative and qualifier ions were used as an additional measure to access proper identification of the analytes using SIM mode. G3P levels were calculated based on the standard curve generated using 500–8,000 ng concentrations of ribitol and G3P run in triplicates (Figure 7).
Figure 7. Standard curve showing response ratio of G3P vs. ribitol. Varying concentrations of G3P and ribitol (500, 750, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, and 8,000 ng) were analyzed in triplicates.
Statistical analysis was performed using Student’s t-test. For pathogen assays, ~16 plants/genotype/treatment were analyzed in a single experiment. For metabolite quantification, ~12 plants/genotype/treatment were analyzed in each experiment. Experiments were repeated at least two/three times with a different set of plants. Unless otherwise mentioned, error bars indicate standard deviation.
This method reliably quantified G3P levels in 10–100 mg fresh weight of Arabidopsis tissue and the GC-MS instrument was able to detect 0.1 ng of G3P. Arabidopsis tissues analyzed with and without ribitol showed ~100% recovery of the internal standard. However, Arabidopsis tissue samples spiked with G3P gave variable results, possibly because exogenous G3P can potentially react with other extracted chemicals or enzymes.
Notes
The protocol (Figure 8) shows excellent reproducibility and has been used to analyze extractions reported in our recent study (Shine et al., 2022).
Figure 8. Simplified flowchart showing steps involved in extraction and analysis of GC-MS-based analysis of G3P
Recipes
10 mM magnesium chloride
Dissolve 20.33 g of magnesium chloride hexahydrate in Milli-Q water to a final volume of 100 mL to obtain 1 M MgCl2. Autoclave 1 M MgCl2 along with 1 L of Milli-Q water at 121 °C for 20 min using a liquid cycle. After the solutions cool down to room temperature, add 1 mL of the 1 M MgCl2 to 99 mL of the autoclaved Milli-Q water, and mix well to obtain 10 mM MgCl2.
Extraction buffer
80% ethanol, containing 1× phosphatase inhibitor.
Internal standard
100 ng/µL ribitol (wt/vol) in Milli-Q water.
Suspend 10 mg of ribitol in 1 mL of autoclaved Milli-Q water and vortex to dissolve. Dilute this stock 100-fold by taking 10 µL of the solution to another vial containing 990 µL of autoclaved Milli-Q water.
Phosphatase inhibitors
Prepare stock solutions as follows:
250 mM monosodium phosphate (25 mM)
Suspend 299.95 mg of monosodium phosphate in a total volume of 10 mL autoclaved Milli-Q water and autoclave the solution at 121 °C for 20 min using a liquid cycle.
100 mM sodium orthovanadate (1 mM)
Suspend 183.9 mg of sodium orthovanadate in a total volume of 10 mL autoclaved Milli-Q water and autoclave the solution at 121 °C for 20 min using a liquid cycle.
100 mM sodium pyrophosphate (10 mM)
Suspend 265.9 mg of sodium pyrophosphate in a total volume of 10 mL autoclaved Milli-Q water and autoclave the solution at 121 °C for 20 min using a liquid cycle.
Prepare 1× phosphatase inhibitor solution from the stock solutions by taking 1 mL of 250 mM NaH2PO4, 100 µL of 100 mM sodium orthovanadate, and 1 mL of 100 mM sodium pyrophosphate to a 15 mL sterile tube. To this, add 7.9 mL of autoclaved Milli-Q water to make up 10 mL of 1× phosphatase inhibitor.
Acknowledgments
This work was supported by grants from National Science Foundation (IOS#051909, IOS#2131400). We thank John Johnson for technical help. The authors would like to acknowledge Duncan et al. (1971) whose work was adapted in the present protocol. This protocol is derived from the original research paper (Shine et al., 2022; DOI: 10.1126/sciadv.abm8791).
Competing interests
The authors declare no conflict of interest.
References
Chanda, B., Venugopal, S. C., Kulshrestha, S., Navarre, D. A., Downie, B., Vaillancourt, L., Kachroo, A. and Kachroo, P. (2008). Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis. Plant Physiol 147(4): 2017-2029.
Chanda, B., Xia, Y., Mandal, M. K., Yu, K., Sekine, K. T., Gao, Q. M., Selote, D., Hu, Y., Stromberg, A., Navarre, D., et al. (2011). Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants. Nat Genet 43(5): 421-427.
Duncan, J. H., Lennarz, W. J. and Fenselau, C. C. (1971). Mass spectral analysis glycerophospholipids. Biochemistry 10(6): 927-932.
Gao, Q. M., Yu, K., Xia, Y., Shine, M. B., Wang, C., Navarre, D., Kachroo, A. and Kachroo, P. (2014). Mono- and digalactosyldiacylglycerol lipids function nonredundantly to regulate systemic acquired resistance in plants. Cell Rep 9(5): 1681-1691.
Lim, G. H., Shine, M. B., de Lorenzo, L., Yu, K., Cui, W., Navarre, D., Hunt, A. G., Lee, J. Y., Kachroo, A. and Kachroo, P. (2016). Plasmodesmata Localizing Proteins Regulate Transport and Signaling during Systemic Acquired Immunity in Plants. Cell Host Microbe 19(4): 541-549.
Lim, G. H., Singhal, R., Kachroo, A. and Kachroo, P. (2017). Fatty Acid- and Lipid-Mediated Signaling in Plant Defense. Annu Rev Phytopathol 55: 505-536.
Mandal, M. K., Chanda, B., Xia, Y., Yu, K., Sekine, K. T., Gao, Q. M., Selote, D., Kachroo, A. and Kachroo, P. (2011). Glycerol-3-phosphate and systemic immunity. Plant Signal Behav 6(11): 1871-1874.
Shen, W., Wei, Y., Dauk, M., Tan, Y., Taylor, D. C., Selvaraj, G. and Zou, J. (2006). Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis. Plant Cell 18(2): 422-441.
Shen, W., Wei, Y., Dauk, M., Zheng, Z. and Zou, J. (2003). Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants. FEBS Lett 536(1-3): 92-96.
Shine, M. B., Gao, Q. M., Chowda-Reddy, R. V., Singh, A. K., Kachroo, P. and Kachroo, A. (2019). Glycerol-3-phosphate mediates rhizobia-induced systemic signaling in soybean. Nat Commun 10(1): 5303.
Shine, M. B., Zhang, K., Liu, H., Lim, G. H., Xia, F., Yu, K., Hunt, A. G., Kachroo, A. and Kachroo, P. (2022). Phased small RNA-mediated systemic signaling in plants. Sci Adv 8(25): eabm8791.
Venugopal, S. C., Chanda, B., Vaillancourt, L., Kachroo, A. and Kachroo, P. (2009). The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling. Plant Signal Behav 4(8): 746-749.
Wang, C., El-Shetehy, M., Shine, M. B., Yu, K., Navarre, D., Wendehenne, D., Kachroo, A. and Kachroo, P. (2014). Free radicals mediate systemic acquired resistance. Cell Rep 7(2): 348-355.
Wang, C., Liu, R., Lim, G. H., de Lorenzo, L., Yu, K., Zhang, K., Hunt, A. G., Kachroo, A. and Kachroo, P. (2018). Pipecolic acid confers systemic immunity by regulating free radicals. Sci Adv 4(5): eaar4509.
Yang, Y., Zhao, J., Liu, P., Xing, H., Li, C., Wei, G. and Kang, Z. (2013). Glycerol-3-phosphate metabolism in wheat contributes to systemic acquired resistance against Puccinia striiformis f. sp. tritici. PLoS One 8(11): e81756.
Yu, K., Soares, J.M., Mandal, M.K., Wang, C., Chanda, B., Gifford, A.N., Fowler, J.S., Navarre, D., Kachroo, A., and Kachroo, P. (2013). A feedback regulatory loop between G3P and lipid transfer proteins DIR1 and AZI1 mediates azelaic-acid-induced systemic immunity. Cell Rep 3(4): 1266-1278.
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Isolation of Immunocomplexes from Zebrafish Brain
JM Jennifer Carlisle Michel
AM Adam C. Miller
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4646 Views: 432
Reviewed by: Xi FengQin Tang Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Apr 2021
Abstract
Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.
Keywords: Immunoprecipitation (IP) Co-immunoprecipitation (co-IP) Protein complex purification Whole tissue Zebrafish Brain
Background
Immunoprecipitation (IP) and, under favorable conditions, co-immunoprecipitation (co-IP) can be used to isolate proteins and associated complexes in vivo (Harlow and Lane, 1999). Unlike column affinity chromatography that requires large amounts of starting material, the immunoprecipitation technique enriches target proteins and possibly their associated complexes from a small amount of material. The challenge of successfully isolating immunocomplexes from in vivo tissue is identifying an antibody that specifically and reproducibly binds the target protein, while also co-precipitating directly and indirectly interacting proteins. While polyclonal and monoclonal antibodies each have their own benefits, with the former recognizing multiple epitopes of a target and the latter recognizing a single epitope, it is not possible to predict success a priori. Therefore, it is crucial to test a variety of antibodies to find one that reproducibly immunoprecipitates and co-immunoprecipitates associated complexes. Additional steps critical to successful isolation of immunocomplexes include identifying a detergent that solubilizes the tissue for optimal release of the target protein and maintaining cold temperatures throughout the isolation to prevent protein degradation. Here, we present a protocol optimized to detect electrical synapse immunocomplexes from zebrafish brain (Lasseigne et al., 2021; see flow chart in Figure 1). The method is useful to identify protein–protein interactions in vivo and, though not validated for other tissues, could be adapted to other zebrafish organs and protein complexes, making this a helpful biochemical tool for the zebrafish community.
Figure 1. Flowchart for isolation of immunocomplexes from zebrafish brain
Materials and Reagents
1.5 mL plastic cuvettes (Fisher, catalog number: 14-955-127)
1.5 mL Eppendorf Snap-Cap Safe-Lock microcentrifuge tubes (Fisher, catalog number: 05-402-25)
0.65 mL graduated microtube (Fisher, catalog number: NC9260727)
Zebrafish brains (from fish ages 3 months–1.5 years) snap frozen in liquid nitrogen and stored at -80 °C
Antibody [This will vary with each experiment. We have successfully used anti-ZO1 monoclonal antibody (ZO1-1A12) (ThermoFisher, catalog number: 33-9100) for co-immunoprecipitation.]
n-Octyl-β-D-Glucopyranoside (Anatrace, catalog number: O311 5 GM)
Pierce protease inhibitor mini tablets, EDTA-free (Fisher, catalog number: PIA32955)
Dithiothreitol (DTT) reducing agent (Bio-Rad, catalog number: 610611)
HEPES (Fisher, catalog number BP310-500)
NaCl (Fisher, catalog number: BP358-212)
EDTA, disodium salt dihydrate (Fisher, catalog number: BP120-1)
EGTA (Sigma, catalog number: E-4378)
NaOH pellets (Fisher, catalog number: M1064621000)
Pierce Protein A/G agarose (Fisher, catalog number: PI20421)
4× Laemmli sample buffer (Bio-Rad, catalog number: 1610747)
Bio-Rad Protein Assay dye reagent concentrate (Bio-Rad, catalog number: 5000006)
1 M HEPES pH 7.5 (see Recipes)
5 M NaCl (see Recipes)
0.5 M EDTA pH 8.0 (see Recipes)
0.5 M EGTA pH 8.0 (see Recipes)
1 M DTT (see Recipes)
Homogenization buffer (see Recipes)
Solubilization buffer (see Recipes)
Preclear buffer (see Recipes)
IP wash buffer (see Recipes)
2× loading buffer (see Recipes)
Note: See Recipes to create working solutions.
Equipment
1 mL dounce tissue grinder with loose and tight pestles (Fisher, catalog number: 50-365-300 or similar)
Benchtop tube rocker (Benchmark Scientific M2100 or similar)
Refrigerated centrifuge (Eppendorf 5430 R or similar)
Spectrophotometer (Eppendorf BioPhotometer or similar)
Procedure
Tissue preparation—Freeze and store brains
Note: Adult fish should be euthanized according to proper guidelines for the ethical care and use of animals in research associated with IACUC protocols of the user’s institution.
Euthanize an adult fish and dissect the brain out as described in Gupta and Mullins (2010; see detailed video at: https://www.jove.com/t/1717).
Transfer each dissected brain to an Eppendorf tube, immediately close the cap tightly, and snap freeze tissue by immersing the tube in liquid nitrogen for at least one minute.
Note: Liquid nitrogen handling should follow appropriate lab safety guidelines.
When all brains are dissected, transfer snap frozen brains to -80 °C freezer until use.
Note: Individual brains are stored separately and combined below dependent upon experimental needs. Frozen brains can be stored at -80 °C for at least two years.
DAY ONE—Homogenize brains
Note: The following protocol describes conditions for two IPs. It is important to maintain all reagents on ice throughout the procedure to prevent protein degradation.
Freshly prepare 5 mL of homogenization buffer by adding one protease inhibitor mini tablet and 5 µL of 1 M DTT; keep on ice.
Note: Homogenization buffer is prepared in bulk and is sufficient for 50 brains. Use within 60 min of preparation to ensure optimal protease inhibitor activity and DTT stability during tissue homogenization.
Freshly prepare 5 mL of solubilization buffer by adding 5 µL of 1 M DTT; keep on ice.
Note: Solubilization buffer is prepared in bulk and is sufficient for 50 brains. Use within 60 min of preparation to ensure DTT stability during tissue solubilization.
Put five frozen zebrafish brains into an ice-cold 1 mL dounce tissue grinder.
Note: The resulting quantity of total protein from each brain will vary depending on the age and size of the adult fish dissected. As a general guideline, using two brains per IP often results in 1–2 mg total protein/IP, though the total quantity required will vary according to protein expression levels and antibody efficiency.
Add 0.5 mL of prepared ice-cold homogenization buffer.
Note: Save unused homogenization buffer at 4 °C for use on DAY TWO, step C10 to mix the blank solution needed for the protein assay.
Homogenize brain tissue with 10–20 strokes using the loose pestle and then 10–20 strokes using the tight pestle.
Note: Homogenizing in the absence of detergent prevents oxidation of proteins. Also, if needed, glass homogenizers and pestles can be decontaminated between samples by rinsing extensively with distilled water, then 70% ethanol, and then distilled water again before drying with air.
Transfer the homogenate (0.5 mL) to a 1.5 mL Eppendorf tube using a 1,000 µL pipette tip.
Add 0.5 mL of prepared solubilization buffer and gently invert to thoroughly mix the solutions.
Note: The prepared solubilization buffer contains 4% detergent, so that when mixed with an equal volume of brain homogenate, the final detergent concentration is 2%. Save unused solubilization buffer at 4 °C for use on DAY TWO, step C10 to mix the blank solution needed for the protein assay.
Solubilize the brain tissue by rocking the tube on a benchtop rocker at 24 rpm, overnight at 4 °C.
DAY TWO—Preclear and set up immunoprecipitations
Prepare pre-washed Pierce Protein A/G agarose by washing a 250 µL bed volume with 1 mL of preclear buffer.
Note: Pre-washed Pierce Protein A/G agarose is prepared in bulk and is sufficient for three preparations.
Spin at 1,000 × g for 3 min at 4 °C.
Remove buffer and repeat wash steps C1 and C2 two more times using new preclear buffer each time.
After the final wash, remove buffer and resuspend agarose in 250 µL of preclear buffer to achieve a 500 µL 50% slurry of pre-washed Pierce Protein A/G agarose. Store pre-washed agarose at 4 °C until use in step C7 below and in DAY THREE, step D2.
Next, centrifuge the overnight solubilized brain homogenate at 20,000 × g for 30 min at 4 °C.
Without disturbing the pellet, transfer the solubilized brain supernatant to a new 1.5 mL Eppendorf tube using a 1,000 µL pipette tip.
Preclear the brain supernatant by adding 100 µL of 50% slurry of pre-washed Pierce Protein A/G agarose (prepared in steps C1–C4), and rock on benchtop rocker at 24 rpm for 1 h at 4 °C.
Spin the supernatant at 1,000 × g for 3 min at 4 °C to pellet the agarose.
Without disturbing the agarose, carefully transfer the precleared supernatant to a new 1.5 mL Eppendorf tube using a 1,000 µL pipette tip.
Measure the relative quantity of total protein using Bio-Rad protein assay according to the manufacturer’s instructions (see detailed protocol at https://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf; 1.5 mL plastic cuvettes and a spectrophotometer are required).
Note: For the blank solution, mix equal quantities of homogenization buffer and solubilization buffer that were saved at 4 °C from DAY ONE, steps B4 and B7.
Save an extract sample by transferring 40 µL of precleared brain supernatant to a new 0.65 mL microtube and adding 40 µL of 2× loading buffer. Save at -20 °C until needed for SDS-PAGE electrophoresis on DAY THREE, step D12.
To set up IPs, transfer 400 µL of precleared brain supernatant to each of two new 1.5 mL Eppendorf tubes (this is approximately 1–2 mg total protein/IP).
Note: If comparing IPs from different samples, be sure to immunoprecipitate an equal quantity of total protein in an equal volume. The minimum volume for an IP is 200 µL. Also, use the precleared extract for immunoprecipitation immediately. Do not freeze the precleared supernatant or the protein complexes will fall apart, and co-IP of associated proteins will fail.
Add 2 µg of antibody per IP.
Note: Anywhere from 0.5–5 µg can be used, depending on the efficiency of the antibody.
Rock the IPs on a benchtop rocker at 24 rpm, overnight at 4 °C.
DAY THREE—Capture immunoprecipitates
Centrifuge the IPs at 1,000 × g for 1 min at 4 °C.
Add 30 µL of 50% slurry of pre-washed Pierce Protein A/G agarose (prepared in DAY TWO steps C1–C4 and stored at 4 °C) to each IP.
Rock IPs on a benchtop rocker at 24 rpm for 1 h at 4 °C.
Centrifuge IPs at 1,000 × g for 3 min at 4 °C.
Remove supernatant with a 1,000 µL pipette tip without disturbing the agarose.
Add 1 mL of IP wash buffer to the agarose and invert several times.
Spin 1,000 × g for 3 min at 4 °C.
Repeat wash steps D5–D7 two more times, using new IP wash buffer each time.
On the last wash, completely remove the wash buffer from the agarose using a 200 µL pipette tip, followed by a flattened 10 µL gel loading tip.
Add 20 µL of 2× loading buffer.
Boil extract sample and immunoprecipitations for 3 min at 95 °C.
Note: Boiling may cause aggregation of membrane proteins. In this case, alternative options include omitting step D11 or heating the samples at 65 °C for 10 min instead. Best results need to be empirically determined per experiment.
Continue with protein analysis as desired, e.g., SDS-PAGE and western blot (Towbin et al., 1979; Sambrook and Russell, 2006; Ni et al., 2017).
Note: The subsequent protein analysis steps are dependent upon the equipment available. For a comprehensive guide, see https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf.
Recipes
1 M HEPES, pH 7.5 (500 mL)
Dissolve 119.15 g of HEPES (MW 238.5 g/mol) in 400 mL of ultrapure water. Adjust pH to 7.5 with 10 N NaOH. Adjust final volume to 500 mL with ultrapure water.
5 M NaCl (500 mL)
Dissolve 146 g of NaCl (MW 58.44) in 400 mL of ultrapure water. Adjust final volume to 500 mL with ultrapure water.
0.5 M EDTA, pH 8.0 (100 mL)
Dissolve 18.61 g of EDTA, disodium salt dihydrate (MW 372.24 g/mol) in 80 mL of ultrapure water. Adjust the pH to 8.0 with NaOH pellets. Adjust the final volume to 100 mL with ultrapure water.
0.5 M EGTA, pH 8.0 (100 mL)
Dissolve 19.02 g of EGTA (MW 380.35 g/mol) in 80 mL of ultrapure water. Adjust the pH to 8.0 with 4 N NaOH. Adjust final volume to 100 mL with ultrapure water.
1 M DTT (10 mL)
Dissolve 1.5 g of DTT in 8 mL of ultrapure water. Adjust volume to 10 mL, dispense into 1 mL aliquots, and store at -20 °C.
Homogenization buffer (50 mL)
20 mM HEPES pH 7.5 1.0 mL 1 M HEPES pH 7.5
150 mM NaCl 1.5 mL 5 M NaCl
5 mM EDTA 0.5 mL 0.5 M EDTA
5 mM EGTA 0.5 mL 0.5 M EGTA
46.5 mL ultrapure water
Solubilization buffer (25 mL)
20 mM HEPES pH 7.5 0.5 mL 1 M HEPES pH 7.5
150 mM NaCl 0.75 mL 5 M NaCl
5 mM EDTA 0.25 mL 0.5 M EDTA
5 mM EGTA 0.25 mL 0.5 M EGTA
4% n-Octyl-β-D-Glucopyranoside 1 g n-Octyl-β-D-Glucopyranoside
Adjust final volume to 25 mL with ultrapure water
Preclear buffer (10 mL)
20 mM HEPES pH 7.5 5 mL homogenization buffer
150 mM NaCl 5 mL solubilization buffer
5 mM EDTA
5 mM EGTA
2% n-Octyl-β-D-Glucopyranoside
IP wash buffer (25 mL)
20 mM HEPES pH 7.5 23.75 mL homogenization buffer
150 mM NaCl 1.25 mL solubilization buffer
5 mM EDTA
5 mM EGTA
0.2% n-Octyl-β-D-Glucopyranoside
2× loading buffer (1 mL)
2× Laemmli sample buffer 0.5 mL 4× Laemmli sample buffer
200 mM DTT 0.2 mL 1 M DTT
0.3 mL ultrapure water
Acknowledgments
This protocol was used in the companion paper Lasseigne et al. (2021) Elife (DOI: 10.7554/eLife.66989). National Institute of Mental Health (RF1MH120016) to Alberto Pereda and Adam C. Miller. National Institute of Neurological Disorders and Stroke (R01NS105758) to Adam C. Miller.
Competing interests
We declare no competing interests.
Ethics
Studies using this protocol were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#AUP 21-42) of the University of Oregon.
References
Gupta, T. and Mullins, M. C. (2010). Dissection of organs from the adult zebrafish. J Vis Exp(37): 1717.
Harlow, E. and Lane, D. (1999). Using antibodies: a laboratory manual. N.Y.: Cold Spring Harbor Laboratory Press.
Lasseigne, A. M., Echeverry, F. A., Ijaz, S., Michel, J. C., Martin, E. A., Marsh, A. J., Trujillo, E., Marsden, K. C., Pereda, A. E. and Miller, A. C. (2021). Electrical synaptic transmission requires a postsynaptic scaffolding protein. Elife 10: e66898.
Ni, D., Xu, P. and Gallagher, S. (2017). Immunoblotting and Immunodetection. Curr Protoc Protein Sci 88: 10.10.1-10.10.37.
Sambrook, J. and Russell, D. W. (2006). SDS-Polyacrylamide Gel Electrophoresis of Proteins. CSH Protoc 2006(4): pdb.prot4540.
Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci U S A 76: 4350-4354.
Article Information
Copyright
Michel and Miller. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite
Category
Neuroscience > Cellular mechanisms > Protein isolation
Biochemistry > Protein > Isolation and purification
Molecular Biology > Protein > Protein-protein interaction
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4,647 | https://bio-protocol.org/en/bpdetail?id=4647&type=0 | # Bio-Protocol Content
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Preparation and Characterization of IL-22 mRNA-Loaded Lipid Nanoparticles
ZA Zahra Alghoul *
JS Junsik Sung *
KW Kenji Wu
GA Gianfranco Alpini
SG Shannon Glaser
CY Chunhua Yang
DM Didier Merlin
(*contributed equally to this work)
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4647 Views: 756
Reviewed by: Oneil Girish BhalalaSrajan Kapoor Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Biomaterials Sep 2022
Abstract
Interleukin-22 (IL-22) has been demonstrated as a critical regulator of epithelial homeostasis and repair; it showed an anti-inflammatory effect against ulcerative colitis. Local microinjection of IL-22 cDNA vector has been shown to be effective in treating ulcerative colitis in mouse models. However, microinjection comes with multiple technical challenges for routine colon-targeted drug delivery. In contrast, oral administration can get around these challenges and provide comparable efficacy. We showed in previous studies that oral administration of new lipid nanoparticles (nLNP)-encapsulated IL-22 mRNA targets the colon region and efficiently ameliorates colitis. This protocol describes the details of preparing and characterizing the nLNP-encapsulated IL-22 mRNA using three major lipids that mimic the natural ginger-derived nanoparticles. It provides an nLNP platform that can be used to orally deliver other types of nucleic acids to the colon.
Keywords: Ulcerative colitis Drug delivery Lipid nanoparticles Interleukin-22 Intestinal inflammation
Background
Interleukin-22 (IL-22) is an anti-inflammatory cytokine that plays a critical role in promoting wound healing during intestinal inflammation. Its expression is decreased in inflamed ulcerative colitis tissue (De Souza and Fiocchi, 2016). Microinjection of IL-22 cDNA into the colon of colitic mice showed its potential in curing ulcerative colitis. Targeted delivery of IL-22 cDNA ameliorated local intestinal inflammation through induction of mucus production, enhancing STAT3 activation in colonic epithelial cells and promoting restitution of goblet cells. Additionally, inhibition of IL-22 activity suppressed goblet cell restitution during the recovery phase of the mouse model (Sugimoto et al., 2008).
Despite its effectiveness, microinjection is an invasive technique that requires trained personnel and specific medical equipment. Contrarily, oral delivery is noninvasive and more convenient for patient self-administration. Due to their excellent biocompatibility and drug-loading capability, nanostructured lipid carriers, such as solid lipid nanoparticles and liposomes, have become an attractive oral drug delivery platform that is starting to replace conventional drug delivery systems (Ahadian et al., 2020). However, challenges exist when the delivery system passes through the gastrointestinal tract, including high enzymatic activity and variation in pH and absorption efficiency across the intestinal epithelium.
We previously used ginger-derived lipid nanoparticles (GDNPs) for orally delivering CD98 siRNA (Zhang et al., 2017) and 6-shogaol (Yang et al., 2021) to treat ulcerative colitis. These studies suggested that GDNPs could overcome the challenges of oral drug delivery. Further, we found that monogalactosyl-diacylglycerol (MGDG), digalactosyl-diacylglycerol (DGDG), and phosphatidic acid (PA) constitute more than 90% of the total lipids of GDNPs (Zhang et al., 2016; Zhang et al., 2017). In this protocol, we will describe how to use these three lipids at the same ratios found in the GDNPs in the synthesis of new lipid nanoparticles (nLNPs) to encapsulate IL-22 mRNA. The nLNP-encapsulated mRNA showed its therapeutic efficacy in one of our recently published studies (Sung et al., 2022).
Materials and Reagents
50 mL single-neck recovery flask (Ace Glass, catalog number: 945808)
Micropipettes, 10–100 µL (Eppendorf, catalog number: 13-684-251)
Pipette tips, 1–300 µL (Fisher Scientific, SureOneTM micropoint pipette tips, catalog number: 02-707-410)
Serological pipettes, 5 mL (VWR, catalog number: 89130-886)
Disposable cuvettes (GMBH + Co KG, catalog number: 759075D)
Disposable folded capillary cell (Malvern, Zetasizer Nano Series, catalog number: DTS1070)
20% glucose solution (Gibco, catalog number: A2494001), stored at 4 °C
DEPC water (Invitrogen, catalog number: AM9916), stored at 4 °C
TurboFectTM transfection reagent (Thermo ScientificTM, catalog number: R0531), stored at 4 °C
Monogalactosyl-diacylglycerol (MGDG) (Avanti Polar Lipids, catalog number: 840523), stored at -20 °C
Digalactosyl-diacylglycerol (DGDG) (Avanti Polar Lipids, catalog number: 840524), stored at -20 °C
L-α-phosphatidic acid (PA) (Avanti Polar Lipids, catalog number: 840074), stored at -20 °C
IL-22 pMRNAXP vector (SBI, sequence XM_006513865.4), stored at -80 °C
mRNA synthesis kit (SBI, catalog number: MR-KIT-1), stored at -80 °C
200 proof ethanol (EtOH) (Decon Labs, catalog number: 2401); mix 350 mL of 100% EtOH with 150 mL autoclaved water for a solution of 70% EtOH
Dichloromethane (DCM) (Sigma, catalog number: 40042)
Equipment
Zetasizer Nano-ZS90 (Malvern Instruments Ltd., ZEN3690, serial number: MAL1181172)
Ultrasonicate cleaner (Branson Ultrasonics Corporation, Bransonic®, model: 3510R-MTH)
Rotary evaporator (BUCHI, model: R-210)
Vacuum pump (BUCHI, model: V-700)
Software
Zetasize software 7.12 Copyright© 2002-2016 (Malvern Instruments Ltd.)
Procedure
Equipment setup
Set the ultrasonic bath temperature to 50 °C prior to the start of the experiment.
Set the rotatory evaporator water bath temperature to 50 °C prior to the start of the experiment.
Clean all glassware with 70% EtOH followed by 100% EtOH.
Sanitize the vacuum hood with UV light for 1 h.
Preparation of lipid solutions
Add 2.5 mL of 100% EtOH to 5.0 mg of MGDG to make a 2.0 mg/mL MGDG solution.
Add 2.5 mL of 100% EtOH to 5.0 mg of DGDG to make a 2.0 mg/mL DGDG solution.
Add 5.0 mL of 100% dichloromethane (DCM) to 25.0 mg of PA to make a 5.0 mg/mL PA solution.
Keep the above solutions stored at -20 °C until use.
Synthesis of IL-22/nLNPs
Mix 99 μL of MGDG, 66 μL of DGDG, and 66 μL of PA solutions in a 50 mL single-neck recovery flask.
Add 2.5 mL of 100 % EtOH to the lipid mixture.
Swirl the flask to mix the lipids and the EtOH mixture.
Attach the flask to the rotary evaporator and rotate for 3–5 min at a 50 torr pressure (Figure 1).
Figure 1. Solvent evaporation by rotary evaporator
Release the vacuum when it reaches 50 torr pressure and transfer the flask to a UV-sanitized vacuum hood.
In a centrifuge tube, prepare a 5% glucose solution by mixing 1 mL of 20% glucose solution with 3 mL of RNase-free DEPC water.
Add 50 μL of in vitro transcription synthesized IL-22 mRNA (generated by using mRNA synthesis kit with custom designed IL-22 pMRNAXP vector as a template, following manufacturer’s instruction) and 50 μL TurboFectTM transfection reagent to the 500 μL of 5% glucose solution and mix. The total volume will be 600 μL.
Incubate the mixture for 15 min at room temperature.
Transfer the solution to the single-neck recovery flask containing the lipid mixture.
Place the bottom of the recovery flask in the water contained in the ultrasonic bath and pipette up and down (approximately 100 times) until the mixture becomes cloudy (Figure 2).
Store the prepared nLNPs at 4 °C.
Figure 2. Sonication of IL-22 mRNA and lipid mixture
Characterization of the nLNPs
Measure particle size
Place approximately 1.5 mL of nLNPs suspension in a cuvette and cover it with a plastic cap.
Place the cuvette in the cuvette holder of Malvern Zetasizer.
Set the software function to measure the size.
Measure zeta potential
Place approximately 1.0 mL of the nLNPs suspension in a capillary cell.
Place the cell in the cuvette holder of the Malvern Zetasizer.
Set the software function to measure the zeta potential.
Data analysis
Dynamic light scattering analysis showed that the average sizes for blank nLNPs (Figure 3) and IL-22/nLNPs (Figure 5) are 189.9 ± 69.3 and 184.2 ± 84.96 nm, respectively, based on triplicate measurement.
The zeta potential of blank nLNPs (Figure 4) and IL-22/nLNPs (Figure 6) are -5.51 ± 3.26 and -17.4 ± 7.63 mV, respectively, based on triplicate measurement.
Figure 3. Measurement of size in blank nLNPs. Particle size was determined to be 189.9 ± 69.3 nm in diameter.
Figure 4. Measurement of zeta potential in blank nLNPs. Surface zeta potential was measured as -5.51 ± 3.26 mV.
Figure 5. Measurement of size in IL-22/nLNPs. Particle size of IL-22/nLNPs was 184.2 ± 84.96 nm in diameter.
Figure 6. Measurement of zeta potential in IL-22/nLNPs. Surface zeta potential of IL-22/nLNPs was -17.4 ± 7.63.
Acknowledgments
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant numbers: RO1-DK-107739 (DM), RO1-DK-116306 (DM), and R01DK132891 (GA and SG). Department of Veterans Affairs (BX002526, DM). Dr. Merlin and Dr. Alpini are recipients of a Senior Research Career Scientist Award from the Department of Veterans Affairs.
Competing interests
The authors declare no conflicts of interest within the work.
References
Ahadian, S., Finbloom, J. A., Mofidfar, M., Diltemiz, S. E., Nasrollahi, F., Davoodi, E., Hosseini, V., Mylonaki, I., Sangabathuni, S., Montazerian, H., et al. (2020). Micro and nanoscale technologies in oral drug delivery. Adv Drug Deliv Rev 157: 37-62.
De Souza, H. S. P. and Fiocchi, C. (2016). Immunopathogenesis of IBD: current state of the art. Nat Rev Gastroenterol Hepatol 13(1): 13-27.
Sugimoto, K., Ogawa, A., Mizoguchi, E., Shimomura, Y., Andoh, A., Bhan, A. K., Blumberg, R. S., Xavier, R. J. and Mizoguchi, A. (2008). IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin Invest 118(2): 534-544.
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.
Yang, C., Long, D., Sung, J., Alghoul, Z. and Merlin, D. (2021). Orally Administered Natural Lipid Nanoparticle-Loaded 6-Shogaol Shapes the Anti-Inflammatory Microbiota and Metabolome. Pharmaceutics 13(9): 1355.
Zhang, M., Viennois, E., Prasad, M., Zhang, Y., Wang, L., Zhang, Z., Han, M. K., Xiao, B., Xu, C., Srinivasan, S., et al. (2016). Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer. Biomaterials 101: 321-340.
Zhang, M., Wang, X., Han, M. K., Collins, J. F. and Merlin, D. (2017). Oral administration of ginger-derived nanolipids loaded with siRNA as a novel approach for efficient siRNA drug delivery to treat ulcerative colitis. Nanomedicine 12(16): 1927-1943.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Biological Engineering > Biomedical engineering
Molecular Biology > Nanoparticle
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4,648 | https://bio-protocol.org/en/bpdetail?id=4648&type=0 | # Bio-Protocol Content
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Analysis of Mouse Brain Sections by Live-cell Time-lapse Confocal Microscopy
TY Tao Yang
TH Ty Hergenreder
BY Bing Ye
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4648 Views: 738
Reviewed by: Miao He Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Jun 2022
Abstract
The developing cerebral cortex of mammals is generated from nascent pyramidal neurons, which radially migrate from their birthplace in the ventral part of the neural tube to the cortical surface. Subtle aberrations in this process may cause significant changes in cortical structure and lead to developmental neurological disorders. During pyramidal neuron migration, we recently showed that the migrating neuron, which bypasses its last preceding neuron, is critical for its proper positioning and contributes to cerebral cortex thickness. Studying this process requires an imaging system with single-cell resolution and a prolonged observation window. Therefore, we built a system to maintain an organotypic brain slice on the stage of a Leica SP5 confocal microscope, which facilitated high-resolution imaging over a 12-hour time-lapse observation period of cellular events during neuron migration. Here, we share our protocol along with guidelines for overcoming difficulties during the setup. This protocol facilitates the observation of, but is not limited to, neurodevelopmental and pathological processes occurring during neuron migration.
Keywords: Organotypic Brain slice Brain section Live cell Imaging Time-lapse
Background
Cortical development is a highly dynamic and precisely orchestrated process (Rakic and Caviness, 1995; Kolk and Rakic, 2022). During development, neural progenitor cells from the dorsal telencephalon proliferate to sequentially generate neurons in the deeper cortical layers, neurons in the upper cortical layers, and then glial cells (Gao et al., 2014). Nascent pyramidal neurons migrate radially to the external border of the developing cortex and generate new cortical layers in an inside-out fashion. How the migrating neurons stop their radial migration and consequently locate at their proper positions in the cerebral cortex is poorly understood (Rakic, 1974; Gongidi et al., 2004; Ohtaka-Maruyama and Okado, 2015). Ex vivo time-lapse imaging can directly reveal the cellular processes occurring during cortical development (Yang et al., 2012). Compared with radial migration and proliferation, which continuously occur during neural development and only require 2–3 h of imaging, terminating radial migration takes much longer. Monitoring termination requires prolonged (≥12 h) time-lapse imaging of neuronal migration at single-cell resolution in well-maintained developing brain slices. However, the stage of a standard microscope is not specifically designed to maintain a living organotypic slice, rendering it less feasible to monitor a highly active live brain slice over a long period of time. Previously published methods of time-lapse imaging can extend several hours (Tsai et al., 2007), which is not sufficient to observe the whole process of radial migration termination.
In our recent paper, Migrating pyramidal neurons require DSCAM to bypass the border of the developing cortical plate, we described the termination process of neuron radial migration and reported a molecular mechanism that underlies this process (Yang et al., 2022). We found that the nascent pyramidal neuron bypassing the last preceding neuron is important for expanding the cortex and thus determines the thickness of the upper cortical layers. Down syndrome cell adhesion molecule (DSCAM) was involved in this process by reducing the strength of N-cadherin-mediated cell-to-cell adhesion in the upper cortical plate, which allowed migrating neurons to traverse the cortical plate border and terminate at the outermost position of the developing cortical plate. Since DSCAM aberrations have been associated with autism spectrum disorder (Wang et al., 2016; Turner et al., 2016; Narita et al., 2020) and Down syndrome (Yamakawa et al., 1998; Agarwala et al., 2000), DSCAM may contribute to these brain disorders by impairing normal cortical development.
In our study, we applied live cell time-lapse imaging on organotypic brain-slice cultures to observe the termination of radial migration in detail (Yang et al., 2022). Organotypic brain slice cultures preserve brain development within an ex vivo environment. The confocal microscope is necessary for imaging fluorescently labeled neurons at single-cell resolution in thick brain slices. Since the brain slice is 300 µm in thickness, 50–100 z-frames of confocal scanning are needed to cover 100–150 µm of tissue in depth to collect z-axis information. Confocal microscopes equipped with high-speed scanners, such as the resonance scanner of Leica SP5 confocal systems, can overcome the speed limit of regular confocal microscopes and minimize laser damage. Together, this system generates single-cell resolution images for long-term (≥12 h) observations.
As we realized that our approach could be used as a standard method for studying cortical development and be extended to other brain regions or tissues, we decided to report our protocol in detail here, so that other researchers can reference, modify, and optimize their own imaging system. For the sake of completeness, we include brief protocols for neuronal labeling and brain slice sectioning in the “animal preparation” section, although these techniques have been previously described (Yang et al., 2022).
Materials and Reagents
15 mL Falcon tube for melting the agarose II (Corning, Falcon, catalog number: 14-959-53A)
3.5 × 1.0 cm Petri dish for mounting the fresh brain (Thermo Fisher Scientific, Nunc Petri Dish, catalog number: 150318)
Extra thick filter paper, precut (Bio-Rad, catalog number: 1703967)
10 cm Petri dish for carrying the new brain dissection (Thermo Fisher Scientific, Nunc Petri Dish, catalog number: 263991)
1/16’’, 3/16’’ inner diameter (i.d.) tubing to connect medium syringe to the sample chamber in the heater (ValveBank 4 II)
5/16’’ i.d. silicone tubing for bubbling
4 mm with medium-grit sandpaper (3M, catalog number: 9002NA-20-CC)
60 mL syringe
Transgenic mouse (Ai14, JAX, catalog number: 007914)
Agarose II (low melting point) (Amresco, catalog number: 17856)
2 L flask for carrying 1× artificial cerebral spinal fluid (ACSF)
Gas cylinder of 95% O2/5% CO2 gas
D-(+)-Glucose monohydrate (Millipore, catalog number: 49159)
NaCl (Fisher, catalog number: BP358-10)
KCl (Fisher, catalog number: BP366-1)
MgCl2·6H2O (Fisher, catalog number: BP214-500)
CaCl2·2H2O (Sigma, catalog number: 223506)
NaH2PO4·H2O (Sigma, catalog number: S9638)
NaHCO3 (Sigma, catalog number: S6014)
Deionized water (Millipore Milli-Q® Integral 5 Water Purification System, catalog number: ZRXQ005US)
Dulbecco’s modified Eagle medium (DMEM), high glucose (Sigma, catalog number: D0822)
Matrigel membrane matrix (Corning, Matrigel, catalog number: 356234)
10× ACSF solution I (see Recipes)
10× ACSF solution II (see Recipes)
1× ACSF (see Recipes)
Equipment
Electroporation (Harvard Apparatus, ECM 830)
Vibratome (Leica VT 1000)
ValveBank 4 II perfusion system (or similar systems)
Tissue culture incubator (37 °C, 5% CO2)
High pressure gas cylinder of 95% O2/5% CO2
Homemade sample dish modified from a 3.5 × 1.0 cm Petri dish. The height of the wall of the dish was reduced from 10 to 4 mm by grinding on a medium-grit sandpaper (e.g., 100 grit). The bottom of the dish was thinned by grinding on a fine-grit sandpaper (e.g., 600 grit).
Stage sample dish heater (Bioscience Tools, TC-E35)
Temperature controller (Bioscience Tools, TC-1-100)
Temperature fiber, miniature 0.87 mm (Bioscience Tools, TC-TP)
Silicon lid for the sample dish (Bioscience Tools, CSC-10P)
Vacuum oil pump (Fisher, VLP-200-115)
Confocal microscope (Leica SP5)
Software
ImageJ (NIH, https://imagej.nih.gov/ij/)
LAS X (Leica Microsystems, https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/)
Procedure
Animal preparation
The migrating pyramidal neurons can be labeled by in utero electroporation (IUE) or induced by tamoxifen injection, which activates nestin-CreERt2 and tdTomato expression in transgenic mouse. IUE can be performed at 14.5 embryonic (E) days to label the upper cortical pyramidal neurons. The IUE electroporation protocol has been published previously (Yang et al., 2022), and is briefly described below:
A timed pregnant mouse is anesthetized.
An incision is made on the belly cavity to expose the uterus.
1–2 µL of DNA solution (plasmids for RFP or GFP expression) is injected into the lateral ventricle of the embryonic head, which can be seen easily across the transparent uterus wall.
Five electric pulses (35–50 V) are delivered across the pup brain, whose neural progenitor cells along the lateral ventricle take up the DNA.
The offspring of these progenitors have fluorescently labeled pyramidal neurons.
Alternatively, the upper cortical pyramidal neurons can also be labeled in an inducible transgenic mouse. At E14.5, intraperitoneal tamoxifen injection into the pregnant mouse induces Cre expression in nestin-CreERt2;Ai14 embryos, which, in turn, induces tdTomato expression in neural progenitor cells and their progeny.
Time-lapse imaging
Embedding and sectioning the brain (Video 1)
Before dissecting the embryonic brain out, prepare 1 L of fresh 1× ACSF from 100 mL of 10× ACSF solution I, 100 mL of 10× ACSF solution II, and 800 mL of deionized water. Some precipitation will occur along with a decrease in transparency. Bubble 95% O2/5% CO2 gas into 1× ACSF on ice until the solution becomes transparent. Before use, add 5–10 g of D-(+)-Glucose monohydrate (glucose) to the 1 L of freshly prepared 1× ACSF and agitate to dissolve.
Add 0.4–0.5 g of agarose II to 12 mL of 1× ACSF and microwave the suspension 4–5 times for 5 s, to melt the agarose without boiling the 1× ACSF solution. Then, let the agarose cool down to 40–45 °C.
During the agarose cooling period, dissect the embryonic brain and keep it in 1× ACSF on ice.
When the temperature of the agarose solution has reached 40–45 °C, pour it into a 3.5 cm Petri dish and transfer the brain into the agarose solution. Use a spatula to stir the agarose around the brain. This step replaces the 1× ACSF surrounding the brain with agarose solution, which permits the agarose to thoroughly attach to the surface of the brain. There is no particular orientation or position in which the brain should be placed in the Petri dish.
Add the lid to the dish and bury the entire dish in ice for 5 min to solidify the agarose quickly.
Trim the brain out of the Petri dish in trapezoid shape gel with the olfactory bulb facing the short side. Glue the long side of the trapezoid onto the ice-cold steel stage of a vibratome. Keep the steel stage on ice.
Prepare the vibratome by filling the space between the inner and outer chambers with ice. Add ice-cold 1× ACSF to the inner chamber. Insert the steel stage to the slot in the inner chamber and ensure the ice-cold 1× ACSF covers the whole brain sample.
Section the brain coronally into 300 µm slices and keep them in ice-cold 1× ACSF until the next step.
Tip 1: For the brain slice to survive, the whole sectioning process should be handled at ice-cold temperatures.
Tip 2: The 1× ACSF should be freshly prepared and always bubbled with 95% O2/5% CO2.
Video 1. Embedding the brain in agarose and sectioning it with a vibratome
Mounting the brain slice into a chamber specially fit for the microscope stage heater
The custom homemade chamber should be prepared ahead of time. Take a 3.5 cm Petri dish and reduce the height of the side wall to approximately 4 mm with medium-grit sandpaper (Figure 1A, middle). Then, use fine-grit sandpaper (Lot Fancy, 600 grit) to grind the bottom to remove 50% thickness. This facilitates heat penetration through the chamber bottom.
Thaw Matrigel on ice before the brain is dissected and dilute it with an equal volume of ice-cold DMEM.
Select the two best labeled brain slices using a fluorescent stereomicroscope.
Cut a thick filter paper into a 0.5 × 1.0 cm rectangle and soak it in ice-cold 1× ACSF. Scoop the filter paper under the brain slices to attach them to the filter paper and lift them out from the 1× ACSF.
Place the filter paper–supported brain slice on the middle of the homemade chamber and then remove the agarose surrounding the brain slice with a spatula (Figure 1A, middle).
Mount the brain slice/filter paper with the DMEM-diluted Matrigel in the middle of the 3.5 cm chamber (Figure 1B). Place the chamber in the tissue culture incubator (37 °C, 5% CO2) to solidify the Matrigel.
Tip 1: The mounting agarose II gel is removed because it may block cortical outgrowth.
Tip 2: Diluted Matrigel is softer and provides the developing brain slice with enough support but more flexibility.
Figure 1. Assembly of the brain slice and maintenance on a Leica SP5 confocal stage. (A) Size of the stage sample heater, sample chamber, and silicon lid. The brain slice on the filter paper is mounted in Matrigel in the middle of the sample chamber. (B) Cover the brain slice with the silicon lid and place the sample chamber into the stage heater. (C) A picture of the perfusion system. (D) Top view of the assembly for imaging the brain slice. (E) Side view of the assembly for imaging the brain slice. The media tubing, which flows DMEM over the brain slice, and temperature fiber, which records the DMEM (brain slice) temperature, are inserted into the sample chamber through openings in the silicon lid. The vacuum pump removes excess media and is inserted into the sample chamber through an opening in the silicon lid. The power cord connects the stage sample heater to the temperature controller.
Setting up the perfusion system
Turn on the Leica SP5 confocal microscope and software (make sure to choose resonance when turning on the software). Manually move all the lens out of position and adjust the stage to the lowest position. This step protects the lens and makes more space for the setup.
Install the heater on the stage and place a regular 3.5 cm Petri dish on it. Turn on the heater to increase the temperature of the Petri dish to 37 °C; the temperature of dish and stage will equilibrate with the confocal microscope lens, which will affect the focus and x-y position. Therefore, the focus and x-y position must be adjusted once the lens equilibrate with the stage temperature.
Add DMEM to a 60 mL syringe held upright and 1 m above the confocal stage (Figure 1C). Bubble the syringe DMEM with 95% O2/5% CO2 and let the media flow down the tubing by gravity into the Petri dish. The tubing is inserted into an opening in the silicon lid covering the Petri dish.
Insert the tubing to the vacuum pump in another opening in the silicon lid (Figures 1D and 1E, see the vacuum pump position). The height of the tubing to the vacuum pump is adjusted to maintain a depth of 5–7 mm of DMEM in the 3.5 cm Petri dish. All the extra media should be removed by the vacuum pump.
Connect the temperature fiber (Figure 1D, see the temperature fiber) to the temperature controller. The detector readout should match the room temperature if it is functioning normally. The temperature fiber is fragile; therefore, it is necessary to confirm it is functioning correctly before proceeding.
Insert the temperature fiber into the DMEM media in the Petri dish (Figure 1D, see the temperature fiber). It is necessary to measure the temperature in the core of the media, where the brain slice will be placed. Adjust the heater to maintain a core temperature of 34–39 °C.
Once the core temperature stabilizes to 34–39 °C, remove the temperature fiber, stop the flow of DMEM, remove the tubing to the vacuum pump, remove the regular 3.5 mm Petri dish, and insert the homemade chamber containing the brain slice/filter paper in Matrigel into the stage heater.
Resume the flow of DMEM, manually move the 25× water lens into position, and adjust the lens to touch the flowing DMEM media. Next, adjust the lens meticulously to focus on the sample.
Reinsert the temperature fiber into the DMEM media between the lens and brain slice to monitor the temperature and adjust the heater output to maintain the DMEM (sample) temperature between 34–39 °C. Reinsert the tubing to the vacuum pump and adjust to maintain a DMEM depth of 5–7 mm, which should completely cover the brain slice.
Tip 1: Bubbling the DMEM with 95% O2/5% CO2 mixed gas helps the brain slice to survive.
Tip 2: The DMEM temperature should be adjusted between 34 and 39 °C to maintain high activity in the brain slice.
Tip 3: The heater’s setting temperature is not the same as the DMEM (sample) temperature; use the temperature fiber to measure the DMEM temperature directly.
Setting up the Leica SP5 confocal microscope and recording images
Switch on the confocal microscope, turn on the confocal software, and check the Resonance box.
Adjust the focus of the 25× water lens to find the right z-position and adjust the x/y-axis to find the best position for imaging.
Confocal settings:
In Configuration, check the box of Line Average during Live Acquisition.
In Acquisition, set Line Average to 5 and Frame Average to 2.
Format (resolution): 512 × 512 pixels; Z-step (optical section thickness): 1.469 µm.
Use bidirectional scanning, which saves 50% scanning time.
Choose z-stack, set the scanning area along the z-axis, and ensure the scanning depth covers all the signal. The scanning depth should also cover 10 µm of extra space above and under the signal area. Total scanning depth is approximately 120 µm.
Choose time-lapse on the software and select a frequency for imaging anywhere between 2 and 30 min. We chose to image once every 5 min, which means scanning duration must be shorter than 5 min.
Ensure the DMEM in the syringe is continuously bubbled with 95% O2/5% CO2 gas. Adjust the flow of the DMEM from the 60 mL syringe into the Petri dish to maintain a flow of perfusion at 50–60 mL/h. Check the level of DMEM in the syringe every hour and refill to keep the flow continuous. It is critical not to allow the culturing system to run out of DMEM during recording.
Monitor the culturing dish on the stage to avoid flooding. Wrap the confocal stage with plastic wrap to protect the confocal microscope from DMEM flooding.
Monitor the sample temperature using the temperature fiber. Maintain the sample temperature at 34–39 °C.
Monitor and adjust the z-scanning, as the confocal microscope may lose focus gradually during long-term time-lapse imaging.
During imaging, the x-y position may change as the stage may drift in various directions, resulting in the sample moving out of focus or view. If imaging for over an hour, adjust the x-y-z axes every hour to prevent compromising the image acquisition. Whenever a problem occurs, e.g., loss of focus or x-y position drift, stop the time-lapse imaging, adjust, and resume time-lapse imaging. This will create another file. Start a new file every 2–3 h; otherwise, the file will become too large to process.
Tip 1: The syringe holds 60 mL of DMEM; adjust the flow speed such that the 60 mL of DMEM can last an hour. Refill the syringe with fresh DMEM every hour.
Tip 2: Since the x-y-z positions must be adjusted to image the brain slice, the images are not consecutive during the whole time-lapse imaging. Images need to be aligned using ImageJ.
Data analysis
Combine the files into one
Export all the images of multiple files from the Leica LAS X software. Rename all the images with a serial number to reflect their sequential time point. Save all these images into one folder. Open ImageJ (or FIJI). Choose Input files and select all these images in the folder. Based on the serial number, ImageJ automatically organizes all the images into one time-lapse file. This file is the original time-lapse file. Save the file.
Image alignment
The original time-lapse file is derived from several different Leica files. During time-lapse imaging, the imaging field may consistently move in one direction, so it is common to stop the program and move the image field back. Therefore, it is important to run image alignment to adjust the position to make the time-lapse imaging consistent. There are multiple plugins designed to improve the file’s consistency and new plugins are published based on different mechanisms. You may try multiple plugins, such as StackReg (http://bigwww.epfl.ch/thevenaz/stackreg/), to improve the continuum of the file.
Montage
To analyze an individual neuron’s behavior, select the area containing a neuron’s entire trajectory (see Video 1 for a migrating wild-type neuron). Right click and choose Duplicate to duplicate the selected area.
Choose Image → Stacks → Make Montage (Movie and Figure 2). Then, a window of Make Montage will pop up. Input a proper number of columns, rows, and the scale factor. Then click OK and ImageJ will create a montage image (Figure 2). The montage image in Figure 2 contains six rows, which represent each 5-min interval, starting from the bottom (+0:00) to the top (+0:25). Therefore, it took 30 min to record all six images in a column. In Figure 2, the second row was chosen to represent the whole process with a time interval of 30 min (labeled in green) between two images.
Equations
Migratory distance (pixels) can be measured based on the start and end position of the neuron based on the montage image. Migratory distance = Migratory distance pixels × Known distance per pixel. Known distance per pixel can be found from the confocal microscope.
Migratory duration = (end serial number – start serial number) × time between two images.
Speed = Migratory distance/Migratory duration (Figure 2, right panel).
Figure 2. Representative data. Download of a wild-type neuron .avi file. Open the file with ImageJ. Select Image on the ImageJ tool bar, choose z-stack, then choose Make Montage. Input the proper number of rows and columns, then click OK. ImageJ will create a montage file like the upper panel. The first image recorded is in the left bottom corner, the second recorded image is in the second row from the bottom and in the first column, the third recorded image is in the third row from the bottom and in the first column, etc. Because the imaging interval is 5 min, each column contains six images that took 30 min to record. Therefore, the columns from left to right can be labeled from 0:00 to 10:00 hours. Then, use rectangle selection to choose the best row (see the area selected in the green rectangle in the upper panel) and copy it in the lower panel to represent the cell behavior process. The right panel shows how to measure the migratory distance and speed. The black lines mark the start and end points of radial migration; the green lines show the distance of the radial migration from the start to the end; the blue lines label the start and end time. The original figure was published previously (Yang et al., 2022).
Recipes
10× ACSF solution I (1 L)
NaCl 73.05 g 1.25 M
KCl 1.86 g 0.025 M
MgCl2·6H2O 2.03 g 0.01 M
CaCl2·2H2O 2.94 g 0.02 M
NaH2PO4·H2O 1.73 g 0.125 M
Dissolve in deionized water to a final volume of 1 L. Store at room temperature.
10× ACSF solution II (1 L)
NaHCO3 21 g 0.25 M
Dissolve in deionized water to a final volume of 1 L. Store at room temperature.
1× ACSF (1 L)
100 mL of 10× ACSF solution I
100 mL of 10× ACSF solution II
Add deionized water to a final volume of 1 L. The final solution should be 310 mOsm, pH 7.4. The solution will be cloudy but will turn clear after 30 min of bubbling with 95% O2/5% CO2. Add 5–10 g of glucose; then, filter before use. 1× ACSF cannot be stored for extended periods after adding glucose.
Acknowledgments
This study was supported by NIH grants (R01EB028159 and R21NS094091), a Seed Grant from the Brain Research Foundation, the Protein Folding Disease Initiative of the University of Michigan to B.Y. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The current protocol is tested in our published work (Yang et al., 2022).
Competing interests
The authors declare no competing financial interests.
Ethics
All animals were handled based on a protocol approved by the Institutional Animal Care & Use Committee (IACUC) committee at the University of Michigan. Animals were housed in groups no larger than the cage limit ordered by IACUC and provided regular chow and water ad libitum. The mice were housed in a dedicated temperature-controlled (20 °C) animal facility with a 12:12 h light/dark cycle. The facility is run by the Unit for Laboratory Animal Medicine, which provides veterinarian staff to monitor the health of the animals onsite daily.
References
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Gao, P., Postiglione, M. P., Krieger, T. G., Hernandez, L., Wang, C., Han, Z., Streicher, C., Papusheva, E., Insolera, R., Chugh, K., et al. (2014). Deterministic progenitor behavior and unitary production of neurons in the neocortex. Cell 159(4): 775-788.
Gongidi, V., Ring, C., Moody, M., Brekken, R., Sage, E. H., Rakic, P. and Anton, E. S. (2004). SPARC-like 1 regulates the terminal phase of radial glia-guided migration in the cerebral cortex. Neuron 41(1): 57-69.
Kolk, S. M. and Rakic, P. (2022). Development of prefrontal cortex. Neuropsychopharmacology 47(1): 41-57.
Narita, A., Nagai, M., Mizuno, S., Ogishima, S., Tamiya, G., Ueki, M., Sakurai, R., Makino, S., Obara, T., Ishikuro, M., et al. (2020). Clustering by phenotype and genome-wide association study in autism. Transl Psychiatry 10(1): 290.
Ohtaka-Maruyama, C. and Okado, H. (2015). Molecular Pathways Underlying Projection Neuron Production and Migration during Cerebral Cortical Development. Front Neurosci 9: 447.
Rakic, P. (1974). Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183(4123): 425-427.
Rakic, P. and Caviness, V. S., Jr. (1995). Cortical development: view from neurological mutants two decades later. Neuron 14(6): 1101-1104.
Tsai, J. W., Bremner, K. H. and Vallee, R. B. (2007). Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue. Nat Neurosci 10(8): 970-979.
Turner, T. N., Hormozdiari, F., Duyzend, M. H., McClymont, S. A., Hook, P. W., Iossifov, I., Raja, A., Baker, C., Hoekzema, K., Stessman, H. A., et al. (2016). Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA. Am J Hum Genet 98(1): 58-74.
Wang, T., Guo, H., Xiong, B., Stessman, H. A., Wu, H., Coe, B. P., Turner, T. N., Liu, Y., Zhao, W., Hoekzema, K., et al. (2016). De novo genic mutations among a Chinese autism spectrum disorder cohort. Nat Commun 7: 13316.
Yamakawa, K., Huot, Y. K., Haendelt, M. A., Hubert, R., Chen, X. N., Lyons, G. E. and Korenberg, J. R. (1998). DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system. Hum Mol Genet 7(2): 227-237.
Yang, T., Sun, Y., Zhang, F., Zhu, Y., Shi, L., Li, H. and Xu, Z. (2012). POSH localizes activated Rac1 to control the formation of cytoplasmic dilation of the leading process and neuronal migration. Cell Rep 2(3): 640-651.
Yang, T., Veling, M. W., Zhao, X. F., Prin, N. P., Zhu, L., Hergenreder, T., Liu, H., Liu, L., Rane, Z., Savelieff, M. G., et al. (2022). Migrating pyramidal neurons require DSCAM to bypass the border of the developing cortical plate. J Neurosci 42(28): 5510-5521.
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Peer-reviewed
Reconstitution of Membrane-tethered Postsynaptic Density Condensates Using Supported Lipid Bilayer
ZF Zhe Feng
MZ Mingjie Zhang
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4649 Views: 296
Reviewed by: David A. Cisneros Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Jul 2022
Abstract
Eukaryotic cells utilize sub-cellular compartmentalization to restrict reaction components within a defined localization to perform specified biological functions. One way to achieve this is via membrane enclosure; however, many compartments are not bounded with lipid membrane bilayers. In the past few years, it has been increasingly recognized that molecular components in non- or semi-membrane-bound compartments might form biological condensates autonomously (i.e., without requirement of energy input) once threshold concentrations are reached, via a physical chemistry process known as liquid–liquid phase separation. Molecular components within these compartments are stably maintained at high concentrations and separated from the surrounding diluted solution without the need for a physical barrier. Biochemical reconstitution using recombinantly purified proteins has served as an important tool for understanding organizational principles behind these biological condensates. Common techniques include turbidity measurement, fluorescence imaging of 3D droplets, and atomic force microscopy measurements of condensate droplets. Nevertheless, many molecular compartments are semi-membrane-bound with one side attached to the plasma membrane and the other side exposed to the cytoplasm and/or attached to the cytoskeleton; therefore, reconstitution in 3D solution cannot fully recapture their physiological configuration. Here, we utilize a postsynaptic density minimal system to demonstrate that biochemical reconstitution can be applied on supported lipid bilayer (SLB); we have also incorporated actin cytoskeleton into the reconstitution system to mimic the molecular organization in postsynaptic termini. The same system could be adapted to study other membrane-proximal, cytoskeleton-supported condensations.
Keywords: Phase separation Synapses Biochemical reconstitution Supported lipid bilayer Actin cytoskeleton
Background
Neurons communicate via synapses that constitute the presynaptic bouton, synaptic cleft, and postsynaptic termini. Postsynaptic density (PSD) refers to a densely packed, protein-enriched area that locates just beneath the postsynaptic membrane. It contains thousands of proteins such as transmembrane receptors and channels, scaffold proteins, protein kinases and other enzymes, and cytoskeletal proteins. PSD serves as a signaling hub that converges signals received from the presynaptic termini and translates them into a series of downstream cellular processes, including dynamic translocation of receptors, re-organization of actin cytoskeleton, and regulation of protein degradation machinery, ultimately leading to altered synaptic morphology and function. The tiny compartmental size of neuronal synapses, the great heterogeneity across synapses, and the redundancy and compensation between different signaling pathways all make it more challenging to investigate mechanistic details behind synaptic organization via conventional imaging techniques. Recent studies have suggested that phase separation might provide an explanation to how synaptic compartments, including the presynaptic active zone and PSD, are assembled (Zeng et al., 2016, 2018 and 2019; Milovanovic et al., 2018; Wu et al., 2019 and 2021; McDonald et al., 2020; Pechstein et al., 2020; Bai et al., 2021; Cai et al., 2021; Hosokawa et al., 2021). In previously published studies, we demonstrated that major excitatory PSD (ePSD) scaffold proteins, when mixed in vitro, could readily condense into molecular assemblies via phase separation (Zeng et al., 2018; Feng et al., 2022). This minimal ePSD system reconstituted in vitro is reminiscent of the ePSD assemblies in vivo in many aspects. ePSD condensates could cluster receptors, exclude inhibitory PSD proteins, and be dispersed in the presence of negative regulators. The biochemically reconstituted minimal PSD system, therefore, provides a powerful platform to bridge in vitro observations to cellular functions in vivo. To better mimic the physiological context of semi-membrane-tethered ePSD assemblies, we developed methods for reconstituting microclusters using purified proteins assembled on supported lipid bilayers (SLBs) (Feng et al., 2022). We attached PSD-95 through the interaction of N-terminal His8 tag with Ni-NTA-functionalized lipids incorporated into the bilayer in order to mimic its membrane proximal localization via N-terminal palmitoylation in a physiological context. We also incorporated phosphatidylinositol 4,5-bisphosphate [PI (4,5) P2] lipids into the bilayer to enable insulin receptor substrate protein 53 (IRSp53), a major PSD scaffold protein as well as a PIP2 binder, to localize to the membrane via its Bin/Amphiphysin/RVS (BAR) domain known to bind negatively charged lipids. To enable visualization, we labeled proteins with amide/maleimide-conjugated fluorophores. PSD-95 was uniformly distributed on membranes and readily assembled into nanodomains when other PSD scaffold proteins, SH3 and multiple ankyrin repeat domains 3 (Shank3), guanylate kinase–associated protein (GKAP), IRSp53, and Homer3, were added to trigger phase separation. We incubated all the components with actin in the experimental system. We observed that rhodamine-labeled actin co-localized with the PSD condensates on the membrane and formed thin filament bundles. This reconstitution system allows us to investigate interactions between lipid membranes, membrane-proximal molecular assemblies, and actin cytoskeleton, as well as to recapture complex cellular behaviors observed in vivo.
Materials and Reagents
Proteins: PSD-95, IRSp53, Shank3, GKAP, and Homer3 [see Feng et al. (2022) for protein production details]
Rabbit skeletal muscle actin (Cytoskeleton, Inc., catalog number: AKL99), rhodamine actin from rabbit skeletal muscle (Cytoskeleton, Inc., catalog number: AR05)
Lipid components:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti, catalog number: 850457P)
1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) [18:1 PI (4,5) P2] (Avanti, catalog number: 850155P)
1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (DGS-NTA) (Avanti, catalog number: 790404P)
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG5000PE) (Avanti, catalog number: 880230P)
Fluorophores:
Alexa FluorTM 647 NHS ester (Thermo Fisher, catalog number: A20006)
Cy3® NHS ester (AAT Bioquest, catalog number: 271)
Alexa FluorTM 488 C5 maleimide (Thermo Fisher, catalog number: A10254)
DiO perchlorate (AAT Bioquest, catalog number: 22066)
Chambered cover glass (Lab-tek®, catalog number: 155409)
Amber glass vials (Thermo Fisher, catalog number: B7800-1-9A)
Hellmanex III (Helma AnalyticsTM, catalog number: Z805939)
Gastight syringes [Agilent, catalog numbers: 5190-1471 (2 μL), 5190-1483 (10 μL), 5190-1493 (25 μL), and 5190-1507 (100 μL)]
Chloroform (Scharlab, product code: 10289473)
NaOH (Scharlab, catalog number: SO04251000)
Sodium cholate (Sigma, catalog number: 27029)
BSA (Goldbio, catalog number: A-421-500)
ATP (Sigma, product number: A6144)
MgCl2 (VWR Chemicals BDH®, catalog number: BDH9244)
NaH2PO4 (Millipore, catalog number: 567545)
KH2PO4 (Sigma, catalog number: P0662)
NaCl (Santa Cruz Biotechnology®, catalog number: SC-203274)
KCl (VWR Chemicals BDH®, catalog number: 26764)
Tris-HCl (Goldbio, catalog number: T-400-5)
DL-Dithiothreitol (DTT) (Sigma, catalog number: D0632)
CaCl2 (Sigma, catalog number: C4901)
HiTrap desalting column (Cytiva, catalog number: 89501-384)
PBS buffer (see Recipes)
Reaction buffer (see Recipes)
G-buffer (see Recipes)
Equipment
Water bath (Shel lab, model number: 1201-2E)
Oven incubator (Binder GmbH, art number: 9010-0002)
High-speed centrifuge (Eppendorf, catalog number: 540600097)
AKTApurifier (GE Healthcare, USA)
Zeiss LSM 800 microscope (Zeiss)
Nanodrop
Software
FIJI (ImageJ)
Procedure
Preparation of small unilamellar vesicles (SUV)
Warm up individual lipid stocks (dissolved in chloroform) to room temperature.
Use glass syringe (see Materials and Methods for details of syringes used here) to prepare lipid mixture with the following composition: 95.9% POPC, 2% DGS-NTA (Ni), 2% PI (4,5) P2, and 0.1% PEG5000PE.
Mix the following components in a glass vial:
POPC (13.16 mM stock) 7.1 μL
DGS-NTA (Ni) (4.74 mM stock) 0.42 μL
PIP2 (500 μM stock) 3.9 μL
PEG5000PE (170 μM stock) 0.57 μL
Note that the above lipid quantities are calculated to coat a single cover glass well (i.e., 75 μg total).
Dry the lipid mix under a stream of nitrogen gas with rotation. You will see multiple white layers adhered to the wall of the vial after the lipids are dried (Figure 1). Note that 1% DiO perchlorate dye is included to label the lipids for a better visualization in Figure 1.
Figure 1. Image showing dried lipid layers adhered to the wall of a glass vial.
1% DiO dye is included to label the lipids for better demonstration.
Continue to dry the lipids under vacuum for at least 1 h to ensure removal of residual chloroform.
Resuspend the lipid mixture with vigorous pipetting and vortexing in ≤300 μL (the resuspension volume/the column injection volume is determined by the column resolution) of PBS buffer supplemented with 1% v/v sodium cholate. Lipids should be completely transparent once fully dissolved in detergent-containing buffer.
Subject the dissolved lipid mixture to a HiTrap desalting column equilibrated with the detergent-free PBS buffer. Use an FPLC system and pool fractions with detected UV absorbance at 280 nm, followed by dilution to the desired volume with PBS buffer. Add 150 μL of lipid solution to coat each well glass to generate SLBs.
Preparation of supported lipid bilayers (SLBs)
Wash the chambered cover glass (well area of 0.7 cm2) with 5% Hellmanex III overnight at room temperature and thoroughly rinse with Milli-Q water the next morning.
Wash the cover glass with 5 M NaOH for 1 h at 50 °C and then rinse thoroughly with Milli-Q water. Repeat this wash step three times.
To each cleaned cover glass, add 500 μL of PBS buffer for equilibration, take out, and discard.
Add 150 μL of SUVs to each cover glass well and incubate for 1 h at 42 °C to generate SLBs.
Wash the SLBs with 750 μL of reaction buffer three times to achieve 216-fold dilution. It should be noted that SLBs should not be exposed to the atmosphere to prevent oxidation.
Block the SLBs with 1 mg/mL BSA (stock concentration of 100 mg/mL) in the reaction buffer at room temperature for 30 min.
Add 2 μM 10% Alexa 647- or Cy3-labeled, His8-tagged PSD-95 protein to the SLBs and leave for incubation with SLBs for 1 h at room temperature.
Wash away the unbound His-PSD-95 with the reaction buffer three times (750 μL per wash).
Image using Zeiss LSM 800 microscope (Figure 2Ai).
Imaging membrane localization of PIP2 binder
Add 1 μM 10% Alexa 488–labeled IRSp53 protein to the SLBs and leave for incubation with SLBs for 1 h at room temperature.
Wash the SLBs with the reaction buffer three times (750 μL per wash) to remove any unbound IRSp53 protein.
Image using Zeiss LSM 800 microscope (Figure 2Aii).
Figure 2. Postsynaptic density (PSD) condensation and actin polymerization on supported lipid bilayers (SLBs). (A) Fluorescence images showing uniform coating of Cy3-labeled, His8-tagged PSD-95 (i) or Alexa 488–labeled IRSp53 (ii) on SLBs before the addition of other PSD scaffold proteins. (B) Representative fluorescence images showing actin filaments assembled from PSD condensates on the SLB. An actin-binding-deficient mutant of IRSp53 (K4E) shows significantly diminished actin bundling to PSD condensates on the membrane. A line plot of the rhodamine-labeled actin intensities along the dashed line is presented. Figures are adapted from Feng et al. (2022) with permission.
Actin reconstitution and labeled actin preparation
Reconstitute lyophilized muscle actin protein to 10 mg/mL with 100 μL of deionized water and then dilute to 0.4 mg/mL in G-buffer supplemented with 0.2 mM ATP and 0.5 mM DTT.
After reconstitution, incubate actin on ice for 1 h to depolymerize actin oligomers that form during storage. Further centrifuge the actin resuspension at 14,000 × g for 15 min at 4 °C.
Transfer the supernatant to a new microcentrifuge tube and determine the total protein concentration with a NanoDrop.
Reconstitute lyophilized rhodamine muscle actin protein to 10 mg/mL with 2 μL of deionized water and then dilute to 2 mg/mL with G-buffer supplemented with 0.2 mM ATP and 1 mM DTT.
Leave actin solution on ice for 1 h to depolymerize actin oligomers.
Centrifuge the rhodamine actin resuspension at 14,000 × g for 15 min at 4 °C.
Transfer the top 90% of the supernatant to a new tube and determine the total protein concentration and percentage of rhodamine-labeled protein with a NanoDrop.
Imaging microcluster formation and actin polymerization
Premix 250 nM of Shank3, GKAP, Homer3, and IRSp53, as well as 1 μM of 10% rhodamine-labeled actin in reaction buffer supplemented with 1 mM ATP and 2 mM MgCl2. The total solution volume in each chambered glass well is 150 μL.
Add the pre-mixture to the Alexa 647–labeled His8-PSD-95-bound SLBs and incubate for 20 min to allow microcluster formation and actin polymerization on the membrane.
Image using Zeiss LSM 800 microscope. Actin polymerization occurs within 20 min after mixture and reaches a plateau after 1 h. All data should be collected within 8 h after lipid coating started.
Data analysis
Analyzing actin recruitment with FIJI (ImageJ)
Open the captured image using FIJI.
To analyze actin enrichment within membrane localized clusters, draw a diagonal line across the image and measure the fluorescence intensity by Plot Profile. Note that it is essential to use identical imaging settings for direct comparison between different experiments.
Recipes
PBS buffer
10 mM NaH2PO4/1.8 mM KH2PO4, pH 7.4
137 mM NaCl
27 mM KCl
Reaction buffer
50 mM Tris-HCl, pH 8.0
100 mM NaCl
2 mM DTT
G-buffer
5 mM Tris-HCl, pH 8.0
0.2 mM CaCl2
0.2 mM ATP
Acknowledgments
This work was supported by grants from the Ministry of Science and Technology of China (2019YFA0508402), the National Science Foundation of China (82188101), Research Grants Council (RGC) of Hong Kong (AoE-M09-12, 16104518 and 16101419), a Human Frontier Science Program research grant (RGP0020/2019) to M. Zhang, and an RGC General Research Fund (GRF) grant (16102120) to Z. Feng. The protocol was adapted from Feng et al. (2022).
Competing interests
The authors declare no competing interests.
References
Bai, G., Wang, Y. and Zhang, M. (2021). Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation. Cell Res 31(3): 312-325.
Cai, Q., Zeng, M., Wu, X., Wu, H., Zhan, Y., Tian, R. and Zhang, M. (2021). CaMKIIalpha-driven, phosphatase-checked postsynaptic plasticity via phase separation. Cell Res 31(1): 37-51.
Feng, Z., Lee, S., Jia, B., Jian, T., Kim, E. and Zhang, M. (2022). IRSp53 promotes postsynaptic density formation and actin filament bundling. J Cell Biol 221(8): e202105035.
Hosokawa, T., Liu, P. W., Cai, Q., Ferreira, J. S., Levet, F., Butler, C., Sibarita, J. B., Choquet, D., Groc, L., Hosy, E., et al. (2021). CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation. Nat Neurosci 24(6): 777-785.
McDonald, N. A., Fetter, R. D. and Shen, K. (2020). Assembly of synaptic active zones requires phase separation of scaffold molecules. Nature 588(7838): 454-458.
Milovanovic, D., Wu, Y., Bian, X. and De Camilli, P. (2018). A liquid phase of synapsin and lipid vesicles. Science 361(6402): 604-607.
Pechstein, A., Tomilin, N., Fredrich, K., Vorontsova, O., Sopova, E., Evergren, E., Haucke, V., Brodin, L. and Shupliakov, O. (2020). Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation. Cell Rep 30(8): 2594-2602 e2593.
Wu, X., Cai, Q., Shen, Z., Chen, X., Zeng, M., Du, S. and Zhang, M. (2019). RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation. Mol Cell 73(5): 971-984 e975.
Wu, X., Ganzella, M., Zhou, J., Zhu, S., Jahn, R. and Zhang, M. (2021). Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates. Mol Cell 81(1): 13-24 e17.
Zeng, M., Chen, X., Guan, D., Xu, J., Wu, H., Tong, P. and Zhang, M. (2018). Reconstituted Postsynaptic Density as a Molecular Platform for Understanding Synapse Formation and Plasticity. Cell 174(5): 1172-1187. e16.
Zeng, M., Diaz-Alonso, J., Ye, F., Chen, X., Xu, J., Ji, Z., Nicoll, R. A. and Zhang, M. (2019). Phase Separation-Mediated TARP/MAGUK Complex Condensation and AMPA Receptor Synaptic Transmission. Neuron 104(3): 529-543 e526.
Zeng, M., Shang, Y., Araki, Y., Guo, T., Huganir, R. L. and Zhang, M. (2016). Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity. Cell 166(5): 1163-1175. e12.
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Assessment of Chemosensory Response to Volatile Compounds in Healthy, Aged, and Neurodegenerative Caenorhabditis elegans Models
CC Cira Crespo
Roberto Grau
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4650 Views: 453
Reviewed by: Juan Facundo Rodriguez AyalaDurai SellegounderRajesh RanjanAyush Ranawade
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Original Research Article:
The authors used this protocol in Journal of Alzheimer's Disease Feb 2020
Abstract
A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy individuals and Caenorhabditis elegans models associated with neurodegenerative diseases.
Keywords: Caenorhabditis elegans Behavior Diacetyl Octanol Neurodegenerative diseases
Background
A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses (Baidya et al., 2014). Caenorhabditis elegans has only fourteen types of chemosensory neurons but responds to dozens of chemicals, because each chemosensory neuron detects a wide variety of volatile (olfactory) and water-soluble (gustatory) cues associated with food, danger signals, or other animals (Troemel et al., 1995; Dosanjh et al., 2010). Much of its nervous system and more than 5% of its genes are devoted to the recognition of environmental chemicals. Chemosensory cues can elicit chemotaxis, rapid avoidance, changes in overall motility, and entry into or exit from the alternative dauer developmental stage. These behaviors are regulated by eleven pairs of chemosensory neurons. Each amphid sensory neuron expresses a specific set of candidate receptor genes and detects a characteristic set of attractants, repellents, or pheromones (Bargmann, 2006). Olfaction is a versatile and sensitive mechanism for detecting volatile odorants. Many of these responses are mediated (at least in part) by a pair of ciliated sensory neurons named ASH, which have sensory openings at the anterior amphid pore at the nose of the animal and are thought to be analogous to polymodal nociceptive neurons in vertebrates; other sensory neurons (e.g., ADL, AWB, ASK) are thought to play minor and auxiliary roles in avoidance responses (Figure 1). The avoidance response to 30% octanol via ASH neurons is enhanced by serotonin; furthermore, altering serotonin levels results in ADL and AWB neurons being recruited to detect 100% octanol redundantly with ASH neurons (Chao et al., 2004). In the nematode, the presence of this compound has a strong modulatory influence on many behaviors. For instance, the olfaction of octanol or diacetyl (DA) stimulates C. elegans mechanosensory neurons to release dopamine, which affects many behaviors including locomotion rate (Sawin et al., 2000), foraging, and response to soluble repellants (Ezcurra et al., 2011). In this protocol, we describe how to evaluate the time response against attractants and repellents in the nematode C. elegans to study the neuronal system and behavioral response of worms, and how this can be translated to neuronal diseases in worms fed on different bacteria. For example, as worms age, they have more difficulty sensing octanol and their avoidance response slows, the same being observed with the DA (Figure 1). This protocol has the advantage of not requiring a centrifugation step, which could severely damage aged and sick worms. As far as we know, this protocol is the first one to be applied to assess neurological damage in C. elegans.
Figure 1. Introduction to C. elegans sensory anatomy. (A) A prominent and well-characterized subset of C. elegans sensory neurons, the amphid sensory neurons. As with most other neuron classes, amphid sensory neuron classes consist of one pair of bilaterally symmetric cells (see also B). Each of the 12 pairs of amphid sensory neurons extends a dendrite to the tip of the nose and an axon into the nerve ring, a nerve bundle where synaptic connections are made (White et al., 1986). Delineated functions of amphid sensory neurons are indicated (Bargmann and Mori 1997). (B) Amphid sensory neuron classes consist of two bilaterally symmetric pairs of neurons, two of which, AWCL/R and ASEL/R, are functionally lateralized. While some other amphid sensory neurons appear to contribute to gustation, ASE is the main gustatory neuron class in C. elegans, mediating responses to salts, amino acids, and small metabolites (Bargmann et al., 1993). ASE mediates not only attractive but also repulsive responses to specific chemicals (Sambongi et al., 1999). The salts sodium, chloride, and potassium are sensed in a left/right asymmetric manner, with ASEL sensing sodium but not chloride and potassium, and ASER sensing chloride and potassium (Pierce-Shimomura et al., 2001). It is not yet known whether other ASE-sensed chemicals may also activate ASEL and ASER differentially. ASE-expressed GCY genes are shown and those that are asymmetric are colored. The AWCL/R neurons can discriminate benzaldehyde and butanone on the basis of the left/right asymmetric expression of str-2 (colored), a G-protein coupled receptor, which is stochastically expressed in either AWCL or AWCR (Troemel et al., 1999; Wes and Bargmann, 2001). Newly identified GCY genes in AWC are shaded. Figure taken from Ortiz et al. (2006).
Materials and Reagents
Glass media bottles 200 mL (Fisher Scientific, catalog number: FB800250).
Petri dishes 60 × 15 mm 500/cs (Fisher Scientific, catalog number: FB0875713A)
Pipette tips 2–200 µL Eppendorf® epT.I.P.S. (Eppendorf, catalog number: 022492039)
Pipette tips 50–1,000 µL Eppendorf® epT.I.P.S. (Eppendorf, catalog number: 022492055)
Eppendorf® Safe-Lock 1.5 mL microcentrifuge tubes (Eppendorf, catalog number: 022363204)
Corning® 15 mL centrifuge tubes (Corning, catalog number: 430791)
C. elegans strains were obtained from Caenorhabditis Genetics Center: N2 Bristol (WormBase ID: WBStrain00000001; dvIs15), CL2122 (WBStrain00005101), and CL2355 [WBStrain00005106; smg-1(cc546); pCL45 [Psnb-1::human Amyloid beta 1-42::3' UTR (long); Pmtl-2::GFP]]
Escherichia coli strain OP50 (University of Minnesota, C. elegans Genetics Center, MN)
Bacillus subtilis strain NCIB3610 (Bacillus Genetic Stock Center, catalog numbers: 3A1 and 1A96)
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M1880)
Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655)
Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P2222)
Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: 7782-85-6)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)
NaClO hypochlorite (Sigma-Aldrich, catalog number: 13440)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045)
Bacto peptone (BD, BactoTM, catalog number: 211677)
Agar (Sigma-Aldrich, catalog number: A1296)
Cholesterol (Sigma-Aldrich, catalog number: C8667)
100% ethanol (Sigma-Aldrich, catalog number: E7023)
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881)
Luria broth (LB) (Sigma-Aldrich, catalog number: L3522)
Octanol (1-octanol) (Sigma-Aldrich, catalog number: 472328)
Diacetyl (DA) (butane-2,3-dione) (Sigma-Aldrich, catalog number: B85307)
M9 buffer (see Recipes)
Nematode growth medium (NGM) agar (see Recipes)
Potassium phosphate buffer (see Recipes)
1 M MgSO4·7H2O (see Recipes)
Cholesterol in absolute ethanol (5 mg/mL) (see Recipes)
1 M CaCl2 (see Recipes)
Potassium phosphate buffer (1 M KPO4, pH 6.0) (see Recipes)
LB media (see Recipes)
Bleaching solution (see Recipes)
1 N NaOH (see Recipes)
0.5% DA (see Recipes)
S-basal buffer (see Recipes)
Equipment
Shaker (Axyos Technologies, catalog number: S04036.tu)
Dissecting microscope (Carl Zeiss, catalog number: 3944010970)
Commercially available paintbrush number: 10/0; 0.2 × 0.2 × 6.5 inches (Princeton art & Brush, catalog number: B0043GCYSI).
Autoclave (Tuttnauer USA, model: 6690)
Worm pick. Worm picks can either be purchased (Genesee Scientific, catalog number: 59-AWP) or made in the lab as described in Wollenberg et al. (2013).
Centrifuge (Eppendorf, model: 5430)
Tabletop centrifuge (Eppendorf, model: 5424)
Bunsen burner (Humbolt, catalog number: H-5870)
Chronometer (Hinotek, catalog number: PS-360A)
Software
Microsoft Excel (Microsoft Corporation, Redmond, USA)
GraphPad Prism 8 (GraphPad Software, Inc.)
Procedure
C. elegans maintenance
Inoculate an E. coli OP50 colony into 50 mL of Luria-Bertani (LB) broth in a 200 mL flask and incubate with gentle agitation at 37 °C overnight.
Seed approximately 200 μL of E. coli OP50 overnight culture (1 × 108 CFU/mL) to a 60 mm NGM agar plate. Incubate plates at 37 °C overnight before use.
Transfer 50–100 mixed-stage larval worms (N2, CL2122, and CL2355 strains) to a new E. coli OP50-seeded NGM agar plate using a heat-sterilized platinum wire pick. Keep worms at 20 °C.
Repeat step A3 every 2–3 days for maintenance.
C. elegans synchronization by bleaching
Allow worms to grow until adult stage.
Recover gravid adults in 15 mL tubes by washing plates with M9 buffer (see Recipe 1).
Pellet worms by centrifuging for 2 min at 400 × g (~1,500 rpm on a standard table centrifuge) at room-temperature and discard supernatant.
Perform 1–3 washes until the buffer appears clear of bacteria (lack of turbidity).
Add 5 mL of bleaching solution (see Recipe 8) into the tube and shake vigorously at room temperature for up to 5 min. Bleaching longer than 5 min will kill the eggs.
Dilute the reaction by adding 10 mL of S-basal buffer to the 15 mL centrifuge tube.
Quickly centrifuge (since treatment may still be active) for 1 min at 400 × g and discard supernatant.
Wash pellet three more times by filling the tube with S-basal buffer. Check using a dissecting microscope.
Place the eggs to unseeded NGM plates and incubate for 18–24 h at 20 °C. Proper aeration should be provided to obtain all animals in stage L1.
Wash each NGM plate with 1 mL of S-basal buffer, transfer the L1 population to NGM agar plates seeded with the corresponding bacterial food, and incubate until they reach the young-adult stage (1-day-old L4), approximately 48 h later. Most of the C. elegans strains are maintained at 20 °C on NGM media seeded with E. coli OP50 or B. subtilis NCIB3610.
Behavior assay
Feed C. elegans N2, CL2122, and CL2355 strains on E. coli OP50 or B. subtilis NCIB3610 bacterial cells from the L1-larval stage to adulthood at 20 °C.
Repulsion and attraction behavioral assays are performed using octanol or DA as repellent or attractant agents, respectively.
Wash E. coli OP50 or B. subtilis NCIB3610 strain-fed adult worms of different ages three times with M9 buffer to remove any residual bacteria and place them in NGM plates without food.
For the repellent assay, 1 h after food starvation, place a paintbrush hair previously dipped in 100% octanol solution in front of a moving animal.
Score the escape response time to octanol as the time (s) from exposure to the repellent to the initiation of a backward or escape movement.
To measure the reaction time against octanol, use a timer that is stopped when the worm begins its regression, making a movement backwards (Figures 3 and 4).
For the attractant assay, 1 h after food starvation, place a paintbrush hair previously dipped in 0.5% DA in ethanol 1.5 cm in front of a moving animal.
Note: Mark the distance of 1.5 cm (with two dots or lines, one for the place where the brush is and another where the worms will be) outside the Petri dish in the attraction test so that the test is reproducible (Figure 2).
Figure 2. Scheme of the response time assay of C. elegans against the repellent octanol or the attractive diacetyl (DA). In the repellent assay, put the paintbrush dipped in octanol in front of the worm’s head and start the chronometer. Stop it when the worm begins its regression. Do the same for the attraction assay, but in this case put the paintbrush dipped in DA 1.5 cm in front of the worm and stop the chronometer when the worm initiates its forward movement in the direction of DA. The movement of the worm can be visualized in the following link: https://www.youtube.com/watch?v=klOJb0DDGGU&ab_channel=NeuroAIUWLaboratory. As can be seen in the video, ALM+AVM neural stimulus is capable of generating the avoidance behavior; the stimulus disturbs forward locomotion and initiates backward locomotion. Inspection of neural activity of motor neurons (DB neurons are anterior -> posterior ordered) indicates that the stimulus induces a change in the directionality of neural activity traveling wave, from anterior -> posterior to posterior -> anterior. The transition is marked through high constant activity in the anterior motor neurons.
Score the DA response time as the time (s) from the DA presentation to the initiation of a forward movement in the direction of DA (Figure 3 and 4).
Figure 3. B. subtilis-mediated cognitive improvement during C. elegans aging. Average response times (in seconds, y-axis) of OP50- and NCIB3610-colonized N2 worms (red and green columns, respectively) of different ages (in days, x-axis) to repellent (octanol, left) and attractant [diacetyl (DA), right] exposition. Results represent mean ± standard deviation of three independent experiments performed in duplicate. A typical result from one of the three independent experiments performed in duplicate is presented (mean ± S.E.M). From Cogliati et al. (2020).
Figure 4. B. subtilis improved the behavioral response of C. elegans expressing pan-neuronal Aβ aggregates. Time of response of OP50- and NCIB3610-colonized (red and green columns, respectively) CL2122 (control or CL2122 wt, left) and CL2355 [CL2355 Alzheimer’s disease (AD) model expressing the pan-neuronal Aβ1-42, right] worms to 100% octanol (repellent), and CL2122 (control or CL2122 wt, left) and CL2355 (AD model expressing the pan-neuronal Aβ1-42, right) worms to 0.5% diacetyl (DA, attractant). A typical result out of three independent experiments (performed in duplicate) is presented (mean ± standard deviation). Asterisks indicate statistical significance (*p < 0.1; ***p < 0.001; ns, no significant difference, p > 0.5).
Data analysis and interpretation
All assays were performed at least three times in duplicate. In the repellent assay, put the paintbrush dipped in octanol in front of the worm’s head and start the chronometer. Stop it when the worms begin their regression. This represents the time of repellent response in seconds. Do the same for the attraction assay, but in this case put the paintbrush dipped in DA 1.5 cm in front of the worm and stop the chronometer when the worms initiate its forward movement in the direction of DA. This represents the time of attraction response in seconds. Calculate the time of attraction response and the time of repellent response and plot the data in Microsoft Excel. Use GraphPad Prism 8 for statistical analysis of data. Combine data from three independent experiments and calculate the mean and standard deviation. Apply a Student’s t-test with a significance cut-off level of P < 0.05 for comparisons between two groups for attraction and repellent time.
Recipes
M9 buffer
3 g of KH2PO4
6 g of Na2HPO4·7H2O
5 g of NaCl
Water up to 1 L. Autoclave, then add 1 mL of 1 M MgSO4.
Nematode growth medium (NGM) agar for 1 L medium
3.0 g of NaCl
20 g of agar
2.5 g of Bacto peptone
Water up to 972 mL. Autoclave to sterilize the agar, cool to 55 °C, then add:
1 mL of 1 M MgSO4·7H2O (see Recipe 3)
1 mL of 1 M CaCl2 (see Recipe 5)
1 mL of 5 mg/mL cholesterol in absolute ethanol (see Recipe 4)
25 mL of 1 M potassium phosphate buffer pH 6.0 (see Recipe 6)
1 M MgSO4·7H2O
Dissolve 6 g of MgSO4 heptahydrate in 50 mL of dH2O
Autoclave (121 °C for 20 min)
Store at room temperature.
Cholesterol in absolute ethanol (5 mg/mL)
Dissolve 0.25 g of cholesterol in 50 mL of 100% ethanol
Do not autoclave.
1 M CaCl2
Dissolve 5.55 g of CaCl2 dihydrate in 50 mL of dH2O
Autoclave (121 °C for 20 min)
Store at room temperature.
Potassium phosphate buffer (1 M KPO4, pH 6.0)
108.3 g of KH2PO4
35.6 g of K2HPO4
Water up to 1 L
Autoclave (121 °C for 20 min)
Store at room temperature.
LB media
Dissolve 20 g of LB broth in 1 L of ddH2O
Autoclave (121 °C for 20 min).
Bleaching solution
1 mL of 3% NaClO hypochlorite
2.5 mL of 1 N NaOH (see Recipe 9)
0.5 mL of dH2O
1 N NaOH
Dissolve 2 g of NaOH in 50 mL of dH2O
0.5% DA
Dissolve diacetyl in 100% ethanol. Diacetyl should be prepared immediately prior to any experiments by dilution in ethanol.
S-basal buffer
5.85 g of NaCl
1 g of K2HPO4
6 g of KH2PO4
H2O to 1 L. Sterilize by autoclaving (121 °C for 20 min). Add 1 mL of cholesterol (5 mg/mL in ethanol).
Notes
When placing the paintbrush in front of the nematode, take care to not touch the worm nor the surface of the agar plate.
In the repellent assay, sterile water was used instead of octanol as a control and assays were halted at 20 s to account for spontaneous reversals.
Only inversions were scored, as determined by observing the backward movement of the tail. An interruption of forward movement or a head-only retreat was not scored.
In the attractant assay, ethanol was used instead of DA as a control.
Acknowledgments
This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) and FONCyT (Fondo para la Investigación Científica y Tecnológica) with the aid of the Pew Latin-nAmerican Program in Biological Sciences (Philadelphia, USA), the Fulbright Committeen (Washington, DC, USA) and former Fundación Antorchas (Buenos Aires, Argentina). We modified the media and NGM plate preparation from Stiernagle (2006). We modified C. elegans synchronization from Montserrat Porta-de-la-Riva et al. (2012). The protocol is based on “Dopamine modulation of avoidance behavior in Caenorhabditis elegans requires the NMDA receptor NMR-1” (Baidya et al., 2014).
Competing interests
The authors declare no conflicts of interest.
References
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Fluorescence Assays for Real-Time Tracking of Cell Surface Protein Internalization and Endosomal Sorting in Axons of Primary Mouse Hippocampal Neurons
TC Tai Chaiamarit
YW Yin Wu
AV Adriaan Verhelle
SE Sandra E. Encalada
Published: Vol 13, Iss 7, Apr 5, 2023
DOI: 10.21769/BioProtoc.4651 Views: 869
Reviewed by: Xi FengElma Frias Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Advances Dec 2021
Abstract
The trafficking and sorting of proteins through the secretory-endolysosomal system is critical for the proper functioning of neurons. Defects in steps of these pathways are associated with neuronal toxicity in various neurodegenerative disorders. The prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored protein that follows the secretory pathway before reaching the cell surface. Following endocytosis from the cell surface, PrP sorts into endosomes and lysosomes for further recycling and degradation, respectively. A few detailed protocols using drug treatments and fluorescent dyes have previously allowed the tracking of PrP trafficking routes in real time in non-neuronal cells. Here, we present a protocol optimized for primary neurons that aims to monitor and/or manipulate the trafficking and sorting of PrP particles at several steps during their secretory-endolysosomal itineraries, including (a) ER export, (b) endocytosis, (c) lysosomal degradation, and (d) accumulation in axonal endolysosomes. These primary neuron live assays allow for the robust quantitation of accumulation and/or degradation of PrP or of other membrane-associated proteins that transition from the ER to the Golgi via the cell surface.
Graphical abstract
Keywords: Primary neurons Axonal transport PrP Trafficking Endosomes Endocytosis ARESTA Endoggresomes
Background
Both wild-type (PrPWT) and mutant prion proteins (PrPmut) exit the ER and Golgi and follow secretory and endolysosomal trafficking pathways en route to somatodendritic and axonal cell surfaces, where PrP attaches via its glycosylphosphatidylinositol (GPI) anchor prior to internalization (Satpute-Krishnan et al., 2014; Zavodszky and Hegde, 2019; Chassefeyre et al., 2021). From the cell surface, PrPWT is endocytosed and sorted into endosomes or lysosomes for recycling or degradation, respectively, while PrPmut accumulates inside non-acidic/non-degradative enlarged endolysosomes where it can form aggregates called endoggresomes (Chassefeyre et al., 2021). PrPmut endoggresome formation in axons occurs via an axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) pathway (Chassefeyre et al., 2021). In ARESTA, vesicles containing luminal PrPmut exit from the Golgi after association with a protein complex composed of the small endolysosomal guanosine triphosphatase (GTPase) ARL8b. ARL8b recruits its effectors SifA and kinesin-interacting protein (SKIP), the kinesin-1 (KIF5C) molecular motor and associated kinesin light chain 1 (KLC1) subunit, and the vacuolar protein sorting 41 (VPS41), a member of the multiunit homotypic fusion and protein sorting (HOPS) complex (Hofmann and Munro, 2006; Khatter et al., 2015), which associate to endosomes carrying PrPmut and promote their entry into the axon and their homotypic fusion to form endoggresomes (Graphical abstract). For aggregation to occur in axons, endosomes carrying PrPmut homotypically fuse within axons after direct trafficking from the Golgi, or after undergoing rapid bouts of exocytosis and endocytosis throughout the axonal surface prior to fusion via the HOPS fusion complex. The rapid and very transient residence at the cell surface of PrPmut compared to PrPWT is key for the formation of endoggresomes vs. their degradation in axons. To understand the biology of misfolded PrPmut endoggresome formation vs. the normal trafficking of PrPWT, it is therefore critical to follow the transit of PrP particles throughout these subcellular itineraries.
Previous studies using drug treatments and fluorescent assays could monitor the trafficking routes of misfolded PrP in non-neuronal cells grown in culture (Satpute-Krishnan et al., 2014; Zavodszky and Hegde, 2019). However, as neurons are primary targets in prion diseases, it is important to follow the subcellular sorting of misfolded PrP in neurons, including in highly polarized compartments such as axons, which are primarily affected in disease. Here, we present live assays to image and quantitate the formation of PrPmut endoggresomes along their subcellular transit inside vesicles at the single-particle level using standard fluorescence microscopy, in either the soma or in axons of primary neurons, in the presence or absence of compounds that can modulate ER export and internalization capacity. These assays also monitor PrPmut accumulation in endolysosomal compartments. These approaches allowed the identification of trafficking and sorting pathways of PrPmut endoggresome biogenesis and would allow the monitoring of the trafficking of various other membrane proteins, including of the amyloid precursor protein (APP) or amyloid beta peptides implicated in the pathophysiology of Alzheimer’s disease, using cultured primary neurons as a model system.
Materials and Reagents
60 × 15 mm NuncTM Cell Culture/Petri Dishes (Thermo Fisher, catalog number: 150288)
150 × 21 mm NuncTM Cell Culture/Petri Dishes (Thermo Fisher, catalog number: 168381)
15 mL Falcon tube (Thermo Fisher, catalog number: 12565268)
#1.5 thickness 12 mm coverslip (Neuvitro, catalog number: NC0319857)
Note: Wash 3× acetone, 3× ethanol, and rinse 3× with sterile water; store in sterile water.
Cell culture plate, size 24-wells, sterile, flat bottom, tissue-culture treated (Sigma, catalog number: SIAL0524)
Syringe filter Millex-GP, 33 mm, PES, 0.22 µm, gamma-sterile, hydrophilic, FDA, CE, 50 pc/PAK (Millipore, catalog number: MPSLGPM33RS)
Eppendorf® Safe-Lock microcentrifuge tubes, 1.5 mL (Sigma, catalog number: T9661)
45 units of papain (Worthington, catalog number: PAPL LS003119)
Hanks' Balanced Salt Solution (HBSS), calcium, magnesium (Gibco, catalog number: 24020-117), store at 4 °C
LipofectamineTM 2000 transfection reagent (Invitrogen, catalog number: 11668030), store at 4 °C
Brefeldin A solution (1,000×) (BioLegend, catalog number: 420601), 5 mg/mL, store at 4 °C
Bungarotoxin (BTX), Alexa Fluor® 647 (AF647) conjugate (Thermo Fisher, catalog number: B35450)
Resuspend in H2O at a concentration of 1 mg/mL
Add sodium azide (Sigma, catalog number: 199931) at 2 mM
Store small aliquots at -20 °C. Avoid freezing and thawing. Protect from light
Tubocurarine hydrochloride pentahydrate (Sigma, catalog number: T2379-100MG)
Resuspend in H2O at 10 mM stock concentration
Store aliquots at 4 °C
Dynasore hydrate (Sigma, catalog number: D7693-5MG)
Resuspend in DMSO to make 80 mM stock solution, store at -20 °C.
LysoTrackerTM Green DND-26 (Invitrogen, catalog number: L7526), 1 mM, store at -20 °C
Magic Red Cathepsin-B Assay kit (ImmunoChemistry Technologies, catalog number: 937), store at 4 °C
Resuspend in 200 µL DMSO to the vial to make 250× stock, store at 4 °C.
Before each use, dilute the 250× DMSO stock in sterile H2O at 1:10 to make 25× sub-stock. Use immediately.
Thrombin from rat plasma (Sigma, catalog number: T5772-100UN), lyophilized powder
Resuspend in 1 mL to make a stock of 100 U/ml (20×), store at -20 °C.
Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D4540), ≥99.5% (GC), suitable for cell culture
Phosphate-buffered saline (PBS), pH 7.2 (Gibco, catalog number: 20012027), suitable for cell culture
Water, cell culture grade (Corning, catalog number: 15363651)
Borate buffer (5 mL) (see Recipes)
Boric acid (Fisher, catalog number: A73-500)
Borax (Sigma, catalog number: B-9876)
1 mL of 0.05% DNaseI (see Recipes)
DNaseI (Boehringer, catalog number: 10104159001, 100 mg from bovine pancreas, grade II)
MgSO4 (Fisher, catalog number: BP213-1)
HBSS (Gibco, catalog number: 24020-117) (without Pen-Strep)
Poly-L-lysine stock solution (see Recipes)
5 mg bottle of Poly-L-lysine hydrobromide, molecular weight = 300,000, lyophilized powder, gamma-irradiated, BioXtra, suitable for cell culture (Sigma, catalog number: P5899-5MG)
Dissection media (see Recipes)
HBSS (Gibco, catalog number: 24020117)
D-glucose (Sigma, catalog number: G6152-100g or G5767-500g)
HEPES (Sigma, catalog number: H4034-100g or H-7523-50g)
Pen-Strep (Gibco, catalog number: 15140148 for 20 mL)
Brain digestion enzyme (Mixture A) (see Recipes)
PBS (Gibco, catalog number: 21600069, or DPBS, catalog number: 14190-144)
DL-cysteine HCl (Sigma, catalog number: C9768)
BSA (Sigma, catalog number: A7906-100G)
D-glucose (Sigma, catalog number: G6152)
Plating media (see Recipes)
DMEM High Glucose (1×) with phenol red + L-Glut w/o Sod. Pyr. (Gibco, catalog number: 11965092)
FBS (Gibco, catalog number: 26050-070)
Growth media (see Recipes)
Neurobasal A (NBA) medium with phenol red (Gibco, catalog number: 10888022)
B-27 Supplement (50×) (Gibco, catalog number: 17504044)
GlutaMAX-I 100× (Gibco, catalog number: 35050061)
Equipment
Surgical scissors (sharp/blunt) 4–6” (Fine Science Tools, catalog number: 14028-10)
Dumont forceps Super Fine #7 (Fine Science Tools, catalog number: 11271-30)
For brain dissections: stereo microscope equipped with dual top and bottom illumination, and with 6× and 12× magnification eyepieces/objectives.
5 mL NuncTM serological pipettes (Thermo Scientific, catalog number: 170355N)
10 mL NuncTM serological pipettes (Thermo Scientific, catalog number: 170356N)
25 mL NuncTM serological pipettes (Thermo Scientific, catalog number: 170357N)
Nikon Ti-E Perfect Focus inverted fluorescence microscope, with a total internal reflection fluorescence (TIRF) setup, equipped with 100×/1.49NA objectives and Andor iXon + DU897 EM Camera
CO2 incubator for live imaging on the microscope
35 mm Dish | No. 1.5 Coverslip | 14 mm glass diameter | uncoated (MatTek, catalog number: P35G-1.5-14-C)
Biosafety cabinet Class II
SciSpin MINI microfuge BLUE, with rotor for 8 × 1.5/2.2 mL, (7,000 rpm/2,680 × g) (SciQuip, catalog numbers: SS-6050 and SS-6058)
Software
ImageJ (Fiji) software (free download from https://imagej.net/software/fiji/)
Microsoft Excel
Procedure
Hippocampal neuron plating preparation
Note: Steps 1–5 should be performed under sterile conditions in a tissue culture room and in a class II biosafety hood, equipped with a vacuum flask system and an aspirator hood.
Dilute poly-L-lysine stock solution to a working solution of 50 µg/mL. Add an appropriate amount of working solution to coat glass coverslips placed inside wells of a 24-well plate (~500 µL/well) or a 35 mm MatTek glass-bottom dish (~1 mL/well) and incubate at 37 °C for at least 2 h or overnight, a range that in our experience coats the coverslip surface for the proper adherence of hippocampal neurons.
Wash coverslips three times in sterile, cell culture grade water.
Fill each well of a 24-well plate containing single coverslips with 500 µL of Plating media (DMEM + 10% FBS) (half the final volume) and incubate at 37 °C and 5.0% CO2 until plating (see Notes #4 below).
Thaw Mixture A and DNase I. Mix 5 mL of Mixture A + 45 units of papain powder in a 15 mL Falcon tube and incubate in a 37 °C water bath for 15 min until papain dissolves completely. Then, add 1 mL of 0.05% DNase I, filter-sterilize the mixture through a 0.22 µm filter, and keep the solution in a 15 mL Falcon tube at room temperature for up to 2 h.
Pre-warm ~25 mL of the Plating media (DMEM + 10% FBS) at 37 °C and 5.0% CO2 in a 150 mm dish.
Mouse hippocampal neuron isolation and plating [adapted from Kaech and Banker (2006)]
Note: Steps 1–8 can be done on a standard laboratory bench. Steps 9–16 should be done in a class II biosafety hood, equipped with a vacuum flask system and an aspirator hood. Steps 4–8 should be done quickly, ideally in under 30 min to avoid deterioration of tissue.
Collect mouse neonates at P0 (Day 0) and up to P2 (Day 2). Keep the pups in contact with a bag of warm water or with a preheated heating pad.
Clean the dissection tools with water and soap, followed by a wash with 70% ethanol.
Dish A and B: Put ~8 mL of Dissection media into two 60 mm dishes and in a 15 mL Falcon tube inside an ice bucket (Figure 1).
Decapitate the pups with surgical scissors into Dish A, containing the Dissection media. Shake gently to clean off the blood (Figure 1A).
Peel off the skin and the skull using two Dumont forceps. Isolate the brain into Dish B (Figure 1B).
Split the brain along the midline with Dumont forceps. Remove the cerebellum on the posterior side and discard. Position the medial side of the hemisphere facing up. Remove the midbrain (ventral part) by inserting the Dumont forceps ~45° and flipping. Peel off meninges (membrane covering cortex) and blood vessels (Figure 1C).
Ensure the hippocampus (seahorse/banana shaped) is visible and intact. To isolate hippocampus, stabilize the hemisphere by spearing the base of the brain and by cutting along the top edges of the hippocampus using Dumont forceps (Figure 1C).
Figure 1. Isolation of hippocampi from mouse P0–P2 neonates for primary hippocampal cultures. (A) Decapitate; (B) Remove the brain; (C) Harvest the hippocampus (outlined by dotted lines in bottom figure). Numbers 4–7 correspond to steps in Procedure B. (adapted from https://www.thermofisher.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/isolation-of-mouse-primary-neurons.html).
Using a 10 mL serological pipette, gently transfer both hippocampi into a 15 mL Falcon tube containing Dissection media. Repeat the steps to harvest more hippocampi, if needed.
Note: Work inside a biosafety hood for the rest of the protocol.
Aspirate the Dissection media and wash the hippocampi twice in ~8 mL of cold HBSS (without antibiotics).
Note: Be careful not to aspirate hippocampi.
Aspirate HBSS. Add ~1 mL of the Brain Digestion Enzyme (5 mL of Mixture A + 45 units of papain + 1 mL of 0.05% DNaseI) per two hippocampi. Incubate for 15 min at 37 °C. Invert the tube gently by hand a couple of times every 2–5 min, so that dead cells on the outer layers detach during this step.
Note: It is critical for viability of plated neurons that temperature is 37 °C and not higher.
Aspirate the Brain Digestion Enzyme slowly and gently. Wash twice with ~6 mL of Plating media (DMEM + 10% FBS), pre-warmed at 37 °C.
Aspirate the Plating media and add back ~1.2 mL of Plating media per two hippocampi. Use a P1000 micropipette to pipette up and down 7–10 times to dissociate the hippocampi (avoid making bubbles). Let the debris sink for 3–5 min.
Pipette ~1 mL of the top part of the supernatant containing dissociated cells into a new 15 mL Falcon tube and add 5 mL of the Plating media for a total volume of 6 mL.
Plating cells in 24-well plates: pipette ~500 µL of the cells into each well of a 24-well plate containing 500 µL of pre-warmed Plating media. Mix each well carefully. The cell density plated for experiments in this protocol is two hippocampi in 12 wells of a 24-well plate, which corresponds to ~ 70,000–125,000 cells per well.
Incubate the cells at 37 °C and 5.0% CO2 (see Notes #4 below), for 1–5 h to allow attachment of the cells to the coverslips.
Gently aspirate the Plating media one well at a time using a P1000 micropipette and replace immediately with 1 mL of Growth media (NBA + B-27 + GlutaMAX) per well. This media should be pre-warmed in the incubator at 37 °C and 5.0% CO2 (see Notes #4 below). Incubate cells at 37 °C and 5.0% CO2 (see Notes #4 below).
Note: Do not let coverslips dry during media change.
Check the plate under the microscope for density, growth, and health of neurons every day. At Days in vitro (DIV) 2, neurites with growth cones should be visible in healthy neurons (Figure 2A). At DIV 4–5, axons (orange arrows) and dendrites (green arrows) are identifiable by morphology (Figure 2B). From this stage forward, neurons are ready for transfection and imaging. By DIV 9–10, axons and dendrites continue to grow and fasciculate extensively (Figure 2C). Researchers might find it difficult to discern a single axon without transfection of axonal fluorescent proteins.
Figure 2. Growth of cultured primary hippocampal neurons at (A) day in vitro (DIV) 2, (B) DIV 4, (C) DIV 9. Neurons were plated at 1:12 dilution, i.e., two hippocampi were plated in 12 wells in a 24-well plate, yielding 75,000–125,000 cells/coverslip. Images were taken with a Nikon Plan Fluor 40×/1.3NA oil objective using Differential Interference Contrast (DIC) optics. Asterisks = soma; arrows point at axons (orange arrows) or dendrites (green arrows). Scale bar = 10 μm.
Transfection of cultured mouse hippocampal neurons
Note: This protocol is modified from one suggested for the use of Lipofectamine 2000 (InvitrogenTM;https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/lipofectamine-2000.html). This protocol is for the transfection of one coverslip in a 24-well plate of neurons at DIV 5–8; adjust accordingly for the number of coverslips required. These steps should be done under a sterile biosafety hood in a tissue culture room.
Prepare two 1.5 mL Eppendorf tubes.
In tube 1, add 50 μL (volume adjusted) of Growth media + 400–800 ng of endotoxin-free purified plasmids that will be transfected. For PrP-mCherry (mCh) plasmids, we added 800 ng. Mix by flicking the tip of the Eppendorf tube and incubate for 5 min.
In tube 2, add 48 μL of Growth media + 2 μL of Lipofectamine 2000. Mix by flicking the tip of the Eppendorf tube and incubate for 5 min.
Spin tube 1 and tube 2 quickly (~5 s) in a MINI microfuge.
After 5 min of incubation, combine the diluted DNA (tube 1) with the diluted Lipofectamine 2000 (tube 2) by dropwise pipetting. Mix by flicking the tip of the Eppendorf tube and incubate the mixture at room temperature for 30 min.
Transfer 500 μL of the media from each well of the 24-well plate, where neurons have been plated for 5–8 days (DIV 5–8), into empty wells of a 24-well plate and add 500 μL of new Growth media to these wells to make a total of 1mL. This will be the media to add back to each well of neurons after transfection to allow the transfer of neurotrophic factors secreted in each well pre-transfection.
Add 100 μL of plasmid-Lipofectamine 2000 mix to each well of the 24-well plate where the neurons are plated. Incubate at 37°C and 5.0% CO2 for 2 h (see Notes #4 below).
After 2 h, replace all the media from each well containing the neurons with the Growth media prepared in step 4. Protein expression from the plasmid transfection depends on the plasmid, protein being expressed, and concentration being transfected, but could occur as early as 45 min to 1 h after transfection.
Notes:
Incubation in Lipofectamine 2000 for longer than 3–4 h will result in toxicity.
This protocol usually yields 1%–5% transfection efficiency, which is useful to observe fluorescent protein expression in single neurons and in single axons.
Brefeldin A (BFA) treatment
Note: Parts D–K will demonstrate the experimental procedures for PrPPG14-mCh expressing neurons. The same procedures apply to PrPWT-mCh (control) or any other membrane protein of interest.
Retention of PrPPG14-mCh in the ER
Prepare the transfection mixture (Lipofectamine 2000 + plasmids) as in Procedure C. In the 100 μL mixture, add 0.5 μL of 1,000× BFA solution (1:200 dilution).
Apply the transfection mixture with BFA to neurons. The final concentration of BFA per well is 5 μg/mL (1×).
Incubate for 1 h at 37 °C and 5.0% CO2 (see Notes #4 below) and replace the media with 5 μg/mL BFA for another 1–5 h (Figure 3A).
Note: Longer incubation time results in higher protein expression but more leakage from ER into other compartments.
Image the PrPPG14-mCh expression pattern in the soma and axons using live imaging. Use the same media with BFA during live imaging.
Note: Neurons can be immunostained with organelle-specific antibodies to confirm protein localization in the cellular compartments at a given time (see examples in Chassefeyre et al., 2021).
Releasing PrPPG14-mCh from the ER
Repeat steps 1a–1c from Procedure D.
After 2–6 h of 5 μg/mL BFA treatment, replace the media with new Growth media pre-warmed at 37 °C and 5.0% CO2 (see Notes #4 below; Figure 3B).
Using a live imaging microscope, image the PrPPG14-mCh expression pattern one or two days post-transfection in the soma and in axons every 10–30 min. The signal of PrPPG14-mCh should appear as big, round punctate aggregates along the length of axons, and as large punctate aggregates in the soma. To confirm that PrPPG14-mCh transits into lysosomes in the soma as observed previously (Chassefeyre et al., 2021), follow steps in Procedure H.
Note: Neurons can be immunostained with organelle-specific antibodies to confirm protein localization in the cellular compartments at a given time (see examples in Chassefeyre et al., 2021).
Figure 3. Timeline of Brefeldin A (BFA) treatment of hippocampal neurons. (A) Hold PrPPG14-mCh in the ER. (B) Release PrPPG14-mCh from the ER.
Imaging BTX-AF647-labeled surface PrP and its subsequent internalization (Figure 4)
Cloning of Bungarotoxin binding sequence (BBS) into PrP was fully described in Chessefeyre et al. (2021). In brief, a 13 amino-acid BBS tag (WRYYESSLEPYPD) was inserted at the BspEI site in the linker between mCh and the C-terminal of the PrP sequence downstream of mCh, which was shown to not alter PrP trafficking or endoggresome formation. Endotoxin-free purification of PrPPG14-mCh-BBS in MoPrP.Xho vector is recommended for transfection of primary neurons.
Transfect neurons at DIV 5-8 with PrPWT-mCh-BBS or PrPPG14-mCh-BBS following the Lipofectamine 2000 protocol outlined in Procedure C.
Two days post-transfection, treat neurons with 150 μM tubocurarine chloride diluted in Growth media and incubate for 2 h at 37 °C and 5.0% CO2 (see Notes #4 below; Figure 4B).
Note: BTX can bind to acetylcholine (ACh) receptors on the surface of neurons. Tubocurarine blocks ACh receptors and thus reduces BTX-AF647 binding and background fluorescence signal.
Replace the Growth media containing tubocurarine with Growth media containing 7 μg/mL of BTX-AF647 and incubate neurons at 37 °C and 5.0% CO2 (see Notes #4 below) for 1 h.
Wash with Growth media and image the live BTX-AF647 signal immediately or one day later.
Figure 4. (A) Schematic and (B) timeline of BTX-AF647 treatment of hippocampal cultured neurons. Schematic for the live imaging of endocytosis of PrPPG14-mCh-BBS into endocytic vesicles and/or the accumulation of PrPPG14-mCh-BBS in endoggresomes one day after transfection.
Dynasore treatment to inhibit clathrin-mediated endocytosis of PrPPG14-mCh from the neuronal cell surface
Follow Procedure E steps 1 and 2 to transfect DIV 5–8 neurons with PrPPG14-mCh-BBS. One or two days post-transfection, add 0.5 μL of 80 nM Dynasore DMSO stock to 500 μL Growth media, mix well, and apply to neurons. Incubate for 30 min at 37 °C and 5.0% CO2 (see Notes #4 below) (Figure 5A).
Replace the media with Growth media + 150 μM Tubocurarine + 80 μM Dynasore and incubate for 1 h at 37 °C and 5.0% CO2 (see Notes #4 below) (Figure 5B).
Replace the media with Growth media + 7 μg/mL BTX-AF647 + 80 μM Dynasore and incubate for 1 h at 37 °C and 5.0% CO2 (see Notes #4 below) (Figure 5B).
Replace the media with Growth media and image neurons starting immediately and then at desired timepoints up to a day later, to image PrPWT/PG14-mCh-BBS internalization and/or aggregation in endoggresomes and the requirement of clathrin-mediated endocytosis in these events (Figure 5B).
Figure 5. (A) Schematic and (B) timeline of BTX-AF647 treatment of cultured hippocampal neurons with Dynasore. Monitoring the inhibition of clathrin-mediated endocytosis on PrPPG14-mCh-BBS internalization and/or on the formation of endoggresomes within one day after PrPPG14-mCh-BBS expression.
Thrombin treatment to cleave off PrP at the cell surface
Cloning of Thrombin cleavage sequence (TCS)-tagged construct MoPrP.Xho PrPPG14-mCh-TCS was described in Chassefeyre et al. (2021). Briefly, the TCS tag (LVPRGS) was inserted at the BspEI site downstream of mCh and was shown not to alter PrP trafficking or PrPPG14-mCh endoggresome formation. Endotoxin-free purification of PrPPG14-mCh-BBS in MoPrP.Xho vector is recommended for transfection of primary neurons (Figure 6A).
Transfect neurons at DIV 5–8 with PrPPG14-mCh-BBS following the Lipofectamine 2000 protocol outlined in Procedure C (Figure 6B).
After 2 h of incubation in Growth media + Lipofectamine 2000 transfection mix at 37 °C, 5.0% CO2 (see Notes #4 below), replace media with Growth media + 5 units/mL of Thrombin protease. Incubate neurons at 37 °C, 5.0% CO2 (see Notes #4 below) up to 2 days prior to live imaging of PrPPG14-mCh endoggresome formation (Figure 6B).
Figure 6. (A) Schematic and (B) timeline of Thrombin treatment of cultured hippocampal neurons. Cleavage of PrPPG14-mCh at the TCS site and its release from the cell surface.
LysoTrackerTMassay to probe for acidic endolysosomes
Grow and transfect neurons with PrPPG14-mCh as described in Procedure C (Figure 7A).
Prepare 50 nM of LysoTrackerTM Green (or other desired LysoTracker with different fluorescent dyes) in Growth media. Incubate DIV 5–8 neurons in LysoTracker for 2 h at 37 °C and 5.0% CO2 (see Notes #4 below). Wash 2–3 times in Growth media and incubate for 30 min at 37 °C and 5.0% CO2 (see Notes #4 below) prior to live imaging. Do not wait longer than 30 min as LysoTrackerTM signal might grow fainter with time (Figure 7B).
Live image LysoTrackerTM Green signal using a microscope equipped with a 488 nm laser (Figure 7B).
Notes:
LysoTrackerTM Green: optimal excitation wavelength is around 504 nm and optimal emission around 511 nm.
LysoTrackerTM fluorescence signal could be weak (Chassefeyre et al., 2021). Image LysoTrackerTM-treated neurons following the recommendations of vendors, which in our protocols is within 30 min to 1 h post-treatment of cells with LysoTrackerTM. This will circumvent the photobleaching that can occur after longer periods of LysoTrackerTM treatment. Imaging using decreased laser power aids in avoiding photobleaching during image acquisition, as well as imaging using other microscopes, e.g., a lattice light sheet microscope. Ideally, this assay should be combined with other lysosomal protein markers (such as LAMP1) or Magic Red® (see Procedure Part I).
Magic Red® Cathepsin B assay to probe the activity of Cathepsin B enzyme in degradative lysosomes (Figure 7)
Grow and transfect neurons with PrPPG14-mCh as described in Procedure C.
Dilute 250× DMSO stock of Magic Red® in sterile H2O. Apply 10 μL into each well of a 24-well plate with plated DIV 5–8 neurons, containing 250 μL of Growth media. Incubate for 2 h at 37 °C and 5.0% CO2 (see Notes #4 below) (Figure 7B).
Wash wells containing Magic Red® 2–3 times with Growth media. Incubate for 30 min at 37 °C and 5.0% CO2 (see Notes #4 below) prior to live imaging (Figure 7B).
Live image Magic Red® signal using a fluorescence microscope equipped with a 561 nm laser (Figure 7B).
Notes:
Magic Red® has an excitation wavelength of 592 nm and an optimal emission of 628 nm.
LysoTrackerTM Green and DeepRed Magic Red® can be combined in the same experiment to cross-validate the observation that degradative and acidic endolysosomal organelles are present in the soma and in axons.
Figure 7. (A) Schematic and (B) timeline of LysoTrackerTM or Magic Red® Cathepsin B Assays. Probing for PrPPG14-mTagBFP2 in degradative endolysosomes.
Image acquisition and axon identification
Pre-warm the incubation chamber on the microscope until the temperature reaches a steady 37 °C. Prepare ahead of time the CO2 chamber.
Live imaging of primary neurons grown on #1.5 glass coverslips in 24-well plates is done on 35 mm MatTek dishes. Add a mixture of ~500 μL of new Growth media and ~500 μL of old Growth media from the culture well to each 35 mm MatTek dish and keep dish at 37 °C and 5.0% CO2 (see Notes #4 below) prior to placing the coverslip inside (step 3).
Using Dumont tweezers with an angled fine tip, pick up the coverslip from the edge. Using care not to drop the coverslip, flip it with the cells side down onto the bottom of the MatTek dish, inside the glass bottom area.
Mount the MatTek dish on the microscope stage within a 37 °C and 5.0% CO2 environment. Let the cells stabilize to that environment for 10–15 min prior to imaging.
Using 60× or 100× objectives with high NA, localize the transfected cells using widefield or fluorescence illumination. Distinguish dendrites vs. axons based on neuronal morphology (Encalada et al., 2011). Find the isolated axon region and acquire images.
Note: Unless otherwise indicated, the axon region imaged is of a mid-axon region, defined as a location at least ~200 μm away from the soma and from the axon termini.
Data analysis
Quantitation of PrPPG14-mCh endoggresome densities in axons using ImageJ
Subtract the background fluorescence from each of the raw images acquired: in ImageJ go to Process – Subtract background, adjust rolling ball radius using ‘preview’ (try rolling ball radius default = 50).
Draw an ROI using the segmented line tool as an outline on top of the axon (Figure 8). Adjust the line width to cover the axon width, which will depend on the width of the axon being analyzed (example below, line width = 1).
Figure 8. Subtraction of background fluorescence from images of axons of neurons expressing PrPPG14-mCh. Screenshot showing the windows in ImageJ with representative images of an axon of a cultured hippocampal mouse neuron expressing PrPPG14-mCh after background subtraction (left), and the window of the same axon image with the segmented line ROI drawn to outline the axon (right). Use brightness and contrast (B&C) tool to help visualize the axon contour.
To identify PrPPG14-mCh endoggresomes:
In ImageJ, go to Analyze – plot profile. Following manual inspection of hundreds of images (Chassefeyre et al., 2021), we defined endoggresomes within axons, as PrPPG14-mCh fluorescence puncta having an intensity of at least five times the intensity of small PrPPG14-mCh vesicles (Figure 9; see Notes #5).
Figure 9. Plot profile of PrPPG14-mCh fluorescent puncta in the axon from Figure 8. A plot profile of fluorescence intensities of the axon in Figure 8, obtained from the drawing of the segmented line ROI feature in ImageJ. The y-axis indicates the fluorescence intensity (gray values) in arbitrary units (AUs) from PrPPG14-mCh point sources along the axon. Following visual examination of fluorescence intensity averages, a threshold was set at AUs = 500 to designate the smallest peaks representing signals from PrPPG14-mCh vesicles. A threshold was then set at five times the vesicle intensity of 500 AUs, to designate these as PrPPG14-mCh endoggresomes. Densities of PrPPG14-mCh endoggresomes are then calculated by manual counts of the peaks. The definitions of thresholds are further described in detail in Chassefeyre et al. (2021).
Calculate PrPPG14-mCh endoggresome densities for each axon image, as the number of peaks above threshold intensity (Figure 9) per length of axon. The length of the analyzed axon fragment is equal to the length of the segmented line (see step 2). Divide the number by length × 100 to get aggregate density/100 μm. Analyze only one axon region per neuron; this will be Naxons.
Statistical analysis: If the dataset is normally distributed, display means ± SEM and perform Student’s t-test or ANOVA/multiple comparison tests as appropriate. If the dataset is not normally distributed, display boxplot, violin plot, or all data points and use non-parametric tests, such as Wilcoxon Rank Sum test or an appropriate multiple comparison test.
Co-transport analysis in axons using ImageJ
This protocol is for the analysis of co-transport of a non-PrP cargo (in this case of Lamp1-EGFP endolysosomes), and of PrPPG14-mCh vesicles or endoggresomes moving (or not) along axons of culture mouse hippocampal neurons.
Co-transfect neurons with desired plasmids following the steps in Procedure C. In this case, neurons were co-transfected with PrPPG14-mCh (400 ng) and Lamp1-EGFP (400 ng). Collect images for both PrPPG14-mCh and Lamp1-EGFP channels, as indicated in Procedure J.
Open an image in ImageJ using split channels. Subtract background fluorescence from each raw image as indicated in step A above (Figure 8, left).
Draw ROI with segmented line tool onto the axon of the first channel as indicated in step A above (Figure 8, right). Adjust the line width to cover the axon width, which will depend on the width of the axon being analyzed (example below, line width = 1).
In ImageJ, go to Image – Stacks – Reslice [/]…This step will draw a kymograph showing the trajectories of Lamp-1-EGFP particles (x-axis) over time (y-axis) (Figure 10).
Figure 10. Transport of Lamp1-EGFP vesicles in axons of cultured hippocampal neurons. Screenshot of ImageJ showing a representative image of a kymograph of an axon showing the trajectories of Lamp1-EGFP moving or stationary vesicles. Movies were 5 min long and collected at a frame rate of 1 frame/s (1 Hz) using a Nikon TIRF microscope. For all images and movies acquired, the exposure time was set to 100 ms (Chassefeyre et al., 2021).
Transfer the ROI drawn from the Lamp1-EGFP image to the PrPPG14-mCh image using command-E or using ROI manager in ImageJ.
In ImageJ, go to Image – Stacks – Reslice [/]… This step will draw a kymograph showing the trajectories of PrPPG14-mCh particles (x-axis) over time (y-axis) (Figure 11).
Figure 11. Transport of PrPPG14-mCh vesicles in axons of cultured hippocampal neurons. Screenshot of ImageJ showing a representative image of a kymograph of an axon showing trajectories of PrPPG14-mCh moving or stationary vesicles. Movies were 5 min long and collected at a frame rate of 1 frame/s (1 Hz) using a Nikon TIRF microscope. For all images and movies acquired, the exposure time was set to 100 ms (Chassefeyre et al., 2021).
Draw lines on kymographs to annotate all identifiable tracks on both channels (Figure 12, left and middle panels). Use brightness and contrast (B&C) tool to help visualize dim tracks.
Note: The designation of trajectories/tracks, including of those that are dim in fluorescence, is determined empirically by the user. Contrast settings can be adjusted to visualize all tracks, if necessary, but it is advised to use consistency in the designation of all tracks including dim ones.
Copy and paste tracks onto the same image to visualize overlapping tracks (Figure 12, right panel).
Figure 12. Analysis of kymographs of PrPPG14-mCh vesicle movement. Representative kymograph images of PrPPG14-mCh (left panel, red), and Lamp1-EGFP (middle panel, green) vesicle tracks/trajectories. Combined (right panel, yellow) tracks/trajectories indicate co-transport events.
Count percentage of co-transport trajectories per kymograph by calculating number of co-transport tracks divided by number of all tracks (of PrPPG14-mCh) × 100. This indicates the degree of PrPPG14-mCh vesicles/endoggresomes co-transport with a Lamp1-EGFP vesicle. Record the numbers in Microsoft Excel. Analyze only one kymograph per axon of a neuron.
Perform statistical analysis. If the dataset is normally distributed, display means ± SEM and perform Student’s t-test or ANOVA/multiple comparison tests as appropriate. If the dataset is not normally distributed, display boxplot, violin plot, or all data points and use non-parametric tests, such as Wilcoxon Rank Sum test or appropriate multiple comparison tests.
Notes
Collect the dataset for three biological replicates, i.e., primary hippocampal neurons plated from three different mice of the same genetic background, plated independently at different time points.
Co-transport analysis requires an appropriate temporal resolution of cargo movement. For example, to image the transport of PrPWT-mCh or PrPPG14-mCh vesicles, a suitable acquisition rate is 10 frames/s for 15 s. In contrast, to image the movement of PrPPG14-mCh endoggresomes, which are largely stationary or move very slowly, use 1 frame/s for 5 min. Consult previous literature on different dynamics of organelles or membrane-bound compartments to get the best time-lapse video quality for quantitative analysis.
Imaging in the cell body requires deconvolution for widefield imaging or a confocal module. In axons, widefield or pseudo-TIRF is sufficient for quantitative imaging, due to little out-of-focus light in thin axonal regions.
For our experiments, incubators for culturing hippocampal mouse neurons were set at 5.5% CO2 to ascertain that small observed fluctuations in CO2 levels from the house lines did not result in CO2 levels dropping below 5.0%, which is the standard level used for hippocampal neuronal culturing (Kaech and Banker, 2006). It is known that mouse hippocampal neurons can grow in >5% CO2 (Geissler et al., 2013). During imaging in the TIRF microscope, CO2 was supplied by a tank that provided continuous stable delivery of 5.0% CO2 directly to the microscope incubation/imaging chamber.
The threshold of gray value for small vesicles and endoggresomes is determined empirically and independently for every image/individual axon.
Recipes
Borate buffer (5 mL)
1.24 g boric acid
1.90 g borax
Dissolve in 400 mL of ddH2O, pH 8.5, filter-sterilize, and store at 4 °C.
1 mL of 0.05% DNaseI
50 mg of DNaseI
0.5 mL of 1.2 M MgSO4
100 mL of HBSS (without pen-strep)
Poly-L-lysine stock solution
Reagent Final concentration Amount
Poly-L-lysine (1 mg/mL) 0.05 mg/mL 0.5 mL
Borate buffer (pH 8.5) n/a 9.5 mL
Total n/a 10 mL
Dissection media
Adjust pH to 7.3, filter-sterilize, and store at 4 °C.
Reagent Final concentration Amount
HBSS n/a 500 mL
D-glucose
HEPES
Pen-Strep
n/a
n/a
n/a
0.4 g
0.834 g
5 mL
Total n/a 505 mL, pH 7.3, filter-sterilize
Brain digestion enzyme (Mixture A)
Filter-sterilize and store aliquots at -20 °C.
Reagent Final concentration Amount
PBS
DL-cysteine HCl
BSA
D-glucose
n/a
n/a
n/a
n/a
4 mL
1 mg
1 mg
25 mg
Total n/a 5 mL
Plating media
500 mL of DMEM High Glucose (1×) with phenol red + L-Glut w/o Sod. Pyr.
50 mL of FBS
Store at 4 °C and pre-warm to 37 °C before plating.
Reagent Final concentration Amount
DMEM n/a 28 mL/brain
Total n/a 28 mL
Growth media
Store at 4 °C and pre-warm to 37 °C before plating
Reagent Final concentration Amount
NBA n/a 48.875 mL
GlutaMAX
B27
n/a
n/a
0.125 mL
1 mL
Total n/a 50 mL
Acknowledgments
We thank Romain Chassefeyre and co-authors of the original research paper (Chassefeyre et al., 2021) for performing experiments. This work was supported by NIH/NIA R01AG049483 and R01AG076745 grants; by the Glenn Foundation for Medical Research Award for Research in Biological Mechanisms of Aging; by a New Scholar in Aging Award from the Lawrence Ellison Foundation; and by a Baxter Family Foundation award to S.E.E. T.C. was supported by a Thai Government Scholarship from the Development and Promotion of Science and Technology Talents Project (DPST). A.V. and Y.W were supported by a Dorris Neuroscience Center Fellowship.
Competing interests
The authors declare no competing interests.
Ethics
Our mouse protocols were reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at The Scripps Research Institute (Scripps Research) and all colonies were maintained following the guidelines recommended by the Department of Animal Resources (DAR) at Scripps Research.
References
Chassefeyre, R., Chaiamarit, T., Verhelle, A., Novak, S. W., Andrade, L. R., Leitao, A. D. G., Manor, U. and Encalada, S. E. (2021). Endosomal sorting drives the formation of axonal prion protein endoggresomes. Sci Adv 7(52): eabg3693.
Encalada, S. E., Szpankowski, L., Xia, C. H. and Goldstein, L. S. (2011). Stable kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles. Cell 144(4): 551-565.
Geissler, M., Gottschling, C., Aguado, A., Rauch, U., Wetzel, C. H., Hatt, H. and Faissner, A. (2013). Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation. J Neurosci 33(18): 7742-7755.
Hofmann, I. and Munro, S. (2006). An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility. J Cell Sci 119(Pt 8): 1494-1503.
Kaech, S. and Banker, G. (2006). Culturing hippocampal neurons. Nat Protoc 1(5): 2406-2415.
Khatter, D., Raina, V. B., Dwivedi, D., Sindhwani, A., Bahl, S. and Sharma, M. (2015). The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes. J Cell Sci 128(9): 1746-1761.
Satpute-Krishnan, P., Ajinkya, M., Bhat, S., Itakura, E., Hegde, R. S. and Lippincott-Schwartz, J. (2014). ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory pathway. Cell 158(3): 522-533.
Zavodszky, E. and Hegde, R. S. (2019). Misfolded GPI-anchored proteins are escorted through the secretory pathway by ER-derived factors. Elife 8: e46740
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4,652 | https://bio-protocol.org/en/bpdetail?id=4652&type=0 | # Bio-Protocol Content
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Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6
Tatsuki Kunoh
EO Erika Ono
TY Tatsuya Yamamoto
IS Ichiro Suzuki
MT Minoru Takeda
NN Nobuhiko Nomura
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4652 Views: 958
Reviewed by: Alberto J. Martin-RodriguezGonzalo Durante-Rodríguez Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Applied and Environmental Microbiology Dec 2022
Abstract
Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes.
Graphical overview
Keywords: Leptothrix cholodnii SP-6 Gene transfer Conjugation Escherichia coli S17-1 Calcium depletion
Background
Leptothrix species are microorganisms with a long history as research targets. The type strain, L. ochracea, was reported in the late 18th century (Spring, 2006) and, since then, has attracted much attention from researchers in the fields of microbiology and environmental science. Leptothrix ubiquitously inhabits seeps, streams of iron-rich groundwater, and activated sludge. Leptothrix divides to produce cell chains encased with entangled nanofibrils termed sheaths and eventually generates mats composed of porous networks of cell chains (Spring, 2006). Over the time of mat aging, sheaths are encrusted with inorganic particles, especially iron oxide, resulting in the production of rigid microtubes (Furutani et al., 2011). Focusing on this innate characteristic, Leptothrix has been utilized for the removal of metal contaminations such as iron and manganese from groundwater in water purification plants (Kunoh et al., 2015a). However, overgrowth of filamentous bacteria, including Leptothrix, often results in clogging of water distribution pipes and bulking of activated sludge in wastewater treatment plants (van der Waarde et al., 2002). Therefore, understanding the functions of key proteins responsible for nanofibril formation and entanglement is significant for the artificial control of their filamentous growth.
The next-generation sequencing technologies that have been developed recently enable us to freely access significant data of the whole genome sequences existing in a wide range of organisms, from prokaryotes to eukaryotes. For Leptothrix species, the complete genome sequence of L. cholodnii SP-6 (hereafter SP-6) was published by the US DOE Joint Genome Institute (Copeland et al., 2008) [GenBank (accession: GCA_000019785.1)], which opened new doors for understanding the gene functions and interactions of this strain.
To date, a gene replacement system for Leptothrix species remains unpublished, except for the sheath-less strain L. discophora SS1 (Bocioaga et al., 2014). Although we attempted to perform gene transfer of SP-6 mediated by conjugation with Escherichia coli S17-1 by following their report, the efficiency of gene replacement was quite low relative to that of SS1. We therefore considered the possibility that the sheath structure of SP-6 might interfere with the conjugation with E. coli cells, and eventually the incorporation of extracellular DNA into SP-6 cells.
We previously reported that sheath structure plays a critical role in holding aligned cell chains of SP-6 (Kunoh et al., 2020), and that calcium depletion frequently results in breakage of cell filaments of SP-6 due to failure in the production of sheath structures encasing the cell chains (Kunoh et al., 2021). From these findings, we hypothesized that preculturing SP-6 cells in a calcium-lacking medium might enhance the accessibility of E. coli cells to SP-6 cells for conjugation, which results in efficient gene transfer and subsequent gene replacement of SP-6 cells.
Here, we describe a protocol for gene replacement in SP-6 covered with sheaths (Graphic abstract), which was modified from that reported previously (Yoshihara, 2002). This protocol is expected to contribute to efficient systematic genetic analyses of SP-6, including genes responsible for sheath formation (Kunoh et al., 2022).
Materials and Reagents
Chemical compounds
Kanamycin sulfate (FUJIFILM Wako Pure Chemical Co., catalog number: 117-00341)
Ampicillin sodium salt (FUJIFILM Wako Pure Chemical Co., catalog number: 012-23303)
Rifampicin (Nacalai Tesque Inc., catalog number: 30259-94)
Ammonium sulfate [(NH4)2SO4] (Nacalai Tesque Inc., catalog number: 02633-15)
Magnesium sulfate heptahydrate (MgSO4·7H2O) (FUJIFILM Wako Pure Chemical Co., catalog number: 131-00405)
Calcium chloride dihydrate (CaCl2·2H2O) (Nacalai Tesque Inc., catalog number: 031-00435)
Potassium dihydrogen phosphate (KH2PO4) (Nacalai Tesque Inc., catalog number: 28721-55)
Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Nacalai Tesque Inc., catalog number: 31725-15)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai Tesque Inc., catalog number: 17514-86)
Iron(II) sulfate (FeSO4) (FUJIFILM Wako Pure Chemical Co., catalog number: 094-02942)
Sodium pyruvate (FUJIFILM Wako Pure Chemical Co., catalog number: 191-03061)
Sodium hydroxide (NaOH) (Nacalai Tesque Inc., catalog number: 31511-05)
Sodium chloride (NaCl) (Nacalai Tesque Inc., catalog number: 31319-45)
Hexadecyltrimethylammonium bromide (CTAB) (Nacalai Tesque Inc., catalog number: 07906-82)
Sodium dodecyl sulfate (SDS) (Nacalai Tesque Inc., catalog number: 30400-85)
Phenol/chloroform/isoamyl alcohol (25:24:1) (FUJIFILM Wako Pure Chemical Co., catalog number: 311-90151)
Chloroform (Nacalai Tesque Inc., catalog number: 08401-65)
Isopropanol (Nacalai Tesque Inc., catalog number: 29113-95)
Ethanol (Nacalai Tesque Inc., catalog number: 14713-95)
Sodium acetate trihydrate (CH3COONa·3H2O) (Nacalai Tesque Inc., catalog number: 31115-05)
Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) (Nacalai Tesque Inc., catalog number: 35434-21)
Ethylenediaminetetraacetic acid tetrasodium salt (EDTA-4Na) (FUJIFILM Wako Pure Chemical Co., catalog number: 349-01885)
35% hydrochloric acid (HCl) (Nacalai Tesque Inc., catalog number: 18321-05)
Acetic acid (Nacalai Tesque Inc., catalog number: 00212-85)
Polyethylene glycol 8000 (PEG8000) (MP Biomedicals LLC., catalog number: 195445)
Glycerol (Nacalai Tesque Inc., catalog number: 17017-35)
Dimethyl sulfoxide (DMSO) (Nacalai Tesque Inc., catalog number: 13407-45)
Potassium chloride (KCl) (Nacalai Tesque Inc., catalog number: 28514-75)
Magnesium chloride (MgCl2) (Nacalai Tesque Inc., catalog number: 20935-05)
d-glucose (Nacalai Tesque Inc., catalog number: 16805-35)
Agarose S (FUJIFILM Wako Pure Chemical Co., catalog number: 312-01193)
1 kb Plus DNA ladder (Thermo Fisher Scientific, catalog number: 10787018)
Ethidium bromide solution (10 mg/mL) (Nacalai Tesque Inc., catalog number: 14631-94)
Green nucleic acid stain (Midori Green Advance, Nippon Genetics Co. Ltd., catalog number: NE-MG04)
Liquid nitrogen (Air Water Inc., YJ code number: 799070CX1020)
Culture media
BD Difco LB broth, Lennox (Thermo Fisher Scientific, catalog number: 240230)
BD Bacto agar (Thermo Fisher Scientific, catalog number: 214010)
BD Bacto yeast extract (Thermo Fisher Scientific, catalog number: 212750)
BD Bacto tryptone (Thermo Fisher Scientific, catalog number: 211705)
BD Difco nutrient broth (Thermo Fisher Scientific, catalog number: 234000)
Vitamins
d-biotin (Nacalai Tesque Inc., catalog number: 04822-04)
Folic acid (Nacalai Tesque Inc., catalog number: 16221-91)
Thiamine hydrochloride (HCl) (Nacalai Tesque Inc., catalog number: 36319-24)
d-calcium pantothenate (Nacalai Tesque Inc., catalog number: 26004-44)
Vitamin B12 (Nacalai Tesque Inc., catalog number: 36323-96)
Riboflavin (Nacalai Tesque Inc., catalog number: 36322-51)
Nicotinic acid (Nacalai Tesque Inc., catalog number: 24326-52)
Pyridoxine hydrochloride (Nacalai Tesque Inc., catalog number: 29611-81)
p-aminobenzoic acid (Nacalai Tesque Inc., catalog number: 01930-32)
Enzymes
Proteinase K (FUJIFILM Wako Pure Chemical Co., catalog number: 166-28913)
Ribonuclease A (FUJIFILM Wako Pure Chemical Co., catalog number: 182-01493)
Lysozyme from egg white (FUJIFILM Wako Pure Chemical Co., catalog number: 122-02673)
Tks Gflex DNA polymerase (Takara Bio Inc., catalog number: R060A)
PrimeStar Max DNA polymerase (Takara Bio Inc., catalog number: R045A)
FastDigest restriction enzymes BamHI, HindIII, and SmaI (Thermo Fisher Scientific, catalog numbers: FD0054, FD0504, FD0663)
Kits
Gel Loading Buffer (10×) (Thermo Fisher Scientific, catalog number: 10816015)
Gel Extraction kit (Promega Co., Wizard® SV Gel and PCR Clean-Up System, catalog number: A9282)
Plasmid Extraction kit (Merck KGaA, GenEluteTM Plasmid Miniprep Kit, catalog number: PLN350-1KT)
DNA Ligation kit <Mighty Mix> (Takara Bio Inc., catalog number: 6023)
Materials
50 mL polypropylene tube (IWAKI AGC Techno Glass Co., Ltd., catalog number: 2345-050)
15 mL polypropylene tube (IWAKI AGC Techno Glass Co., Ltd., catalog number: 2425-015)
1.5 mL centrifuge tube (Midland Scientific, catalog number: 311-08-052)
PCR microtube (Bio Regenerations Co., Ltd., catalog number: BRG-DC-01)
ϕ 90 × 15 mm sterilized dish (RIKAKEN Holdings Co., Ltd, catalog number: RSU-SP915-2)
10 and 30 mL syringes (TERUMO Co., Ltd., catalog numbers: SS-10ELZ, SS-30ESZ)
25 G × 1 inch injection needle (TERUMO Co., Ltd., catalog number: NN-2525R)
Stericup quick release-HV sterile vacuum filtration system (Merck Millipore, catalog number: S2HVU05RE)
MF-Millipore membrane filter, 0.22 µm pore size (Merck Millipore, catalog number: GSWP04700)
Toothpicks (e.g., Yanagi Products Co., Ltd., DN Tsumayouji L)
0.22 μm syringe filter (Sartorius AG, catalog number: S7597-FXOSK)
Vacuum filtration device (Merck Millipore, catalog number: SCGPS02RE)
2.0 mL stock tube (Sarstedt AG, catalog number: 72.694.107.03)
IWAKI 20 mL glass test tube (ϕ 16.5 × 165 mm) (AGC Techno Glass Co., Ltd., catalog number: TEST16.5NP)
Test-tube closures (ϕ 17–18 mm) (LÜDI SWISS AG, catalog number: 08418)
Erlenmeyer flask (200 mL) (AGC Techno Glass Co., Ltd., catalog number: 4980FK200)
Silicon stopper (Shin-Etsu Polymer Co., Ltd., catalog number: T-38)
100–1,000 mL medium bottles (AGC Techno Glass Co., Ltd., catalog numbers: 1516/04D, 1516/08D, 1516/10D)
Cuvette for electroporation (2 mm gap) (NEPA GENE Co. Ltd., catalog number: EC-002S)
Bacterial strains
Leptothrix cholodnii SP-6 (ATCC strain 51168)
Bacillus subtilis 168 (Lab stock)
Escherichia coli S17-1 (Lab stock)
Escherichia coli DH5α (Lab stock)
Escherichia coli SN1187 [National BioResource Project – E. coli Strain (National Institute of Genetics, Mishima, Japan)]
Plasmids
pSUP5011::Tn5-mob (Lab stock, DSMZ collection 5167)
pUC18 (Takara Bio Inc., catalog number: 3218)
Prepared recipes (see Recipes below)
MSVP (ATCC 1917) medium
LB medium
SOC medium
NB medium
1 M MgSO4 and 1 M MgCl2 solutions
1 M glucose solution
NaCl solutions (0.9%, 5 M)
TE buffer
TEN buffer
10% SDS solution
CTAB/NaCl solution
3 M sodium acetate (CH3COONa) solution
50× TAE buffer
2× TSS solution
TSS solution
50% glycerol solution
Rifampicin solution
Kanamycin solution
Ampicillin solution
Equipment
Reciprocating shakers (TAITEC Co., Ltd., models: personal-11 SM, BR-41FM)
Centrifuges (Eppendorf AG., models: 5430R, 5425R)
Thermostat (Yamato Scientific Co., Ltd., model: DG400)
Spectrophotometer (ERMA INC., model: MP-1200)
Water purification systems (ELGA LabWater, model: PURELAB Chorus 1 Complete, PURELAB Chorus 2+)
pH meter (HORIBA, Ltd., model: F-74)
Electronic scale (A&D Co., Ltd., model: HR-202i)
Magnetic stirrer with hot plate (Kenis, model: MI10 33220962)
Autoclave (TOMY SEIKO Co., Ltd., model: LBS-245)
Safety cabinet (DALTON Co., Ltd., model: Class 2 A1)
Thermal cycler (BIO-RAD, model: T-100)
Microwave (for example, Sharp Co., Ltd., model: RE-21G)
Submarine electrophoresis system (Mupid.com, model: Mupid-2plus)
Gel documentation system (ATTO Co., model: WSE-5400 Printgraph Classic)
Heat block incubator (Anatech Co., Ltd., model: 5520a)
NanoDrop UVVis spectrophotometer (Thermo Fisher Scientific, model: 2000c)
Mixer (Vortex Co., Ltd., model: VORTEX-GENIE 2)
Adjustable air displacement pipette (Gilson Inc. model: PIPETMAN P series)
Electroporation system (Bio-Rad Laboratories Inc., model: Genepluser Xcell)
-30 freezer (for example Nihon Freezer Co., Ltd., model: GS-5210HC)
-80 freezer (for example Thermo Fisher Scientific, model: TSX600G)
LED illuminator (Optocode Co., Ltd., model: LED-EXTA/GFP＋RFP)
Ceramic tweezer (ASONE, catalog number: 7-166-03)
Microscope (for example Carl Zeiss, model: Axio observer Z1)
Software
ApE (Davis and Jorgensen, 2022) used as a DNA plasmid sequence editor
R software version 4.2.2 [R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (https://www.R-project.org/)] used for statistics
Procedure
Preparation of genomic DNA from SP-6 cells
This protocol was essentially described in Kunoh et al. (2015b).
Pick SP-6 cells from a glycerol stock with a sterilized toothpick, spread on MSVP agar plate, and incubate for 3–5 days at room temperature (RT).
Pick several colonies of SP-6 cells on an MSVP plate with a sterilized toothpick, inoculate into 25 mL of MSVP liquid medium in a 50 mL polypropylene tube, and incubate for 2–3 days at RT by shaking at 70 rpm.
Collect exponentially growing SP-6 cells (total 50–100 mL) by centrifugation at 7,000 × g for 5 min.
Wash the resultant cell pellet twice in 25 mL of TE buffer, resuspend in 5 mL of TE buffer containing 20 μg/mL proteinase K and 0.6% SDS, and transfer to a 15 mL polypropylene tube.
After incubation at 65 for 1 h, add 1 mL of 5 M NaCl and 800 μL of CTAB/NaCl solution to the cell suspension and incubate at 37 °C for one additional hour by shaking at 190 rpm.
To eliminate proteins, add an equal volume (~7 mL) of phenol/chloroform/isoamyl alcohol (25:24:1) to the cell suspension, mix by inversion, and centrifuge at 7,000 × g for 15 min.
Transfer the resultant aqueous phase to a new 15 mL polypropylene tube and perform chloroform extraction as described in step A5.
For precipitation of nucleic acids, transfer the resultant supernatant to a new 15 mL polypropylene tube, add an equal volume (~5 mL) of isopropanol, and incubate at -20 °C for 1 h.
Then, collect the pellet by centrifugation (7,000 × g, 30 min) and discard the supernatant, followed by rinsing the pellet in ~5 mL of 70% ethanol.
After drying up the pellet at RT for 15 min, resuspend it in 500 μL of TE buffer containing 20 μg/mL ribonuclease A by pipetting, transfer it to a 1.5 mL centrifuge tube, and incubate the suspension at 37 °C for 30 min.
To remove ribonuclease A from the suspension, add an equal volume (~500 μL) of phenol/chloroform/isoamyl alcohol (25:24:1), mix by inversion, and centrifuge (15,000 × g, 30 min). Then, transfer the resultant aqueous phase to a new 1.5 mL centrifuge tube. Repeat this step once more.
For precipitation of nucleic acids, add an equal volume (~500 μL) of ethanol and 1/10 volume (~50 μL) of 3 M CH3COONa solution to the resultant aqueous phase.
After incubation at -20 °C for ~30 min, centrifuge the mixture (15,000 × g, 30 min) and remove the supernatant, followed by rinsing the pellet in 70% ethanol.
After drying at RT for 5 min, suspend the resultant pellet in ~100 μL of TE buffer. Then, determine DNA concentration of the suspension by using a NanoDrop 2000c.
Preparation of genomic DNA from B. subtilis 168 cells
Pick B. subtilis 168 cells from a glycerol stock with a sterilized toothpick, spread on LB agar plate, and incubate at 37 °C overnight.
Inoculate a single colony into 4 mL of LB medium in a test tube and incubate by shaking at 37 °C overnight.
Transfer cell culture to 1.5 mL centrifuge tubes and harvest the cells by centrifugation (20,000 × g, 1 min).
Resuspend the resultant cell pellet in 300 μL of TEN buffer containing 10 μg/mL ribonuclease A and 30 μg/mL lysozyme and incubate at RT for 20 min.
To lyse the cells, add 9 μL of 10% SDS solution to the cell suspension, mix gently by inversion, and incubate at 50 °C for 10 min.
Add 200 μL of TEN buffer and 500 μL of phenol/chloroform/isoamyl alcohol (25:24:1) to the cell lysate, mix gently by inversion, and centrifuge at 20,000 × g for 10 min.
Transfer the upper aqueous phase to a new centrifuge tube, add 1 mL of ethanol, and mix by inversion, followed by centrifugation at 20,000 × g for 10 min.
Discard the supernatant, add 1 mL of 70% ethanol for rinsing, and centrifuge at 20,000 × g for 5 min.
Discard the supernatant, dry the DNA pellet, and resuspend it in 200 μL of TE buffer. Dilute approximately 10-fold by TE buffer and use the resultant DNA solution as a template for PCR.
Construction of plasmids pUC18-Kmr and pUC18-mob
To construct pUC18-Kmr, digest 20–50 ng of pSUP5011 and pUC18 with SmaI and HindIII at 37 °C for 30 min. To construct pUC18-mob, digest these plasmids with BamHI.
Purify the insert DNAs from pSUP5011 (1.3 kb HindIII-SmaI and 1.8 kb BamHI fragments, respectively) and digest pUC18 by agarose gel extraction.
Clone the purified insert DNA into pUC18 by using DNA Ligation kit according to the manufacturer’s instructions.
Transform E. coli DH5α cells with the ligation mixtures by electroporation, spread the cells on LB plates containing 50 μg/mL ampicillin sodium salt, and incubate at 37 °C overnight.
Pick the resultant single colonies, re-streak them on LB agar with 50 μg/mL ampicillin sodium salt, and incubate at 37 °C overnight.
Inoculate the resultant colonies into 4 mL of LB medium containing 50 μg/mL ampicillin sodium salt in test tubes and incubate by shaking at 37 °C overnight.
Harvest the cells by centrifugation (20,000 × g, 1 min) and extract the plasmid DNA (pUC18-mob and pUC18-Kmr) using the GenEluteTM Plasmid Miniprep kit according to the manufacturer’s instructions.
Confirm the plasmid DNAs by digesting the restriction enzymes.
Construction of the plasmid pUC18-mob-ΔLcho_0008::Kmr for Lcho_0008 gene deletion (see Figures 1 and 2)
Standard overlap extension PCR, DNA extraction from agarose gel, and iVEC3 system are described in Sections H, G, and I, respectively.
Figure 1. An overview of plasmid construction (see Kunoh et al. 2022). To construct a plasmid for the gene replacement of Lcho_0008 with the kanamycin resistance gene (Kmr), we first inserted fragments containing Kmr and mob from pSUP5011 into pUC18, respectively. Then, the upstream and downstream of Lcho_0008 (approximately 1.5 kb) and the spoVG terminator (approximately 0.1 kb) were amplified using genomic DNAs isolated from L. cholodnii SP-6 and B. subtilis 168, respectively. In parallel, Kmr was amplified using the pUC-Kmr plasmid as a template. These four fragments were stepwise joined by overlap extension PCRs (also see Figure 2) and cloned into the pUC-mob plasmid by the iVEC3 system, resulting in construction of the plasmid pUC18-mob-ΔLcho_0008::Kmr. Now, the resultant plasmid can be used not only for the gene replacement of Lcho_0008 but for the amplification of the Kmr-spoVG terminator cassette as the template to construct other plasmids, such as pUC18-mob-ΔLcho_3510::Kmr. Lcho_0008_5′ and Lcho_0008_3′, and Lcho_3510_5′ and Lcho_3510_3′ represent upstream and downstream fragments of these genes, respectively. spoVG represents the spoVG terminator.
Figure 2. An overview of overlap extension PCR. The amplified PCR products contain overlapping 40 bp regions at both ends, whose Tm values were designed to be 65–76 °C (Ave. 70). These overlapping 40 bp regions are important for the overlap extension PCRs. Lcho_0008_5′ and Lcho_0008_3′ represent upstream and downstream fragments of Lcho_0008, respectively. spoVG represents the spoVG terminator.
Amplify the upstream (approximately 1.5 kb) and downstream (approximately 1.5 kb) regions of Lcho_0008 gene by PCR using SP-6 genomic DNA as a template and Lc0008_F_out/Lc0008_Km_2 and term-Lc0008-1/Lc0008-R-out primer sets, respectively (see Table 1).
Table 1. Primers for the construction of pUC18-mob-ΔLcho_0008::Kmr
Name Primer sequence
Lc0008-F-out 5'-AGAAGTAGTGCAGGTGCTTGAAATTG-3'
Lc0008-R-out 5'-TGGTCGACGAGCTGAACCGCAAATG-3'
pUC-Lc0008-1 5'-ACTCATTAGGCACCCCAGGCACAGCTGCAGCCCTTCGACTG-3'
pUC-Lc0008-2 5'-AGTCGAAGGGCTGCAGCTGTGCCTGGGGTGCCTAATGAGTGAG-3'
Lc0008-Km-1 5'-GCATGAACATCCTGCCCTGAGCCGCAAGCACTCAGGGCGCAAG-3'
Lc0008-Km-2 5'- CCCTGAGTGCTTGCGGCTCAGGGCAGGATGTTCATGCGTCGGCAG-3'
Km-term-1 5'-CGTTTTCCGGGACGCCGGCTAAAATAACCAAAAAGCAAGGAC-3'
Km-term-2 5'-CCTTGCTTTTTGGTTATTTTAGCCGGCGTCCCGGAAAACG-3'
term-Lc0008-1 5'-TTTCAAACTTAGTTGCACTCCCCGAGCGTGGCGGGCGGGTAC-3'
term-Lc0008-2 5'-ACCCGCCCGCCACGCTCGGGGAGTGCAACTAAGTTTGAAAAATCAG-3'
Lc0008-pUC-1 5'-TCGCAGCTTCAGCTCCGTGTTTTCACACAGGAAACAGCTATGACCATG-3'
Lc0008-pUC-2 5'-TAGCTGTTTCCTGTGTGAAAACACGGAGCTGAAGCTGCGATGAC-3'
Amplify the kanamycin resistance gene (Kmr) (approximately 1.2 kb) and the transcriptional terminator of B. subtilis spoVG gene (approximately 0.1 kb) by PCR using the primers Lc0008-Km-1/Km-term-2 and Km-term-1/term-LC0008-2 from pUC18-Kmr and the genomic DNA of B. subtilis 168, respectively.
Purify the four resultant PCR products (upstream and downstream regions of Lcho_0008, Kmr, and spoVG terminator) by agarose gel extraction.
Ligate these purified products of the Kmr and spoVG terminator by overlap extension PCR with Lc0008-Km-1 and term-Lc0008-2 primers, resulting in the production of Kmr-spoVG fragment (see Figure 2).
Purify the resultant PCR product by agarose gel extraction and ligate Kmr-spoVG fragment with upstream and downstream regions of Lcho_0008 (steps D3, D4) by overlap extension PCR with pUC-Lc0008-1 and Lc0008-pUC-2 primers, resulting in the production of the upstream Lcho_0008 + Kmr-spoVG + downstream Lcho_0008 fragment.
Purify the resultant PCR product by agarose gel extraction and use as an insert DNA in the subsequent iVEC3 procedure in step D9.
Amplify the plasmid backbone by inverse PCR with pUC-Lc0008-2 and Lc0008-pUC-1 primers using pUC18-mob plasmid as a template.
Purify the resultant PCR product by agarose gel extraction and use it as a vector DNA for the iVEC3 method in step D9.
Clone the insert DNA (upstream Lcho_0008 + Kmr-spoVG + downstream Lcho_0008) into the pUC18-mob vector by the iVEC3 system.
Pick the resultant single colonies, re-streak them on LB agar with 50 μg/mL ampicillin sodium salt, and incubate at 37 °C overnight.
Inoculate the resultant colonies into 4 mL of LB medium with 50 μg/mL ampicillin sodium salt in test tubes and incubate by shaking at 37 °C overnight.
Harvest the cells by centrifugation (20,000 × g, 1 min) and extract the plasmid DNA (pUC18-mob-ΔLcho_0008::Kmr) using the GenEluteTM Plasmid Miniprep kit according to the manufacturer’s instructions.
Confirm the construction by PCR using the extracted plasmid DNA and primer sets listed in Table 1. Sequencing especially the insert DNA of the plasmid is recommended.
Now, we are ready to use the resultant plasmid pUC18-mob-ΔLcho_0008::Kmr for the gene replacement of the Lcho_0008 gene. In addition, we can employ this plasmid as a template for the construction of other plasmid(s). For example, the plasmid pUC18-mob-ΔLcho_3510::Kmr is constructed as follows.
Construction of the plasmid for Lcho_3510 gene deletion (see Figure 1)
Detailed protocols of standard overlap extension PCR, DNA extraction from agarose gel, and iVEC3 system are described in Sections H, G, and J, respectively.
Amplify the upstream and downstream regions of Lcho_3510 gene by PCR using L. cholodnii SP-6 genomic DNA as a template and the Lc3510-F-out/Lc3510-Km-2 and Km-Lc3510-1/Lc3510-R-out primer sets, respectively (see Table 2).
Amplify the Kmr gene and the transcriptional terminator of B. subtilis spoVG gene region by PCR using the pUC18-mob-ΔLcho_0008::Kmr as a template and Lc3510-Km-1 and Km-Lc3510-2 primers.
Purify the three resultant PCR products (upstream and downstream regions of Lcho_3510 and the Kmr-terminator) by agarose gel extraction.
Ligate these purified products by overlap extension PCR with the pUC18-Lc3510-1 and Lc3510-pUC18-2 primers.
Purify the resultant PCR product by agarose gel extraction and use it as an insert DNA for the iVEC3 method in step E8.
Amplify the plasmid backbone by inverse PCR with the pUC18-Lc3510-2 and Lc3510-pUC18-1 primers using pUC18-mob plasmid as a template.
Purify the resultant PCR product by agarose gel extraction and use it as a vector DNA for the iVEC3 method in step E8.
Clone the insert DNA (upstream Lcho_3510-Kmr-spoVG terminator-downstream Lcho_3510) into the pUC18-mob vector by the iVEC3 method.
Pick the resultant single colonies, re-streak them on LB agar with 50 μg/mL ampicillin sodium salt, and incubate at 37 °C overnight.
Inoculate the resultant colonies into 4 mL of LB medium with 50 μg/mL ampicillin sodium salt in test tubes and incubate with shaking at 37 °C overnight.
Harvest the cells by centrifugation (20,000 × g, 1 min) and extract the plasmid DNA (pUC18-mob-ΔLcho_3510::Kmr) using a GenEluteTM Plasmid Miniprep kit according to the manufacturer’s instructions.
Confirm the construction by PCR using the extracted plasmid DNA and primer sets listed in Table 2. Sequencing especially the insert DNA of plasmid is recommended.
Table 2. Primers for the construction of pUC18-mob-ΔLcho_3510::Kmr
Name Primer sequence
pUC18-Lc3510-1 5'-ACTCATTAGGCACCCCAGGCTCGCACGCATCCGTCCCACCGCCTTG-3'
pUC18-Lc3510-2 5'-GGTGGGACGGATGCGTGCGAGCCTGGGGTGCCTAATGAGTGAG-3'
Lc3510-Km-1 5'-CACGGATGTTTCGAGTTTTTTGAGCCGCAAGCACTCAGGGCGCAAG-3'
Lc3510-Km-2 5'-GCGCCCTGAGTGCTTGCGGCTCAAAAAACTCGAAACATCCGTGATCCCCCTC-3'
Km-Lc3510-1 5'-TTTCAAACTTAGTTGCACTCCCGCGCGTTTTCCATCCTCGGTCG-3'
Km-Lc3510-2 5'-CGAGGATGGAAAACGCGCGGGAGTGCAACTAAGTTTGAAAAATCAG-3'
Lc3510-pUC18-1 5'-GTTGAATCGGAGAACCGCCTTTTCACACAGGAAACAGCTATGACCATG-3'
Lc3510-pUC18-2 5'-TAGCTGTTTCCTGTGTGAAAAGGCGGTTCTCCGATTCAACCAGTTTC-3'
Lc3510-F-out 5'-TTCGATCGACCGACCCTTCAGATGAG-3'
Lc3510-R-out 5'-TCCTGATTGGAGGTTGACGCAACGGTC-3'
DNA amplification by PCR
Mix 1 μL of template DNA, 0.15 μL of 100 μM forward primer, 0.15 μL of 100 μM reverse primer, and 25 μL of PrimeStar Max Premix (x2) and fill up to 50 μL with ultrapure water.
Perform the PCR with the initial denaturation at 94 °C for 1 min, followed by 30 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s/kb.
DNA extraction from agarose gel
Separate 10–50 μL of plasmid DNA or PCR products by agarose gel electrophoresis on 0.8%–1.5% agarose gel in 1× TAE buffer.
Stain the DNA in the agarose gel with Midori Green Advance for ~15 min to visualize it with a 470 nm LED illuminator.
Excise the appropriate DNA band and extract the DNA from the agarose gel using the Wizard® SV Gel and PCR Clean-Up System according to the manufacturer’s instructions.
Overlap extension PCR
Mix 50–100 ng of gel-extracted PCR products with homologous ends and 20 μL of PrimeStar Max Premix and adjust the volume to 40 μL with ultrapure water.
Perform the PCR with an initial denaturation at 94 °C for 1 min, followed by 15 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s/kb.
Mix 1 μL of the resultant PCR solution, 0.15 μL of 100 μM forward primer, 0.15 μL of 100 μM reverse primer, and 25 μL of PrimeStar Max Premix and adjust the volume to 50 μL with ultrapure water.
Perform the PCR with an initial denaturation at 94 °C for 1 min, followed by 30 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s/kb.
Preparation of competent cells of E. coli SN1187 for the iVEC3 (in vivo E. coli cloning) system
Inoculate colonies of E. coli SN1187 on an LB plate into 4 mL of LB liquid medium and incubate at 37 °C by shaking at 190 rpm overnight.
Transfer 1 mL of the cell culture to 60 mL of LB medium in a 200 mL Erlenmeyer flask and further incubate for ~90 min.
Place the Erlenmeyer flask containing the cell culture on ice for ~10 min at the time of OD600 = 0.4–0.5.
Then, transfer the cell culture to 50 mL polypropylene tubes and centrifuge at 5,000 × g for 5 min.
Discard the medium and suspend the resultant cell pellets in 2 mL of ice-cold LB medium.
Add 1.6 mL of ice-cold 2× TSS solution to the cell suspension and mix gently by pipetting.
Add 0.4 mL of DMSO to the resultant suspension and mix gently at RT by pipetting.
Dispense the cell suspension (~4 mL) in 0.1 mL aliquots in centrifuge tubes.
Then, freeze the cell suspensions immediately in liquid nitrogen and keep at -80 °C.
Circularization of PCR products by the iVEC3 system
The iVEC3 system was essentially reported in Nozaki and Niki (2019). Incorporated PCR products of the vector and insert are circularized in vivo using the overlapping 40 bp regions at both ends.
Thaw the frozen competent E. coli SN1187 cells in a 1.5 mL centrifuge tube on ice.
Add 300 fmol of the insert DNA and 60 fmol of the vector DNA to the competent cells, mix gently by pipetting, and incubate for 20 min on ice.
Add the suspension to 1 mL of LB medium, mix by inversion, and incubate at 37 °C for 45 min.
Then, centrifuge the cell suspension at 20,000 × g for 1 min, discard 1 mL of the supernatant, resuspend the cell pellet with the remaining supernatant, and spread it on an LB plate containing 50 μg/mL of ampicillin sodium salt.
Preparation of competent cells of E. coli S17-1 and DH5α and transformation
Streak E. coli S17-1 or DH5α cells on an LB plate from a glycerol stock using a sterilized toothpick and incubate at 37 °C for 12 h.
Pick a single colony, inoculate into 5 mL of LB medium, and incubate at 37 °C for 12 h by shaking at 190 rpm.
Transfer 200 μL of the cell culture to 80 mL of fresh LB medium in a 200 mL Erlenmeyer flask and incubate at 37 °C for an additional ~3 h by shaking at 190 rpm.
Place the Erlenmeyer flask containing the cell culture on ice for ~10 min at the time of OD600 = 0.6–0.7.
Then, transfer the cell culture to 50 mL polypropylene tubes and centrifuge at 3,000 × g for 10 min.
For washing, suspend the resultant cell pellet in 80 mL of ice-cold sterile ultrapure water by pipetting and centrifuge at 3,000 × g for 10 min.
Repeat step K6 once more.
Suspend the resultant cell pellet in 1.4 mL of ice-cold 10% glycerol solution and transfer the suspension to a 1.5 mL centrifuge.
Then, centrifuge the cell suspension at 7,000 × g for 10 min, discard the supernatant, and suspend the resultant cell pellet in 220 μL of ice-cold 10% glycerol solution.
Dispense the cell suspension in aliquots of 42 μL in centrifuge tubes and keep them at -80 °C until use.
Thaw the competent cells in a centrifuge tube on ice.
Add 1 μL of plasmid DNA to suspension of the competent cell and mix gently by pipetting.
Transfer the resultant mixture to an electroporation cuvette.
Perform electroporation using a GenePulser X cell at 2.5 kV.
Add ~1.0 mL of SOC medium, transfer the cell suspension to a centrifuge tube, and incubate at 37 °C for 1 h.
Spread the resultant cell suspension on a LB plate containing 50 μg/mL of ampicillin sodium salt (the Kmr on the constructed plasmid optionally allows us to use kanamycin for E. coli colony selection).
Isolation of rifampicin-resistant mutant of SP-6 (hereafter WT*)
Pick several colonies of SP-6 cells from an MSVP plate with a sterilized toothpick, inoculate into 25 mL MSVP liquid medium containing 5 μg/mL rifampicin in a 50 mL polypropylene tube, and incubate for 2–3 days at RT by shaking at 70 rpm.
Spread 100 μL of the cell culture on MSVP plates containing 5 μg/mL rifampicin and incubate for 5–7 days.
Pick a single colony with a sterilized toothpick, inoculate into 25 mL of MSVP medium containing 10 μg/mL rifampicin in a 50 mL polypropylene tube, and incubate for 2–3 days at RT by shaking at 70 rpm.
Repeat steps L2 and L3 three times using MSVP medium containing 10, 15, and 20 μg/mL rifampicin in sequence.
Isolate a single colony on an MSVP plate containing 20 μg/mL rifampicin and define it as WT*.
Mix a 1-day culture of WT* with an equal volume of 50% glycerol solution, dispense the resultant cell suspension to stock tubes, and store them at -80 °C.
Gene transfer to WT* cells by conjugation with E. coli S17-1 (see Figure 3)
Preparation of WT* cells
Streak WT* cells on a MSVP plate from a glycerol stock with a sterilized toothpick and incubate at 25 °C for 3–5 days.
Pick several WT* colonies from an MSVP plate with a sterilized toothpick, inoculate into 25 mL of MSVP medium in 50 mL polypropylene tubes (25 mL × four tubes), and incubate for 2–3 days at RT by shaking at 70 rpm.
Collect exponentially growing WT* in 2-days MSVP culture by centrifugation at 7,000 × g for 5 min, wash twice in MSVP lacking Ca (MSVP-Ca), and culture in 25 mL of MSVP-Ca for additional 6 h (see Figures 7 and 8A).
Collect exponentially growing WT* cells by centrifugation at 7,000 × g for 5 min. Discard the medium and resuspend the cell pellet in 1 mL of 0.9% NaCl.
Pass the cell suspension through a needle syringe (23 G × 1 inch) using a relatively high pressure to break apart cell aggregates composed of sheathed cell chains into single cells.
Adjust the volume of the resultant cell suspension to 2 mL with 0.9% NaCl and measure the optical density at 600 nm (OD600) to determine the cell density.
Figure 3. A detailed step-by-step chart of the culture and maintenance of L. cholodnii and E. coli
Preparation of E. coli S17-1 cells containing a plasmid
Streak E. coli S17-1 cells containing a plasmid such as pUC18-mob-ΔLcho_3510::Kmr on an LB plate containing 50 μg/mL ampicillin sodium salt from a glycerol stock with a sterilized toothpick and incubate at 37 °C for 12 h.
Pick a single colony, inoculate into 4 mL of LB medium containing 50 μg/mL ampicillin sodium salt (4 mL × two tubes), and incubate at 37 °C for 16 h by shaking at 190 rpm.
Collect exponentially growing E. coli cells by centrifugation at 3,000 × g for 1 min.
Discard the medium and wash the collected cells three times with 0.9% NaCl.
Resuspend the washed cells in 2 mL of 0.9% NaCl and measure the OD600 to determine cell density.
Note: To prevent damage of pilus excreted from the cell surfaces of E. coli cells, which are important for conjugation, handle the E. coli cells gently (steps M9–M11).
Conjugation and colony selection
Drop 500 μL of the WT* suspension (from step M6) onto a membrane filter placed on an NB plate and incubate for 1 h until there is no liquid on the membrane surface (see Figure 4).
Figure 4. Image of mixing WT* and E. coli cells on a membrane
Drop 500 μL of the suspension of E. coli cells (from step M11) onto the same membrane filter incubated with SP-6 and incubate for 16 h.
Transfer the membrane filter into a 15 mL polypropylene tube with a tweezer.
Remove bacterial cells from the nitrocellulose membrane filter by pipetting with 10 mL of 0.9% NaCl and collect them by centrifugation at 7,000 × g for 5 min.
Resuspend the bacterial cells in 1 mL of 0.9% NaCl, plate 100 µL each onto MSVP plates containing 20 μg/mL rifampicin and 50 μg/mL kanamycin, and incubate at RT for 5–7 days.
Pick single colonies and streak them on the same plate to allow for single-colony isolation.
Confirmation of gene replacement and preparation of glycerol stock
Colony PCR (see Figure 5)
Mix 0.15 μL of 100 μM forward primer, 0.15 μL of 100 μM reverse primer, 10 μL of 2× Gflex PCR Buffer (Mg2+, dNTP plus), and 0.4 μL of Tks Gflex DNA Polymerase (1.25 units/μL) and adjust the volume to 20 μL with ultrapure water in PCR tubes.
Pick single colonies (~0.5 mm in diameter) from MSVP plates containing 20 μg/mL rifampicin and 50 μg/mL kanamycin (Recipes 17, 18) with sterilized toothpicks and suspend them in the colony PCR mixture.
Perform the PCR with the following steps: initial denaturation at 94 °C for 1 min, followed by 30 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 1 min/kb.
Apply and electrophorese 10 mL of PCR products into an 0.8% agarose gel.
After staining the agarose gel with the ethidium bromide solution, image amplified bands using a gel documentation system.
Figure 5. Confirmation of gene replacement of the Lcho_3510 gene with Kmr. Lc3510-F-out/Km-Lc3510-2 (left), Lc3510-Km-1/Lc3510-R-out (middle), and Lcho1694-F-BamHI/Lcho1694-R-BamHI (right) primer sets were employed for colony PCR (see Tables 2, 3 and the reference Kunoh et al., 2022). Size markers (kb) determined by 1 kb Plus DNA ladder (indicated as M) are indicated by numbers and arrowheads on the left.
Table 3. Primers for colony PCR
Name Primer sequence
Lc1694-F-BamHI 5'-CGGGATCCATGTCGAGATCGACCGCCAGCG-3'
Lc1694-R-BamHI 5'-CGGGATCCTTACTTGCGTGACGAGCGCAGC-3'
Sequencing
Perform colony PCR using primers located outside the replaced gene as described in steps N1–N3.
Purify the resultant PCR products by agarose gel extraction.
Sequence the PCR products using sequencing primers listed in Table 2 (outsourced to Eurofins).
Glycerol stock
Pick the confirmed colonies (from step M17), inoculate into 25 mL of MSVP, and incubate at RT for one day.
Transfer 0.75 mL of the cell culture to a 1.5 mL tube, mix with an equal volume of 50% glycerol solution, and store at -80 °C.
Data analysis
Carry out statistical analyses by unpaired Welch's t-tests and Bonferroni-Holm method using the R software version 4.1.2 [R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (https://www.R-project.org/)] (see Figures 6, 8B, and 9).
Notes
The genomic DNA from B. subtilis 168 cells can be prepared using kits such as Wizard® Genomic DNA Purification kit (Promega).
To fully stop the transcription of the kmr gene at downstream of the stop codon, we employed the Bacillus subtilis spoVG transcriptional terminator because it was shown to form the stable configuration (Hudspeth et al., 1992). However, other terminators can be used for this purpose.
For in vivo cloning (the iVEC3 system), E. coli strain (SN1187) is engineered to employ the exonuclease III activity of XthA for exposure of ~40 bp of the homologous region at each end that enables annealing of DNAs, while DNA polymerase I activity of PolA is used for the gap repair of annealed DNAs (Nozaki and Niki, 2019). Although we recommend this system due to its cost-effectiveness, other cloning kits such as In-Fusion® HD Cloning kit (Clontech) can be used.
The plasmid pUC18-mob-∆Lcho_3510::Kmr is available from the RIKEN BRC Japan collection (RDB No.19817 pUC18_delLcho3510). Please contact us if the L. cholodnii strain WT* is needed.
As far as we examined by colony PCR and the following agarose gel electrophoresis, no long band corresponding to the size when the whole plasmid was integrated by single crossover recombination was detected (see Figure 5), indicating most of obtained colonies received double crossover recombination.
We employed E. coli S17-1 as a donor in this protocol. However, the S17-1 strain is known to transfer bacteriophage Mu silently. Therefore, we encourage using Mu-free donor strains such as MFDpir (Ferrières et al., 2010).
We examined the conjugation efficiencies with different donor-recipient ratios. The cell suspensions of WT* (step M6) and S17-1 (step M11) were diluted prior to mixing on a membrane filter. After conjugation, the washed bacterial suspension (step M16) was serially diluted and plated on MSVP containing rifampicin and rifampicin/kanamycin to count the total WT* cells and their transconjugates, respectively. Figure 6 indicates that the efficiency tends to be highest when the WT*:S17-1 ratio is 1:1.
Figure 6. Conjugation efficiency with different donor-recipient ratios. Statistical analysis was performed by unpaired Welch's t-tests and Bonferroni-Holm method, suggesting no significant differences.
We considered the conjugation between WT* and S17-1 to be efficient when the cell surfaces of WT* cells were free from sheaths (Figure 7). Since we previously reported that SP-6 cells become sheath-less when cultured in calcium-free conditions (Kunoh et al., 2021), WT* cells cultured in routine MSVP were harvested and further cultured in MSVP lacking Ca (MSVP-Ca) for an additional 6 h (step M3) (Figure 8A). As expected, Figure 8B indicates that the conjugation efficiency with additional culturing in MSVP-Ca is approximately 2-fold higher than that without additional culturing (MSVP+Ca).
Figure 7. A strategy for enhancement of conjugation efficiency by suppressing sheath formation
Figure 8. Conjugation efficiency with suppressing sheath formation. The brightfield images indicate that aggregate formation is reduced when cultured in the Ca-deficient condition (A). Conjugation efficiency with or without culturing cells in calcium-free medium (B). Statistical analysis was performed by unpaired Welch's t-tests. *p < 0.04.
To validate whether the described method is universal for gene transfer, we employed three additional plasmids designed to target the Lcho_1694, Lcho_2352, and Lcho_2734 loci. As Figure 9 shows that the conjugation efficiencies are comparable among the representative plasmids, we consider our method to be useful for constructions targeting different loci.
Figure 9. Conjugation efficiencies of plasmids targeting different loci. Statistical analysis was performed by Welch's t-tests and Bonferroni-Holm method, suggesting no significant differences.
In experiments shown in Figure 9, approximately 250 colonies on average were obtained when 25 × 4 and 8 × 2 mL cultures of L. cholodnii and E. coli, respectively, were employed for conjugation. As far as confirmed by colony PCR, the gene replacement occurred correctly in most candidate colonies. These results implicate that we can set up the mutagenesis experiments as described in this protocol.
Recipes
MSVP (ATCC 1917) medium
Prepare MVSP medium (Emerson and Ghiorse, 1993) for culturing SP-6 cells (see Tables 4, 5). Omit CaCl2·2H2O from the original recipe of MSVP for a calcium-lacking medium (MSVP-Ca). To isolate spontaneous rifampicin-resistant mutant (WT*), sequentially spread the cell suspension on MSVP plates containing 5, 10, and 20 μg/mL rifampicin. MSVP plates containing 20 μg/mL rifampicin and 50 μg/mL kanamycin sulfate can be used for the selection of recombinants of WT* containing the Kmr gene, because E. coli S17-1 cells are sensitive to rifampicin, while non-recombinant WT* cells are sensitive to kanamycin but not to rifampicin.
Table 4. Composition of MSVP medium
Component Amount (g/L) Concentration (mM)
(NH4)2SO4 0.24 1.81
MgSO4·7H2O 0.06 0.24
CaCl2·2H2O 0.06 0.41
KH2PO4 0.02 0.15
Na2HPO4 0.03 0.14
HEPES 2.383 10.00
Adjust to pH 7.0 with 0.1 N NaOH, fill up to 1 L with ultrapure water, and autoclave
Component Amount (mL/L) Concentration (mM)
10 mM FeSO4 1.00 0.01
20% sodium pyruvate 5.00 9.09
Vitamin stock solution (see Table 5) 1.00
Sterilize these solutions by filtration and add prior to use
Table 5. Composition of vitamin stock solution
Component Amount (mg/L) Concentration (mM) Working concentration (nM)
Biotin 20.00 0.04 0.04
Folic acid 20.00 0.05 0.05
Thiamine·HCl 50.00 0.15 0.15
D-(+)-Calcium pantothenate 50.00 0.10 0.10
Vitamin B12 1.00 0.74 (nM) 0.74 (pM)
Riboflavin 50.00 0.13 0.13
Nicotinic acid 50.00 0.41 0.41
Pyridoxine hydrochloride 100.00 0.49 0.49
p-Aminobenzoic acid 50.00 0.36 0.36
Sterilize by filtration and add prior to use
LB medium
Dissolve 20 g of LB broth in 1,000 mL of distilled water and autoclave at 121 °C for 15 min. For the LB plate, add agar at 1.5%. After autoclaving, add 50 μg/mL of kanamycin sulfate and ampicillin sodium salt as needed.
SOC medium
Dissolve 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl, and 0.186 g of KCl in distilled water, adjust the pH to 7.0 by 5 N NaOH solution, and fill up to 1,000 mL with distilled water. After autoclaving at 121 °C for 15 min, add 10 mL of 1 M MgSO4 and 1 M MgCl2 solutions (see Recipe 5), and 20 mL of 1 M glucose solution (see Recipe 6).
NB medium
Dissolve 8 g of nutrient broth in 1,000 mL of distilled water and autoclave at 121 °C for 15 min. For the NB plate, add agar at 1.5%.
1 M MgSO4 and 1 M MgCl2 solutions
Dissolve 24.65 g of MgSO4·7H2O and 9.52 g of MgCl2 in 100 mL of distilled water and autoclave at 121 °C for 15 min.
1 M glucose solution
Dissolve 18.0 g of d-glucose in 100 mL of distilled water and sterilize using a 0.22 μm syringe filter.
NaCl solutions (0.9%, 5 M)
Dissolve 0.052 or 29.22 g of NaCl in ultrapure water, adjust the volume to 100 mL, and autoclave at 121 °C for 15 min, respectively.
TE buffer
Add 0.61 g of Tris-HCl (10 mM) and 0.23 g EDTA-4Na (1 mM) in ultrapure water, adjust pH to 8.0 with hydrochloric acid (HCl), adjust the volume to 500 mL with ultrapure water, and autoclave at 121 °C for 15 min.
TEN buffer
Mix 5 mL of 1 M Tris-HCl buffer (pH 8.0), 1 mL of 0.5 M EDTA-4Na (pH 8.0), 10 ml of 5 M NaCl, and 484 mL of ultrapure water, followed by autoclaving at 121 °C for 15 min.
10% SDS solution
Dissolve 10 g of SDS in ultrapure water and adjust the volume to 100 mL.
CTAB/NaCl solution
Dissolve 10 g of CTAB and 4.09 g of NaCl (0.7 M) in ultrapure water, adjust the volume to 100 mL, and autoclave at 121 °C for 15 min.
3 M sodium acetate (CH3COONa) solution
Dissolve 40.82 g of CH3COONa·3H2O in ultrapure water, adjust the volume to 100 mL, and autoclave at 121 °C for 15 min.
50× TAE buffer
Dissolve 242 g of Tris and 22.6 g of EDTA-4Na in ultrapure water containing 57.1 mL of acetic acid and adjust the volume to 1,000 mL.
2× TSS solution
Dissolve 4 g of PEG8000, 0.49 g of MgSO4·7H2O, and 4 mL of glycerol in LB medium (see Recipe 2), adjust the volume to 20 mL, and autoclave at 121 °C for 15 min.
TSS solution
Mix 25 mL of sterilized LB medium (see Recipe 2) and 20 mL of 2× TSS solution (see Recipe 14) with 5 mL of DMSO.
50% glycerol solution
Mix 50 mL of glycerol with 50 mL ultrapure water and autoclave at 121 °C for 15 min.
Rifampicin solution
Mix 50 mg of rifampicin with 1 mL of DMSO and stock at 4 °C.
Kanamycin solution
Mix 50 mg of kanamycin sulfate with 1 mL of ultrapure water and stock at 4 °C.
Ampicillin solution
Mix 50 mg of ampicillin sodium salt with 1 mL of ultrapure water and stock at 4 °C.
Acknowledgments
We acknowledge NBRP-E. coli at NIG for the E. coli strain SN1187. We also thank Drs. Tamaki H. and Morinaga K. (AIST) for providing the pSUP5011 plasmid. This study was financially supported by the Japan Science and Technology Agency (JPMJER1502, JPMJMI21G8) and a scholarship donation from Bridgestone Corporation.
Competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.
Ethics
This study was approved by the committee for the safe handling of living modified organisms at the University of Tsukuba and was carried out according to the guidelines of the committee.
References
Bocioaga, D., El Gheriany, I. A., Lion, L. W., Ghiorse, W. C., Shuler, M. L. and Hay, A. G. (2014). Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1. Microbiology (Reading) 160(Pt 11): 2396-2405.
Copeland, A., Lucas, S., Lapidus, A., Glavina del Rio, T., Dalin, E., Tice, H., Bruce, D., Goodwin, L., Pitluck, S., Chertkov, O., et al. (2008). Complete sequence of Leptothrix cholodnii SP-6. Submitted to the EMBL/GenBank/DDBJ databases.
Davis, M. W. and Jorgensen, E. M. (2022). ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. Front Bioinform 2: 818619.
Emerson, D. and Ghiorse, W. C. (1992). Isolation, Cultural Maintenance, and Taxonomy of a Sheath-Forming Strain of Leptothrix discophora and Characterization of Manganese-Oxidizing Activity Associated with the Sheath. Appl Environ Microbiol 58(12): 4001-4010.
Ferrières, L., Hémery, G., Nham, T., Guérout, A.-M., Mazel, D., Beloin, C., Ghigo, J.-M. (2010). Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J Bacteriol 192(24): 6418-6427.
Furutani, M., Suzuki, T., Ishihara, H., Hashimoto, H., Kunoh, H., and Takada, J. (2011). Initial assemblage of bacterial saccharic fibrils and element deposition to form an immature sheath in cultured Leptothrix sp. strain OUMS1.Minerals 1(1): 157-166.
Hudspeth, D. S. and Vary, P. S. (1992). spoVG sequence of Bacillus megaterium and Bacillus subtilis. Biochim Biophys Acta 1130(2): 229-231.
Kunoh, T., Kunoh, H., and Takada, J. (2015a). Perspectives on the biogenesis of iron oxide complexes produced by Leptothrix, an iron-oxidizing bacterium and promising industrial applications for their functions. J Microb Biochem Technol 7(6): 419-426.
Kunoh, T., Suzuki, T., Shiraishi, T., Kunoh, H., and Takada, J. (2015b). Treatment of Leptothrix cells with ultrapure water poses a threat to their viability. Biology (Basel) 4(1): 50-66.
Kunoh, T., Morinaga, K., Sugimoto, S., Miyazaki, S., Toyofuku, M., Iwasaki, K., Nomura, N. and Utada, A. S. (2020). Polyfunctional Nanofibril Appendages Mediate Attachment, Filamentation, and Filament Adaptability in Leptothrix cholodnii. ACS Nano 14(5): 5288-5297.
Kunoh, T., Yamamoto, T., Sugimoto, S., Ono, E., Nomura, N., and Utada, A. S. (2021). Responses of Leptothrix cholodnii cells to nutrient limitation.Front Microbiol 12, 691563.
Kunoh, T., Yamamoto, T., Prasad, M., Ono, E., Li, X., Sugimoto, S., Iida, E., Obana, N., Takeda, M., Nomura, N. and Utada, A. S. (2022). Porous Pellicle Formation of a Filamentous Bacterium, Leptothrix. Appl Environ Microbiol 88(23): e0134122.
Nozaki, S. and Niki, H. (2019). Exonuclease III (XthA) Enforces In Vivo DNA Cloning of Escherichia coli To Create Cohesive Ends. J Bacteriol 201(5): e00660-18.
Spring, S. (2006). The genera Leptothrix and Sphaerotilus. In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. H. and Stackebrandt, E. (Eds.). The Prokaryotes (pp. 758–777). New York, Springer.
van der Waarde, J., Krooneman, J., Geurkink, B., van der Werf, A., Eikelboom, D., Beimfohr, C., Snaidr, J., Levantesi, C. and Tandoi, V. (2002). Molecular monitoring of bulking sludge in industrial wastewater treatment plants. Water Sci Technol 46(1-2): 551-558.
Yoshihara, H. (2002). Identification of genes essential for sheath formation in Leptothrix cholodnii. Master thesis, Graduate School of Engineering, Yokohama National University.
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Cell Biology > Cell engineering
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A Quick DNA Extraction Method for High Throughput Screening in Gram-positive Bacteria
NC Nuo Chen
XY Xiaoming Yuan
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4653 Views: 1227
Reviewed by: Emilia KrypotouMarcelo S. da Silva Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in ACS Nano Jun 2022
Abstract
In this study, a sonication-based DNA extraction method was developed, in which the whole process can be finished within 10 min. This method is almost zero cost and time-saving, which is useful for high throughput screening, especially in the screening of mutants generated in random mutagenesis. This method is effective in genomic DNA extraction for PCR amplification in several Gram-positive bacteria, including Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Listeria monocytogenes.
Keywords: Gram-positive Bacteria DNA Extraction Ultrasound High Throughput Screening PCR
Background
Colony PCR is commonly used to verify the expected genome DNA in Escherichia coli, but it fails in Gram-positive bacteria such as Bacillus subtilis even when treated with chemical reagents (Packeiser et al., 2013; Azevedo et al., 2017). The reason colony PCR fails in these cases is the thick cell wall that exists in most Gram-positive bacteria, which impedes cell lysis and subsequent DNA release. Enzymes such as lysozyme, proteinase K, and other chemical reagents are used to lyse the Gram-positive bacterial cell wall (Mertens et al., 2014); however, these methods are costly, time consuming, and not eco-friendly for the DNA extraction of a huge quantity of samples. For example, it is very difficult to achieve a fast screen towards thousands of mutants generated in random mutagenesis. It is therefore necessary to set up a convenient and economical method.
Sonication is a common physical method employed to disrupt cells by transmitting ultrasonic energy to the liquid region (Taylor et al., 2001). Sonication-based DNA extraction is considered as an efficient and cost-effective method. In the intestinal microflora and compost microbes of fish, the ultrasonic lysis method combined with lysozyme was used to extract DNA (Yang et al., 2006; Han et al., 2018). Moreover, direct extraction of DNA from sputum clinical sample (containing diverse microbes) for real-time PCR proved to be convenient and suitable (Bello et al., 2020). In this study, a sonication-based DNA extraction method was developed, which could screen large samples in just one day. Despite not being as pure as the commercial kit, this method can be used for high-throughput screening purposes, which requires extracting great amounts of DNA, in Gram-positive bacteria such as Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Listeria monocytogenes.
Materials and Reagents
Materials
1.5 mL tube (Axygen, catalog number: MCT-150-C)
0.2 mL PCR tube (Axygen, catalog number: PCR-02-C-P)
10 and 100 μL pipettes (Eppendorf, catalog numbers: 3120000224 and 3120000828)
10 and 100 μL micropipettes (Axygen, catalog numbers: TGL-10FT-17-R and TF-100)
Petri dishes (Guangdong Huankai, catalog number: 20221700)
Inoculation loop (Guangdong Huankai, catalog number: 00416571)
Strains
Bacillus cereus ATCC 14579 (ATCC, catalog number: ATCC 14579)
Bacillus thuringiensis ATCC 10792 (ATCC, catalog number: ATCC 10792)
Bacillus subtilis ATCC 6633 (ATCC, catalog number: ATCC 6633)
Listeria monocytogenes ATCC 19115 (ATCC, catalog number: ATCC 19115)
Reagents
Nutrient agar plate (Guangdong Huankai, catalog number: 1024089)
Taq DNA polymerases (Thermo Fisher Scientific, catalog number: 10966018)
Marker D, 100–2,000 bp (Sangon Biotech, catalog number: B600335-0050)
GoldViewTM (Coolaber, catalog number: GD001-1mL*5)
50× TAE buffer (Sango Biotech, catalog number: B548101-0500)
Equipment
Incubator (SHANGHAI MINQUAN INSTRUMENT, model: LAB-0001-0004-SHMQ)
Benchtop centrifuge (Eppendorf, model: 5415D)
Ultrasonic cleaning machine (LANGEE, model: UC-9120)
Thermal cycler (Bio-Rad, model: C1000 Touch)
Electrophoresis imaging system (Bio-Rad, model: GelDoc XR Biorad)
Spectrophotometer (Thermo Fisher, model: NanoDropTM One Microvolume UV-Vis)
Autoclave (ZEALWAY, model: GR60DA)
Software
Image Lab (Bio-Rad)
Procedure
Cultivate the strains (listed in materials) conserved in glycerol at 37 °C, at 200 rpm overnight (for approximately 8–12 h).
Dip a small amount of the overnight culture using a 10 μL inoculation loop and streak lightly on the nutrient agar plate. Then, cultivate the plate at 37 °C overnight.
Suspend a single colony into 30 μL of ddH2O in a 1.5 mL tube.
Note: The size of the colonies of B. cereus ATCC 14579, B. thuringiensis ATCC 10792, B. subtilis ATCC 6633, and L. monocytogenes ATCC 19115 is recommended in Figure 1 and Table 1, and the quality of DNA is listed in Table 2.
Figure 1. B. cereus (A), B. thuringiensis (B), B. subtilis (C), and L. monocytogenes (D) colonies formed on Petri dishes. Scale bars represent 10 mm.
Table 1. Colony size of B. cereus, B. thuringiensis, B. subtilis, and L. monocytogenes
Microorganism Colony size
B. cereus ATCC 14579 2–4 mm
B. thuringiensis ATCC 10792 1–2 mm
B. subtilis ATCC 6633 1–3 mm
L. monocytogenes ATCC 19115 0.2–0.5 mm
Treat the bacterial suspension by sonication at 40 kHz and 120 W at room temperature for 5 min and then centrifuge at 10,000 × g for 1 min at room temperature.
Note: No need to set pauses between the pulses.
Carefully transfer the upper aqueous phase to a new 1.5 mL tube without disturbing the pellet.
The DNA samples are tested using 1 μL of supernatant as DNA template by PCR. Primers for PCR are listed in Table 3. Taq DNA polymerases are added and parameters for PCR are as follows: initial denaturation: 95 °C, 3 min; denaturation: 95 °C, 30 s; annealing: 55 °C, 30 s; extension: 72 °C, 1 min; final extension: 72 °C, 10 min; number of cycles: 25×. After electrophoresis on 1.2% agarose gels (in 1× TAE diluent), all amplicons are visualized under UV light with GoldViewTM staining. The results are shown in Figure 2.
Note: The bands are brighter if you increase the PCR reaction cycles.
Figure 2. B. cereus, B. thuringiensis, B. subtilis, and L. monocytogenes DNA analyzed by electrophoresis. Lane M: 100–2,000 bp DNA ladder (Marker D). B. c: B. cereus; B. t: B. thuringiensis; B. s: B. subtilis; L. m: L. monocytogenes. DNA: DNA extracts by ultrasonic method for 5 min. Positive control: DNA extracted with a commercial kit (1 μL DNA template). CK: Check control (no template).
Table 2. The quality of DNA sample by spectrophotometry analysis
Microorganism Nucleic Acid (ng/μL) A260/A280 A260/A230
B. cereus ATCC 14579 20.343 1.517 0.641
B. thuringiensis ATCC 10792 19.43 1.5 0.605
B. subtilis ATCC 6633 21.935 1.303 0.634
L. monocytogenes ATCC 19115 6.321 1.808 1.018
Table 3. Primer sequences used in B. cereus, B. thuringiensis, L. monocytogenes, and B. subtilis, respectively
Microorganism Target gene Primer sequence (5′ to 3′)
B. cereus ATCC 14579 gmk
F-TTAAGTGAGGAAGGGTAGG
R-AATGTTCACCAACCACAA
B. thuringiensis ATCC 10792 pycA
F-GTGAAAGCAAGAACACAAGC
R-ATAGTTTTTGTATCCAACTGCG
B. subtilis ATCC 6633 SigB
F-ATGACACAACCATCAAAAACTACGA
R-TTACATTAACTCCATCGAGGGATCT
L. monocytogenes ATCC 19115 prfA
F-GTCAAAACATACGCTCTTATC
R-ACATAATCAGTCCAAAGTAGATGC
Acknowledgments
This protocol was adapted from previous work (Chen et al., 2022; Li et al., 2022). This research was supported by Guangdong Major Project of Basic and Applied Basic Research (2020B0301030005), Guangdong Provincial Key Laboratory (2020B121201009), and Guangdong Province Academy of Sciences Special Project for Capacity Building of Innovation Driven Development (2020GDASYL-20200301002).
Competing interests
There are no conflicts of interest or competing interests.
References
Azevedo, F., Pereira, H. and Johansson, B. (2017). Colony PCR. Methods Mol Biol 1620: 129-139.
Bello, G. L., Morais, F. C. L., Wolf, J. M., Gehlen, M., Soares, T. D. S., Halon, M. L., Barcellos, R. B. and Rossetti, M. L. R. (2020). Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication. Braz J Infect Dis 24(5): 398-404.
Chen, M., Wei, X., Zhang, J., Zhou, H., Chen, N., Wang, J., Feng, Y., Yu, S., Zhang, J., Wu, S., Ye, Q., Pang, R., Ding, Y. and Wu, Q. (2022). Differentiation of Bacillus cereus and Bacillus thuringiensis Using Genome-Guided MALDI-TOF MS Based on Variations in Ribosomal Proteins. Microorganisms 10(5): 918.
Han, Z., Sun, J., Lv, A., Sung, Y., Sun, X., Shi, H., Hu, X., Wang, A. and Xing, K. (2018). A modified method for genomic DNA extraction from the fish intestinal microflora. AMB Express 8(1): 52.
Li, Y., Chen, N., Wu, Q., Liang, X., Yuan, X., Zhu, Z., Zheng, Y., Yu, S., Chen, M., Zhang, J., Wang, J. and Ding, Y. (2022). A Flagella Hook Coding Gene flgE Positively Affects Biofilm Formation and Cereulide Production in Emetic Bacillus cereus. Front Microbiol 13: 897836.
Mertens, K., Freund, L., Schmoock, G., Hänsel, C., Melzer, F. and Elschner, M. C. (2014). Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples. Int J Food Microbiol 170: 29-37.
Packeiser, H., Lim, C., Balagurunathan, B., Wu, J. and Zhao, H. (2013). An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. Appl Biochem Biotechnol 169(2): 695-700.
Taylor, M. T., Belgrader, P., Furman, B. J., Pourahmadi, F., Kovacs, G. T. and Northrup, M. A. (2001). Lysing bacterial spores by sonication through a flexible interface in a microfluidic system. Anal Chem, 73(3), 492–496.
Yang, Z. H., Xiao, Y., Zeng, G. M., Liu, Y. G. and Deng, J. H. (2006). [DNA extraction methods of compost for molecular ecology analysis]. Huan Jing Ke Xue 27(8): 1613-1617.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Microbiology > Microbial genetics > Transformation
Microbiology > Microbial genetics > DNA
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Can this be scaled up to a cell pellet as opposed to a colony?
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A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS
SM Sree I. Motipally
BM Boyden Myers
ES Emily R. Sechrest
David Sokolov
JM Joseph Murphy
SK Saravanan Kolandaivelu
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4654 Views: 1043
Reviewed by: Ansul LokdarshiRicardo Urquidi Camacho Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in International Journal of Molecular Sciences Sep 2022
Abstract
Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3H- or 14C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective.
Graphical overview
Keywords: Palmitoylation Acyl-RAC PTM Retina Photoreceptors Agarose S3 resin Thiopropyl Sepharose resin
Background
The retina is a thin layer of neural tissue lining the back of the eye, containing six different cell types organized together for visual perception. Vision is initiated through retinal photoreceptors, which are polarized and compartmentalized neurons containing ciliated outer segments (OS). Photoreceptor OS contains thousands of double membranous discs that harbor phototransduction proteins, which are responsible for capturing light and converting it into an electrical signal. The OS discs, along with the proteins that mediate phototransduction within the ciliated structures, are phagocytized in a circadian rhythm by pigmented epithelial cells that abut the retinal layer. On the basal side of the photoreceptor neurons lay the ribbon synapses that are responsible for the transmission of light-dependent electrical signals to downstream neurons, which ultimately transmit the signals to the visual cortex. The proteins present at the synapse and in the OS disc membranes are synthesized in the inner segment (IS) of the photoreceptor neurons and trafficked to their destination. The constant renewal of the OS discs necessitates continual protein synthesis and efficient sorting machinery (Burns and Arshavsky, 2005; Baker et al., 2008).
The protein trafficking machinery in photoreceptor neurons is greatly aided by the lipid modification of proteins (Kolandaivelu et al., 2009;Maeda et al., 2010;Yang and Wensels, 1992). Among the lipid modifications, palmitoylation is the most common form of protein acylation, which adds a 16-carbon palmitic acid to a cysteine residue in the protein. Unlike most lipid modifications, palmitoylation is unique and reversible, and therefore plays a dynamic role in dictating the localization of a protein both spatially and temporally. Defects in the sorting of proteins due to the absence/loss of palmitoylation have been associated with various inherited blinding diseases, suggesting an important role of this lipid modification. Several studies including ours described the importance of palmitoylation in retinal health and function. We showed that lack of palmitoylation in a retinitis pigmentosa–associated protein—progressive rod-cone degeneration (PRCD)—leads to protein mistargeting to the photoreceptor IS where it undergoes severe destabilization (Murphy and Kolandaivelu, 2016). Likewise, mutating palmitoylation sites in rhodopsin leads to severe light-induced photoreceptor degeneration (Maeda et al., 2010). However, a paucity of information on retinal palmitoylated proteins hinders our progress toward understanding the significance of this crucial modification in retinal structure and function. Our modified acyl resin–assisted capture (Acyl-RAC) method efficiently identifies the global palmitoyl proteome in retinal neurons and other tissues that are compatible with downstream mass spectrometry (LC-MS/MS) analyses for further validation (Forrester et al., 2011;Murphy and Kolandaivelu, 2016; Myers et al., 2022). Using our protocol, we show the efficient isolation of palmitoylated proteins and downstream validation of known palmitoylated proteins by immunoblotting. Using this protocol, we show that several proteins that are critical for proper retinal function, and proteins linked with blinding diseases, are post-translationally lipid-modified by palmitoylation. Additionally, we show that our protocol can be reliably used to identify palmitoylated proteins in other tissues.
Materials and Reagents
Eppendorf 1.5 mL tubes (Thermo Fisher Scientific, catalog number: 05-408-129)
1 M HEPES, pH 7.5 (Thermo Fisher, catalog number: 15630080)
5 M NaCl (Thermo Fisher Scientific, catalog number: 00622643)
500 mM EDTA (Thermo Fisher Scientific, catalog number: AM9260G)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 8170341000)
Hydroxylamine-HCl (Thermo Fisher Scientific, catalog number: 270100010)
Pierce protease inhibitor tablet, EDTA free (Thermo Fisher Scientific, catalog number: A32955)
Phosphatase inhibitor (Thermo Fisher Scientific, catalog number: J63907.AA)
HDSF (Santa Cruz, catalog number: sc-221708A)
Methyl methanethiosulfonate (MMTS) (Sigma-Aldrich, catalog number: 2949920)
100% acetone (Thermo Fisher Scientific, catalog number: 186947)
2-Mercaptoethanol (β-ME) (Calbiochem, catalog number: UN2966)
1 M dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)
Agarose S3 high-capacity acyl-RAC capture resin (Nanocs, catalog number: AR-SS-2)
0.5 M Tris-HCl, pH 6.8 (Thermo Fisher Scientific, catalog number: J63735.K2)
100% glycerol (Thermo Fisher Scientific, catalog number: 15548224)
Bromophenol blue (Sigma-Aldrich, catalog number: B5525)
3 kD cutoff centrifugal filter (Millipore, catalog number: Z677094)
Trichloroacetic acid (TCA) (Thermo Fisher Scientific, catalog number:184151000)
Coomassie stain (Bio-Rad, catalog number:1610786)
Triton X-100 (Sigma-Aldrich, catalog number: 9036195)
Sodium hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: A16037.36)
Lysis buffer base (see Recipes)
Lysis buffer – working solution (see Recipes)
Binding buffer (see Recipes)
Blocking buffer (see Recipes)
5× Laemmli sample buffer (see Recipes)
1× Laemmli sample buffer (see Recipes)
1 M dithiothreitol (DTT) (see Recipes)
100% TCA stock preparation (w/v) (see Recipes)
Tris-glycine SDS running buffer (Thermo Fisher Scientific, catalog number: LC26755) (see Recipes)
70% acetone (see Recipes)
2 M hydroxylamine (see Recipes)
2 M sodium chloride (see Recipes)
5 M sodium hydroxide (See Recipes)
10% Triton X-100 (see Recipes)
Equipment
Sonicator (Fisher, Microson Ultrasonic cell disruptor, model: CL-18)
Heat block (Thermo Fisher Scientific, catalog number: 11715125D)
Vortex (Scientific Industries, model: G-560)
Centrifuge, (Eppendorf, catalog number: 0011627)
Nutator (Thermo Fisher Scientific, model: 88881001)
Software
Excel 2016 (Microsoft)
GraphPad Prism (Version 9.0)
Adobe Illustrator 2023
CSS-Palm 4.0 – Palmitoylation Sites Prediction tool (Ren et al., 2008)
Procedure
Tissue lysis
Take four freshly collected or flash-frozen mouse retinae in a 1.5 mL Eppendorf tube and add 300 μL of lysis buffer (see Recipes) (Murphy and Kolandaivelu, 2016).
Sonicate the tissue at 12–15 psi thrice, four seconds each, with 30 s of incubation on ice between pulses.
To solubilize the proteins, add 33 µL of 10% Triton X-100 (to a final concentration of 1% Triton X-100) and nutate the samples at 4 °C for 15 min.
Remove cell and nuclear debris by centrifuging at 200 × g and 4 °C for 3 min and transfer the supernatant into a fresh 1.5 mL tube.
Blocking free thiol groups
Prepare blocking buffer containing MMTS (add 4 µL of MMTS per every 300 µL of blocking buffer).
Separate supernatant from step A4 into 100 µL aliquots (in 1.5 mL tubes) and add three volumes of blocking buffer containing MMTS (300 µL).
Note: Briefly vortex the reaction mixture before each use to keep the solution homogeneous.
Incubate samples in the heat block at 40 °C for 16 min with a 5 s vortex every 2 min.
Protein precipitation
Add 1.1 mL of 100% ice-cold acetone into each tube, invert thrice, and incubate at -20 °C for 20 min.
Centrifuge samples at 20,000 × g and 4 °C for 15 min to pellet the proteins.
Discard the supernatant and wash the protein pellet four times with ice-cold 70% acetone by gentle vortexing followed by centrifugation at top speed for 1 min at 4 °C.
Note: Dislodge the pellet during the first wash to remove the residual MMTS between the pellet and the Eppendorf tube wall.
Cleaving thioester bonds with hydroxylamine (HA) & capturing palmitoylated proteins using agarose S3 high-capacity acyl-RAC resin
Air-dry the protein pellet at room temperature (RT) for ~30 min to remove residual acetone.
Note: Do not over-dry the protein pellet to avoid issues with resuspension.
Resuspend each pellet in 200 µL of binding buffer by brief sonication and pool samples into one tube (e.g., 200 µL × three tubes = 600 µL).
Pipette 40 µL of agarose S3 high-capacity resin into each of the two clean 1.5 mL Eppendorf tubes labeled as Elute + HA (E +HA) and Elute - HA (E -HA) and wash to equilibrate the resin with 500 µL of binding buffer once. Allow the beads to pellet by gravity and discard the wash buffer.
Note: Make sure the resin is homogeneously resuspended before use.
Pipette 75 µL of the pooled sample to a clean 1.5 mL Eppendorf tube labeled as Total.
Add 250 µL of sample to each of the E +HA and E -HA tubes (if there is more sample volume, add additional beads accordingly and split equal sample volume for +HA and -HA; if there is less sample volume, make it up to 600 µL with elution buffer).
Add 45 µL of 2 M hydroxylamine to the E +HA tube and add 45 µL of 2 M sodium chloride to the E -HA tube.
Nutate all samples, including the total fraction sample at RT for 2 h.
Let the beads fall by gravity and collect 75 µL of the supernatant from each of the E +HA and E-HA tubes into clean 1.5 mL Eppendorf tubes labeled as Unbound +HA (UB +HA) and Unbound -HA (UB -HA), respectively.
Elution of bound protein
Wash E +HA and E -HA beads by adding 1 mL of binding buffer and gently inverting the tubes approximately six times. Let the beads fall by gravity and discard the supernatant. Repeat the wash step three more times.
Add 75 µL of binding buffer containing 10 mM DTT to each of the E +HA and E -HA tubes and incubate at RT for 10 min to elute the bound proteins.
Note: To make the above elution buffer, add 10 µL of 1M DTT in 990 µL of binding buffer and mix thoroughly by vortexing. Scale up as needed.
Repeat the elution step 2–3 times and pool the eluates.
Note: The majority (90%) of proteins are eluted in the first fraction (i.e., elute 1). Check each eluted fraction by western blotting with known palmitoylated proteins, e.g., Rhodopsin, Goα, and PRCD. If the second and third elute show significant amounts of known palmitoylated proteins, pool the eluates and use TCA precipitation or suitable Microcon cutoff filters to concentrate the samples.
To concentrate the samples, transfer the pooled eluates to Pierce Concentrator 3K MWCO filter and centrifuge at 6,000–9,000 × g for 30 min at RT.
Notes:
Samples can be stored at -20 °C for approximately one month until further processing.
Optional: Eluates can be further concentrated by using TCA precipitation. See the protocol below.
TCA precipitation
Add one volume of TCA [50 µL of 100% TCA stock preparation (w/v)] to four volumes of protein samples (200 µL of eluted protein samples) and incubate the tubes for 15 min on ice.
Centrifuge the tubes at 18,400 × g and 4 °C for 5 min to pellet the proteins.
Carefully remove the supernatant, leaving the protein pellet intact.
Wash the pellet with 200 µL of ice-cold acetone and centrifuge the tube at 18,500 × g and 4 °C for 5 min. Discard the supernatant and repeat the wash step.
Air-dry the pellet for 5–10 min to remove residual acetone.
Identification of palmitoylated protein by SDS-PAGE/ LC-MS/MS
To perform immunoblotting, add 18.75 µL of 5× Laemmli sample buffer containing a final concentration of 5% 2-mercaptoethanol (β-ME) and 10 mM DTT to 75 µL of Total and Unbound samples.
Add 70 µL of 1× Laemmli sample buffer containing 5% β-ME and 10 mM DTT to both +HA and -HA eluted fractions.
Let the tubes sit at RT for 20 min with intermittent mixing every 5 min.
Boil the samples for 5 min on a floating rack in the water bath and centrifuge at 6,000 × g for 30 s at RT.
To detect palmitoylated proteins by mass spectrometry, run the samples on a 12% SDS-PAGE gel at 80 V for 30 min (Figures 1 and 2).
Note: Ensure the electrophoresis equipment is thoroughly cleaned with ddH2O and use fresh SDS-PAGE buffer.
Transfer the gel onto a clean glass tray and perform Coomassie staining (Figure 3A).
After overnight destaining with Milli-Q water (autoclaved), cut the protein bands to perform in-gel digestion (see Figure 3A) and process the samples for LC-MS/MS analysis (Edmonds et al., 2017)
For additional validation, perform immunoblotting and probe the blots with antibodies against the proteins of interest to identify/show their palmitoylation status (Figures 1 and 2).
Figure 1. Acyl resin–assisted capture (Acyl-RAC) validation by immunoblotting of known palmitoylated proteins. Purification and detection of known palmitoylated proteins Goα (top panel) and rhodopsin (middle panel) in the wild-type mice retina using thiopropyl Sepharose 6B beads. Phosphodiesterase-6 (PDE6α), a prenylated protein, is not palmitoylated and serves as a control (bottom panel). HA = Hydroxylamine, T = Total, U = Unbound, E = Eluted. Elution in the presence of HA (+HA) represents the isolation of palmitoylated proteins fraction. Elution in the absence of HA (-HA) is used as an internal control to validate the purification scheme.
Figure 2. Western blots demonstrating the purification of palmitoylated proteins using agarose S3 beads. A. Palmitoylated proteins isolated from retinal extracts using agarose S3 high-capacity beads were separated by 4%–20% SDS-PAGE and immunoblotted with known palmitoylated proteins, namely progressive rod-cone degeneration (PRCD, top panel) and Goα (middle panel). β-tubulin (bottom panel) is not palmitoylated and serves as a control. B. Western blots showing efficient isolation of palmitoylated proteins from other tissues (brain and lung) using the present Acyl-RAC protocol. Blots are probed with Goα (positive control, top panel) and β-tubulin (negative control, bottom panel).
Figure 3. Retinal palmitoyl proteins identified through LC-MS/MS. A. SDS-PAGE followed by Coomassie staining: elutes from -HA (left lane, control) and +HA (right lane) beads were separated on a 10% polyacrylamide gel and stained with Coomassie blue for 1 h at RT, followed by overnight destaining with autoclaved Milli-Q water to visualize the protein bands. The protein bands were cut (as indicated by dotted lines) with a clean blade to perform in-gel digestion, and samples were sent to Harvard Mass spec core for LC-MS/MS to identify retinal palmitoyl proteins. B. Venn diagram showing the total number of unique proteins detected in -HA and +HA fractions. The overlap indicates non-specific proteins detected in both fractions.
Data analysis
Using Microsoft Excel, we identified potential palmitoylated proteins exclusively present in the +HA fraction. Proteins detected in both +HA and -HA fractions are considered non-specific proteins and were excluded from the analyses. To validate the protocol, we show the list of previously known palmitoylated proteins (Table 1) and uncharacterized palmitoylated proteins (Table 2) that are captured by the assay, based on comparisons with published literature. CSS Palm 4.0 – Palmitoylation Sites Prediction software was used to predict the potential palmitoylation sites, with high/medium confidence thresholds, for all the proteins listed in the tables (Ren et al., 2008).
Table 1. Known palmitoylated proteins captured by Acyl-RAC. A list of retinal palmitoylated proteins detected in the +HA fraction by LC-MS/MS that were compared with published literature to confirm the palmitoylation status
Accession # Description Symbol Mol. Wt (kDa) CSS-Palm Predicted palmitoylation residue(s) position Coverage Peptides Function
254039605
Progressive rod-cone degeneration protein homolog precursor
[Mus musculus]
PRCD 6 2 (Medium) 35.8 1 Photoreceptor disc formation
21717805
Rhodopsin
[Mus musculus]
Rho 39 322, 323 (High) 31.9 13 Phototransduction
71892412 Retinol dehydrogenase 8 [Mus musculus] RDH8 34.67 None 21.5 6 Converts all-trans-retinal to all-trans-retinol
568921542 Ankyrin-2 isoform X8 [Mus musculus] ANK-2/ANK-B 441 1451 (High) 22.6 60 Lens architecture and biomechanics
9055356 Syntaxin-8 [Mus musculus] STX8 26.9 214 (Medium) 22.9 5 Vesicle trafficking protein
12025532 V-type proton ATPase 116 kDa subunit a isoform 1 isoform 1 [Mus musculus] V-ATPase 96.23 24, 310 (High) 31.7 24 ATP hydrolysis-driven proton pumps
569019533 Flotillin-1 isoform X1 [Mus musculus] Flot-1 47.84
5, 17 (High)
Cys34; experimentally proved
64.5 24 Cell proliferation, T-cell activation, and phagocytosis
31543797 Synaptotagmin-2 [Mus musculus] SYT2 47.21
82, 83, 85, 87,
90, 91, 92 (High)
42.9 17 Trigger Ca2+-dependent neurotransmitter release
239985643 Paralemmin-1 isoform 2 [Mus musculus] PALM 36.69 335, 336 (High) 77 27 Regulator of filopodia induction and spine maturation
568916068 Synaptosomal-associated protein 23 isoform X2 [Mus musculus] SNAP23 24.53 79, 80, 83, 85 (High) 69.2 13 Vesicular membrane fusion machinery
31982233 Lyric [Mus musculus] AEG-1 63.8 None 42.8 18 NF-kappaB binding activity; double-stranded RNA binding activity; and transcription coactivator activity
6677873 Stathmin-3 [Mus musculus] STMN3 20.94 22, 24 (High) 25 5 Microtubule destabilizing activity, regulation of GTPase activity
194328695 Phospholipid scramblase 1 [Mus musculus] PLSCR1 35.89 157, 162, 163, 190, 193, 194, 197, 198, 248, 249 (High) 18.6 6 Transcriptional activator activity, EGFR binding, DNA binding, apoptosis, immune response
194363764 GTPase HRas isoform 1 [Mus musculus] HRAS 21.28 181, 184 (High) 62.4 11 GTPase activity, cell cycle, signaling
19526820 Protein XRP2 isoform a [Mus musculus]
XRP2
39.35 3, 4, 127 (High) 23.6 9 GAP activity, protein transport
11596855 Transferrin receptor protein 1 [Mus musculus] TfR1 85.67 98 (medium) 16.9 10 Cellular iron uptake
11612509 Ras-related protein Ral-B precursor [Mus musculus] RALB 23.33 203, 204 (high) 23.3 5 Cell cycle, apoptosis
304307779 Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 [Mus musculus] PAG1 46.52 39, 42 (High) 24 7 SH2 domain binding, immune response, signal transduction
30520123 Phospholipid scramblase 4 [Mus musculus] PLSCR4 36.55 193, 194, 195, 197, 198, 200 (High) 11 3 Ion binding, migration of bilayer
6680878 Lysosome membrane protein 2 [Mus musculus] LIMP2 54 4, 5 (High) 22.6 8 Lysosomal receptor for GBA-targeting, cell adhesion, protein targeting to lysosome
Table 2. Potentially palmitoylated retinal proteins captured by Acyl-RAC.
A list of uncharacterized retinal palmitoylated proteins detected in the +HA fraction by LC-MS/MS.
Accession # Description Symbol Mol. Wt (kDa) CSS-Palm Predicted palmitoylation residue(s) position Coverage Peptides Function
306482605 Cyclic nucleotide-gated cation channel beta-1 isoform 1 [Mus musculus] CNGAb1 148.62 707, 708 (High) 32.8 34 Phototransduction
10946800 Syntaxin-6 [Mus musculus] STX6 28.97 236 (Medium) 45.9 7 Trans-Golgi network vesicle trafficking
755565489
Plasma membrane calcium-transporting ATPase 3 isoform X1
[Mus musculus]
ATPase3 137.5 None 28.2 32 Ca2+ extrusion
254675320 Prominin-1 isoform s8 precursor [Mus musculus] Prom1 94.41 14, 129, 134, 136, 137, 154, 308 (High)
24.5
19
Required for retinal development and photoreceptor disc morphogenesis
113680348 Fascin [Mus musculus] FSCN2 54.47 19 (High) 24.7 9 Actin binding, cytoskeletal
115749432 Grifin [Mus musculus] GRIFN 15.96 9 (Medium) 16 2 Lens-specific protein that binds to alpha-crystallin
568889996 Ubiquitin-conjugating enzyme E2 L3 [Mus musculus] UBE2L3 17.85 17 (high) 21.4 2 Critical role in the UPS
568986868 Kinectin isoform X1 [Mus musculus] KTN1 152.44 None 10.6 8 Facilitates ER transport along microtubules in association with kinesin to support focal adhesion growth
21313162 Ras-related protein Rab-1B [Mus musculus] Rab1B 22.17 200 (High) 61.2 10 Intracellular membrane trafficking, protein transport, autophagy, vesicle fusion
568928433 Sodium- and chloride-dependent glycine transporter 1 isoform X2 [Mus musculus] SLC6A9 72.312 490 (High) 17.5 9 Amino acid transporter, neurotransmitter transport
Recipes
Lysis buffer base
25 mM HEPES (1.25 mL of 1 M HEPES, pH 7.5)
25 mM NaCl (250 µL of 5 M NaCl)
1 mM EDTA (100 µL of 500 mM EDTA)
Up to 50 mL with Milli-Q water
Store at RT
Lysis buffer – working solution
10 mL of lysis buffer base
1 Pierce protease inhibitor mini tablet, EDTA-free
10 µL of phosphatase inhibitor
10 µL of HDSF (depalmitoylation inhibitor)
Store at -20 °C
Binding buffer
100 mM HEPES (5 mL of 1 M HEPES, pH 7.5)
1 mM EDTA (100 µL of 500 mM EDTA)
1% SDS (5 mL of 10% SDS solution)
Up to 50 mL with reverse osmosis (RO) water
Store at RT
Blocking buffer
100 mM HEPES (5 mL of 1 M HEPES, pH 7.5)
1 mM EDTA (100 µL of 500 mM EDTA)
2.5% SDS (12.5 mL of 10% SDS solution)
Up to 50 mL with RO water
Store at RT
5× Laemmli sample buffer
3.0 g of SDS
15 mL of 0.5 M Tris-HCl, pH 6.8
15 mL of 100% glycerol
0.02% bromophenol blue
Aliquot 950 µL per tube and store at -20 °C
Before use, add 50 µL of β-ME and 10 mM DTT (10 µL of 1 M DTT) to the tube and vortex to mix thoroughly.
1× Laemmli sample buffer
Dilute 5× Laemmli sample buffer (with β-ME and DTT) with autoclaved distilled/Milli-Q water. Vortex to mix thoroughly. Store at -20 °C.
1 M dithiothreitol (DTT)
154.2 mg of DTT
1 mL of autoclaved Milli-Q water
Aliquot and store at -20 °C
100% TCA stock preparation (w/v)
Dissolve 27.5 g of TCA in 20 mL of Milli-Q water.
After dissolving, make up to 50 mL with Milli-Q water
Store at 4 °C
1× SDS-PAGE running buffer
500 mL of 10× Tris-glycine SDS running buffer
4500 mL of RO water
Mix thoroughly and store at RT
70% acetone
70 mL of 100% acetone + 30 mL of autoclaved Milli-Q water
Store at RT
2 M hydroxylamine (freshly prepare for every assay)
69.49 mg of hydroxylamine-HCl
Dissolve with 300 µL of autoclaved Milli-Q water
Adjust to pH 7.5 with NaOH
Make up to 500 µL with autoclaved Milli-Q water
2 M sodium chloride
200 µL of 5 M NaCl + 300 µL of autoclaved Milli-Q water
Store at RT
5 M sodium hydroxide
Dissolve 10 g of NaOH pellets in 40 mL of autoclaved Milli-Q water and let it cool
Bring the final volume to 50 mL
Store at RT in the corrosive cabinet
10% Triton-X 100
Dissolve 5 mL of Triton X-100 in 45 mL of autoclaved Milli-Q water
Mix thoroughly and store at 4 °C
Acknowledgments
This research was supported by the National Institutes of Health, grant number RO1EY028959 (SK), and West Virginia University Bridge funding (SK). Also, we thank Dr. Ramamurthy for his valuable suggestions and support in developing this acyl-RAC protocol.
Competing interests
The authors declare no competing interests.
Ethics
The experimental procedures involving animals were performed in agreement with National Institutes of Health (NIH) guidelines. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at West Virginia University.
References
Baker, S. A., Haeri, M., Yoo, P., Gospe, S. M., Skiba, N. P., Knox, B. E. and Arshavsky, V. Y. (2008). The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors. J Cell Biol 183(3): 485-498.
Burns, M. E. and Arshavsky, V. Y. (2005). Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction. Neuron 48(3): 387-401.
Edmonds, M. J., Geary, B., Doherty, M. K. and Morgan, A. (2017). Analysis of the brain palmitoyl-proteome using both acyl-biotin exchange and acyl-resin-assisted capture methods. Sci Rep 7(1): 1-13.
Forrester, M. T., Hess, D. T., Thompson, J. W., Hultman, R., Moseley, M. A., Stamler, J. S. and Casey, P. J. (2011). Site-specific analysis of protein S-acylation by resin-assisted capture.J Lipid Res 52(2): 393.
Kolandaivelu, S., Huang, J., Hurley, J. B. and Ramamurthy, V. (2009). AIPL1, a protein associated with childhood blindness, interacts with α-subunit of rod phosphodiesterase (PDE6)and is essential for its proper assembly. J Biol Chem 284(45): 30853-30861.
Maeda, A., Okano, K., Park, P. S. H., Lem, J., Crouch, R. K., Maeda, T. and Palczewski, K. (2010). Palmitoylation stabilizes unliganded rod opsin. Proc Natl Acad Sci U S A 107(18): 8428-8433.
Murphy, J. and Kolandaivelu, S. (2016). Palmitoylation of Progressive Rod-Cone Degeneration (PRCD) Regulates Protein Stability and Localization. J Biol Chem 291(44): 23036-23046.
Myers, B., Sechrest, E. R., Hamner, G., Motipally, S. I., Murphy, J. and Kolandaivelu, S. (2022). R17C Mutation in Photoreceptor Disc-Specific Protein, PRCD, Results in Additional Lipidation Altering Protein Stability and Subcellular Localization. Int J Mol Scis 23(18): 10802.
Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y. and Yao, X. (2008). CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein Eng Des Sel 21(11): 639-644.
Yang, Z. and Wensels, T. G. (1992). N-Myristoylation of the Rod Outer Segment G Protein, Transducin, in Cultured Retinas. J Bio Chem 267(32): 23197-23201.
Article Information
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© 2023 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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Biochemistry > Protein > Posttranslational modification
Neuroscience > Sensory and motor systems > Retina
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Anti-tumor Efficacy of CD19 CAR-T in a Raji B Cell Xenografted Mouse Model
QX Qian Xiao
XS Xiaolei Su
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4655 Views: 1612
Reviewed by: Aleksei TikhonovShengwen Calvin Li Anonymous reviewer(s)
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Bio-protocol journal peer-reviewed
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Preprint
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Original Research Article:
The authors used this protocol in Science Immunology Aug 2022
Abstract
Chimeric antigen receptor (CAR)-T therapy launched a new era for cancer treatments, displaying outstanding effectiveness in relapsed or refractory B-cell malignancies. Demonstrating the tumor-killing ability of CAR-Ts in mouse xenograft models serves as a golden criterium in preclinical research. Here, we describe a detailed method for evaluating CAR-T’s function in immune-deficient mice bearing Raji B cell–induced tumors. It includes generating CD19 CAR-T cells from healthy donors, injecting tumor cells and CAR-T cells into mice, and monitoring tumor growth and CAR-T state. This protocol provides a practical guide to evaluate CAR-T’s function in vivo within eight weeks.
Graphical overview
Keywords: Chimeric antigen receptor CD19 CAR-T Raji B cell Xenografted tumor model In vivo tumor killing assay
Background
Chimeric antigen receptor (CAR)-T-cell therapy exploits an antibody-dependent, major histocompatibility complex–independent antigen recognition paradigm to specifically target cancer cells using genetically engineered T cells. It is one of the most sought-after tools for treating hematologic malignancies (Daniyan and Brentjens, 2016; Posey et al., 2016). Recently, extensive studies have revealed the molecular mechanisms of CAR-T activation, such as charged lipids and ions–regulated CD28 conformation and signaling (Yang et al., 2017) and LAT-independent formation of CAR microclusters (Dong et al., 2020); SILAC-based phosphoproteomics revealed unique signaling circuits of CAR-Ts, and the distance of synapse from CAR-T/tumor cells was found to affect T-cell function (Xiao et al., 2022). Understanding CAR-T signaling further paved the way to develop new strategies to improve its function (Davenport et al., 2018; Majzner et al., 2020).
Generating CAR-Ts of high quality is a crucial step for validating their function in vitro and in vivo. Virus-based gene delivery systems are commonly used and can achieve high transduction efficiencies (Vormittag et al., 2018). Lentiviral and gamma-retroviral vectors are the most frequently used viral vector systems (Vannucci et al., 2013). Compared to retrovirus, which only infect proliferating cells, lentivirus could infect both proliferating and non-proliferating cells. Thus, lentivirus can infect a broader range of cell types than retroviruses. Meanwhile, lentiviruses need regulatory genes to neutralize the host's anti-viral response and regulate viral replication (Vannucci et al., 2013). They also require a large amount of plasmid for transient transfection to produce a high titer of lentivirus (Merten et al., 2016).
Immunodeficient mice (e.g., NSG, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJl) are widely used to study the interactions between the human immune system and cancer. They offer a low-barrier platform for evaluating immunotherapeutic effects in the context of human immune cells and tumors (Shultz et al., 2014). Although some of those immunodeficient aged mice develop T and B cells, which causes the leakiness of immune cells (Allen et al., 2019), these mice are the gold standard host for engraftment of human tumors or for establishing human immune components following a hematopoietic stem cell transplant. Furthermore, they allow precise preclinical evaluations of antibody-based therapeutics, cancer vaccines, checkpoint inhibitor therapies, and adoptive cancer immunotherapies (Shultz et al., 2014). NSG mice xenografted with Raji (lymphoma) tumor cells are among the most used and recognized models for evaluating CAR-Ts' efficacy and toxicity targeting CD19 (Pegram et al., 2015; Majzner et al., 2020).
Here, we provide a detailed step-by-step protocol describing how to evaluate CD19 CAR-Ts tumor cell–killing effect in vivo. The protocol covers the generation of CD19 CAR-T cells from healthy donors, injection of tumor cells and CAR-T cells into mice, and monitoring of tumor growth and CAR-T state.
Materials and Reagents
T-25 or T-75 flask (Thermo Fisher, catalog number: 156499 or 156367)
15 mL conical centrifuge tubes (Thermo Fisher, catalog number: 14-959-53A)
50 mL conical centrifuge tubes (Thermo Fisher, catalog number: 14-959-49A)
Non-tissue culture–treated 24-well plates (Sigma, catalog number: CLS3738)
Tissue culture–treated 24-well or 6-well plates (StemCell, catalog number: 38017 or 38016)
Plastic wrap (GLAD Flex’N Seal Food Storage Plastic Bag)
1.5 mL polypropylene microcentrifuge tubes (Fisher Scientific, catalog number: 07-000-243)
5 mL polypropylene snaplock microcentrifuge tube (Fisher Scientific, catalog number: 14-568-101)
5 mL disposable pipettes (VWR, catalog number: 76201-710)
VWR individual tubes (FACS tube) (VWR, catalog number: 83009678)
1 mL syringe (Covidien, catalog number: 1180125158)
Alcohol prep pad (Dukal, catalog number: 853)
Blades (VWR, catalog number: 55411-050)
Micro-hematocrit capillary tubes (Thermo Fisher, catalog number: 22-363-566)
0.22 µm filter (Sigma Millipore, catalog number: GSWP04700)
NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJl) (The Jackson Laboratory, Strain #005557)
Cell line: Raji B cell (ATCC, catalog number: CCL-86)
Human peripheral blood mononuclear cells (PBMCs), FRESH (Zenbio, #SER-PBMC-200)
CD19 CAR constructs: pHR-SP-CD19 ScFv (FMC63) – myc-CD8 Stalk-CD8 (CD28-41BB-CD3) cyto-sfGFP (Su lab, plasmid #XSB848) (Xiao et al., 2022)
Lentiviral packaging plasmids (second generation): pMD2.G and psPAX2 (Addgene, #12259 and #12260)
Polybrene (Sigma, catalog number: H9268)
OKT-3 antibody (Thermo Fisher, catalog number: 11161D)
Recombinant human IL-2 (PeproTech, catalog number: 200-02)
1× Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher, catalog number: 10010023)
GibcoTM HEPES (1 M) (Thermo Fisher, catalog number: 15-630-080)
1 mg/mL retronectin (Takara, catalog number: T100A/B, stock solution in DPBS)
BSA (Sigma, catalog number: A7030)
AIMV (Thermo Fisher, catalog number: 12055083)
RPMI 1640 (Thermo Fisher, catalog number: 11875093)
Human serum (GemCell, catalog number: 100-512)
Penicillin-Streptomycin-Glutamine (PSG) (Life Tech, catalog number: MT30009CI)
Heat-inactivated fetal bovine serum (FBS) (Life Tech, catalog number: 16140071)
PSG (Life Tech, catalog number: MT30009CI)
EDTA (Thermo Fisher, catalog number: 15-575-020)
Human TruStain FcXTM (Fc receptor blocking solution) (BioLegend, catalog number: 4223020)
Anti-human CD3 antibody (BD Biosciences, catalog number: 558117)
Anti-human CD8 antibody (BioLegend, catalog number: 344750)
Anti-human CD4 antibody (BioLegend, catalog number: 300508)
Anti-human CD279 (PD-1) antibody (BioLegend, catalog number: 621608)
Anti-human CD366 (Tim-3) antibody (BioLegend, catalog number: 345012)
Anti-human CD223 (LAG-3) antibody (BioLegend, catalog number: 345012)
Monoclonal Anti-FMC63 scFv antibody (ACROBiosystems, catalog number: FM3-Y45)
Red blood cell lysing buffer (Sigma, catalog number: R7757-100ML)
UltraComp eBeads Plus compensation beads (Thermo Fisher, catalog number: 01-3333-42)
Matrigel (VWR, catalog number: 47743-716)
DPBS/2% BSA (see Recipes)
T-cell culture media (T-cell media) (see Recipes)
Cell culture media for Raji B cells (Raji media) (see Recipes)
FACS staining buffer (see Recipes)
Equipment
Water bath (Thermo Scientific Precision, model: UX-12150-00)
Cell culture incubator, 37 °C with 5% CO2 (Thermo Fisher, catalog number: 51033776)
Flow cytometer analyzer (BD, LSR II Flow Cytometer or Thermo Fisher, Attune Flow Cytometers)
Centrifuge with a swinging rotor and plate holder (Thermo Fisher, catalog number: 75009210)
Digital Electronic Caliper (Fine Science Tools, catalog number: 30087-00)
EasySepTM Magnet (StemCell, catalog number: 18000)
Automated Cell Counters (Thermo Fisher, model: Countess 3)
Software
FlowJo software (BD, https://www.flowjo.com/solutions/flowjo)
Prism software (GraphPad by Dotmatics, https://www.graphpad.com/)
Procedure
Generation of CD19 CAR-T cells
Prepare T-cell media as in Recipes. Store the medium at 4 °C and warm it to 37 °C before usage.
Note: The T-cell medium should be used within two months.
(Day 1) Count donor PBMC number. Plate 7.5 × 106 cells per well of a 24-well plate in 2 mL of T-cell media containing 300 IU/mL of IL2 and 50 ng/mL of OKT-3.
Note: OKT-3 antibody could be replaced by DynabeadsTM Human T-Activator CD3/CD28 (Thermo Fisher, #11161D or equivalent). Prepare the beads according to the user manual.
(Day 2) Coat non-tissue culture–treated 6-well plates with retronectin at a final concentration of 20 µg/mL in 1.5 mL of DPBS per well. Wrap the plate with plastic wrap and incubate the plate overnight at 4 °C.
Note: Retronectin incubation could be done at 37 °C in a 5% CO2 incubator for 4–6 h. Typically, two 6-well plates (12 wells, 18 mL of retronectin buffer) will be used for each CAR-T transduction.
(Day 3) Warm up centrifuge to 32 °C.
Remove retronectin solution from the 6-well plate using 5 mL disposable pipettes, and then block the plate with 2 mL of DPBS/2% BSA for 30 min at room temperature.
Thaw a lentivirus stock on ice.
Wash each well with 2 mL of DPBS and leave DPBS in the well of the retronectin-coated plates.
Calculate the volume of lentivirus for transduction based on T-cell number (0.5–1 million) and multiplicity of infection (MOI) (10–50).
Note: MOI could be adjusted (5–100) based on your virus titer and total amount. When transducing T cells with lentivirus, the MOI should be calculated. In general, the protocol for the viral transduction must be optimized for each virus independently. The presented step-by-step instruction offers a starting point.
Here is an example:
- Number of T cells to be transduced: 106 cells
- Viral titer: 2 × 108 TU/mL
- MOI: 20
Example:
(Number of cells/viral titer) × MOI = Volume of viral vector
(1 × 106 cells/2 × 108 TU/mL) × 20 = 100 μL
Add the above calculated amount of lentivirus to the T-cell culture medium. Then, remove DPBS from the plate and add 2 mL/well of viral medium.
Wrap the plate with plastic wrap and spin the plate at 2,000 × g for 2 h at 32 °C.
During the spin, harvest, wash, and count T cells to be transduced.
Note: It is recommended to include a negative control of un-transduced wells and a positive control (e.g., GFP) of transduced wells.
When the centrifugation of plates is finished, remove the viral medium.
Add 0.5 × 106–1 × 106 T cells per well in the T-cell culture media (300 IU/mL of IL-2) at the concentration of 0.25 × 106 cells per well containing 8 µg/mL polybrene.
Wrap the plate and spin at 500 × g for 10 min with the acceleration and brake set to the lowest setting at 32 °C.
Gently remove the plate from the centrifuge and leave it in the incubator overnight.
(Day 4) Transfer transduced cells onto a new tissue culture–treated 6-well plate to get rid of retronectin. Cells from the identically treated wells can be combined into a T-25 or T-75 flask at a concentration of 0.5 × 106 cells per well.
Verify CAR-GFP expression using fluorescence microscopy, if available (see Figure 1 for representative data).
Figure 1. Detection of T-cell population (A), CAR expression by GFP (B), and anti-FMC63 antibody (C). Un-transduced T cells and CD19 CAR-T cells were pre-gated on the CD3+ population; CD4+ and CD8+ were gated from CD3+; CAR-T cells were gated based on GFP expression or anti-FMC63 staining. Data were first published in Xiao et al. (2022) and presented in a modified way.
(Day 6) Perform flow cytometry analysis of CAR-GFP expression, three days post-transduction (see Procedure B) (see Figure 1 for representative data).
Continue to inspect T-cell culture daily. Cell numbers were calculated using an Automated Cell Counter. Split culture when cells reach a density of 1 × 106 cells/mL.
Note: A 9–12-day expansion period should be adequate to obtain enough CAR-T cells for mouse injection. Upon stimulation with DynabeadsTM, expression of CD3 will be transiently downregulated, which might affect the staining of CD3 on CAR-T.
Flow cytometry analysis of CAR-GFP expression
Count the cell number of transduced CD19 CAR-T cells and un-transduced T cells. Spin down 0.5 million cells for 5 min (300–400 × g) at 4 °C and discard the supernatant.
Resuspend the cell pellet in FACS staining buffer and centrifuge cells as in Step B1. Then, resuspend the pellet in 50 μL of FACS staining buffer to reach a final cell density of 1 × 107/mL.
[Optional] Block non-specific Fc-mediated interactions: pre-incubate the cells with 5 μL of human TruStain FcXTM per 100 μL for 5–10 min on ice before staining.
Add the recommended amount of fluorochrome-labeled antibody to cells and incubate in the dark for 30 min on ice (e.g., 2.5 μL of anti-human CD3 antibody, 2.5 μL of anti-human CD8 antibody, and 2.5 μL of anti-human CD4 antibody for 0.5 million cells in 100 μL of FACS staining buffer).
Note: Higher temperatures and/or a longer incubation time may lead to non-specific cell labeling.
Staining performed on ice requires increased incubation times.
Add 1 mL of FACS staining buffer to each 1.5 mL Eppendorf tube. Centrifuge samples at 300–400 × g for 5 min at room temperature and then discard the supernatant.
Resuspend stained cells in an appropriate volume (400–600 μL) of FACS staining buffer.
Prepare single-color compensation controls and set up the compensation following the manual (MAN0019374_UltraComp_eBeads_and_UltraComp_eBeads_Plus_Compensation_Beads_PI).
Once the compensation is set up, acquire data on the flow cytometer LSRII or Attune Flow Cytometers.
Note: Cells must be analyzed within 1 h after staining.
The T-cell purity and CAR expression were analyzed by FlowJo software (see Figure 1 for representative data).
Note: We recommend using CAR-T cells with higher transduction (~50%) for in vivo study. Please increase the volume of virus to achieve higher transduction.
In vivo Raji B tumor killing assay
Order 6–7-week-old NSG mice from The Jackson Laboratory. Once the mice are delivered, let them adapt to the local facility for one week before performing tumor cell injection.
Thaw and culture Raji B cells in Raji media (as in Recipes) for 3–5 days before injecting them into mice.
Count Raji cell numbers and centrifuge them at 300–400× g for 10 min at 4 °C. Discard the supernatant.
Resuspend Raji cells with pre-cooled PBS. Centrifuge cell suspension at 300–400 × g for 10 min at 4 °C and discard the supernatant. Repeat once.
Resuspend Raji cells with pre-cooled PBS (1 × 106 Raji cells in 50 µL for each mouse) and transfer the cells into a 1.5 mL Eppendorf tube.
Mix cells with an equal volume of Matrigel (Matrigel is stored in a -80 °C freezer. It needs to be thawed on ice), e.g., 50 µL of cells with 50 µL of Matrigel. This process needs to be performed on ice.
Transfer the mixed cells on ice to the animal facility for tumor injection.
Before injection, pellet mixed cells and avoid bubbles. Take 100 µL of mixture via 1 mL syringe.
Perform a subcutaneous injection. Wet the right flank of mice with an alcohol prep pad and directly inject the cells into the mouse.
Seven days later, prepare for CAR-T cell infusion. The tumor size should be between 20 and 100 mm3 at the time of the T-cell injection.
Count CD19 CAR-T cell number and centrifuge CAR-T cells at 300–400× g for 10 min at 4 °C. Discard the supernatant.
Resuspend CD19 CAR-T cells with pre-cooled PBS and centrifuge cell suspension at 300–400 × g for 10 min at 4 °C; then, discard the supernatant. Repeat once.
Resuspend CD19 CAR-T cells with pre-cooled PBS (1 × 107 cells in 100 µL for each mouse) and transfer cells into a 1.5 ml Eppendorf tube.
Infuse 1 × 107 CD19 CAR-T cells in 100 µL of PBS to tumor-bearing NSG mice intravenously; the control mice will be treated with 100 µL of PBS via tail-vein injection.
Monitor mice's body weight and tumor size every other day. Measure the tumor size using a caliper. Tumor volume is calculated with the formula: V = ½ (length × width2) (see Figure 2 for representative data).
Figure 2. Monitoring body weight (A) and tumor size (B) in PBS-treated and CD19 CAR-T treated Raji-bearing mice. (A) Mouse body weight was measured after CAR-T cell infusion. (B) Individual tumor growth from the untreated PBS group (blue, seven mice) and CD19 CAR-Ts treated group (red, six mice) is presented. Data were first published in Xiao et al. (2022) and presented in a modified way.
On day 7, 14, and 21 post-CAR-T cells infusion, tail blood is taken by blade and capillary to verify CAR-T cell percentage and exhaustion status (see Procedure D) (representative data is shown in Figures 3 and 4).
Figure 3. Representative data of CAR-T cell percentage in circulation. On days 7, 14, and 21 after CAR-T infusion, the tail vein blood of mice from untreated and treated groups were harvested to examine CAR-T cell percentage by FACS. The percentage of CAR-Ts in circulation could be used as a parameter to determine CAR-Ts persistence in vivo. Data were first published in Xiao et al. (2022) and presented in a modified way.
Figure 4. Representative data of T-cell exhaustion markers expressed on CD19 CAR-Ts. On days 14 and 21 after CAR-Ts infusion, the tail vein blood of mice was harvested to examine the expression of T-cell exhaustion markers, including PD-1, TIM-3, and LAG-3 by FACS (isotype antibody served as staining negative control). Data were first published in Xiao et al. (2022) and presented in a modified way.
Euthanize mice using CO2 inhalation when tumor size reaches ∼1,500 mm3.
Monitoring CAR-T cell percentage and exhaustion status in vivo
On days 7, 14, and 21 after CAR-T cell infusion, use a sterile scalpel blade to cut the tail. Clip no more than 1 mm of tail tissue and collect blood by gently milking the tail through the capillary.
Stop blood flow by applying pressure with sterile gauze.
Transfer blood to 100 µL of PBS containing 3 mM EDTA in a 1.5 mL Eppendorf tube.
Centrifuge cell suspension at 300–400 × g for 10 min at 4 °C and discard supernatant.
Incubate cells with 0.2 mL of red blood cell lysis buffer at room temperature for 5 min to remove erythrocytes. Top up the tube with 1 mL of FACS staining buffer and pellet cells (300–400 × g) at 4 °C for 5 min.
Repeat Step D5 once or twice if necessary.
Resuspend cells with 100 µL of FACS staining buffer.
Add fluorochrome-labeled antibodies to cells and incubate in the dark for 30 min on ice. Prepare single-color compensation controls and set up the compensation.
Add 1 mL of FACS staining buffer to each sample. Centrifuge samples at 300–400 × g for 5 min at room temperature and discard the supernatant.
Resuspend stained cells in 400–600 µL of FACS staining buffer and acquire data on the flow cytometer LSRII or Attune Flow Cytometers (for representative data see Figures 3 and 4).
Recipes
DPBS/2% BSA
10 g of BSA in 500 mL of DPBS
Filter through a 0.22 µm filter
T-cell culture media (T-cell media)
45% AIMV
45% RPMI 1640
10% human serum
1% PSG
2.5% HEPES (1 M)
Cell culture media for Raji B cells (Raji media)
RPMI 1640, 10% heat-inactivated FBS, 1% PSG
FACS staining buffer
DPBS with 2% FBS and 1 mM EDTA
Acknowledgments
X.S. was supported by an American Cancer Society Research Scholar Grant, the Charles H. Hood Foundation Child Health Research Awards, the Andrew McDonough B+ Foundation Research Grant, the Gilead Sciences Research Scholars Program in Hematology/Oncology, the Rally Foundation and Bear Necessities Foundation A Collaborative Pediatric Cancer Research Awards Program, the Rally Young Scholar Program, the Yale SPORE in skin cancer DRP Award CA121974, the Yale DeLuca Pilot Award, the NIGMS MIRA (R35) program GM138299, and the Human Frontier Science Program Early-Career Research Grant. FACS Services, results, and/or products in support of the research project were generated by the Rutgers Cancer Institute of New Jersey Immune Monitoring Shared Resource, supported in part by funding from the NCI-CCSG P30CA072770-5920. This protocol is derived from the original research paper (Xiao et al., 2022; 10.1126/sciimmunol.abl3995).
Competing interests
X.S. is a co-applicant for a provisional patent on CAR, which is not based on the specific results in this manuscript. The other authors declare that they have no competing interests.
Ethics
Mice were housed under specific pathogen–free conditions and cared for in accordance with U.S. National Institutes of Health guidelines, and all procedures were approved by the Rutgers University Animal Care and Use Committee (PROTO202100020).
References
Allen, T. M., Brehm, M. A., Bridges, S., Ferguson, S., Kumar, P., Mirochnitchenko, O., Palucka, K., Pelanda, R., Sanders-Beer, B., Shultz, L. D., et al. (2019). Humanized immune system mouse models: progress, challenges and opportunities. Nat Immunol 20(7): 770-774.
Daniyan, A. F. and Brentjens, R. J. (2016). At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies. J Leukoc Biol 100(6): 1255-1264.
Davenport, A. J., Cross, R. S., Watson, K. A., Liao, Y., Shi, W., Prince, H. M., Beavis, P. A., Trapani, J. A., Kershaw, M. H., Ritchie, D. S., et al. (2018). Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc Natl Acad Sci U S A 115(9): E2068-E2076.
Dong, R., Libby, K. A., Blaeschke, F., Fuchs, W., Marson, A., Vale, R. D. and Su, X. (2020). Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR). EMBO J 39(16): e104730.
Majzner, R. G., Rietberg, S. P., Sotillo, E., Dong, R., Vachharajani, V. T., Labanieh, L., Myklebust, J. H., Kadapakkam, M., Weber, E. W., Tousley, A. M., et al. (2020). Tuning the Antigen Density Requirement for CAR T-cell Activity. Cancer Discov 10(5): 702-723.
Merten, O. W., Hebben, M. and Bovolenta, C. (2016). Production of lentiviral vectors. Mol Ther Methods Clin Dev 3: 16017.
Pegram, H. J., Purdon, T. J., van Leeuwen, D. G., Curran, K. J., Giralt, S. A., Barker, J. N. and Brentjens, R. J. (2015). IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia. Leukemia 29(2): 415-422.
Posey, A. D., Jr., Schwab, R. D., Boesteanu, A. C., Steentoft, C., Mandel, U., Engels, B., Stone, J. D., Madsen, T. D., Schreiber, K., Haines, K. M., et al. (2016). Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity 44(6): 1444-1454.
Shultz, L. D., Goodwin, N., Ishikawa, F., Hosur, V., Lyons, B. L. and Greiner, D. L. (2014). Human cancer growth and therapy in immunodeficient mouse models. Cold Spring Harb Protoc 2014(7): 694-708.
Vannucci, L., Lai, M., Chiuppesi, F., Ceccherini-Nelli, L. and Pistello, M. (2013). Viral vectors: a look back and ahead on gene transfer technology. New Microbiol 36(1): 1-22.
Vormittag, P., Gunn, R., Ghorashian, S. and Veraitch, F. S. (2018). A guide to manufacturing CAR T cell therapies. Curr Opin Biotechnol 53: 164-181.
Xiao, Q., Zhang, X., Tu, L., Cao, J., Hinrichs, C. S. and Su, X. (2022). Size-dependent activation of CAR-T cells. Sci Immunol 7(74): eabl3995.
Yang, W., Pan, W., Chen, S., Trendel, N., Jiang, S., Xiao, F., Xue, M., Wu, W., Peng, Z., Li, X., et al. (2017). Dynamic regulation of CD28 conformation and signaling by charged lipids and ions. Nat Struct Mol Biol 24(12): 1081-1092.
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In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network
TL Tong Liu
MX Mengbo Xu
NM Na Mi
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4656 Views: 747
Reviewed by: Oneil Girish BhalalaZhongmin Liu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Cell Research Jul 2022
Abstract
In cells, p62/SQSTM1 undergoes liquid–liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.
Keywords: p62 SQSTM1 Phase separation Actin Cytoskeleton Arp2/3 Reconstitution
Background
Liquid–liquid phase separation (LLPS) plays critical roles in orchestrating biomolecular function and assembly of membrane-less organelles during diverse cellular processes (Banani et al., 2017). How LLPS is spatiotemporally regulated in the context of complicated cellular structures and components remains largely unknown. We recently revealed that cytoskeleton, as a solid phase, can actively participate in the phase separation of liquid-phase protein condensates, by using p62 bodies as a model system (Feng et al., 2022). P62/SQSTM1, a selective autophagy receptor, mediates LLPS of poly-ubiquitinated proteins into large condensates, which are also known as p62 bodies (Sun et al., 2018; Zaffagnini et al., 2018). The phase-separated p62 bodies can act as nucleation site for autophagosome formation, being then degraded by autophagy. Failure or inappropriate formation of p62 bodies is associated with pathological development of neurodegenerative diseases and Paget's disease, a chronic bone disease characterized by abnormal and excessive bone loss (Wong and Cuervo, 2010; Zaffagnini et al., 2018).
Despite p62 protein and polyubiquitin chain being shown to form large condensates in vitro (Sun et al., 2018), we reveal that the dendritic branched actin network is required for large p62 body formation in vivo. By establishing an in vitro reconstitution assay, we further demonstrate that such cytoskeleton components can greatly lower the protein concentration threshold during p62 body formation (Feng et al., 2022). The implication of cytoskeleton in LLPS represents an important and probably general way to achieve precise spatiotemporal regulation of biomolecular phase separation. Therefore, establishing an in vitro phase separation assay on cytoskeleton network is necessary and critical for these research fields; however, such protocol has not been clearly described before. Here, we present a detailed protocol to in vitro reconstitute p62 bodies along with branched actin network components in Lab Tek Chambered cover glasses. Actin polymerization is initiated by adding actin monomer, Arp2/3 complex, capping protein (CapZ), and PWCA [a domain consisting of a conserved C-terminal proline-rich region and WASP-homology 2 peptides plus a connector and acidic segments, which promotes Arp2/3-mediated actin polymerization (Campellone et al., 2008)]. Motor protein Myo1D is also added to facilitate phase separation of p62 and poly-ubiquitin (Ub8). The in vitro–reconstituted p62 bodies can be visualized by total internal reflection fluorescence (TIRF) and scanning electronic microscopy. This protocol provides an instruction on how to set up an in vitro phase separation assay involving cytoskeletal components.
Materials and Reagents
HEK293F cells (ATCC, catalog number: CRL-1573)
Sf9 cells (ATCC, catalog number: CRL-1711)
BL21 (DE3) E. coli cells (TransGen Biotech, catalog number: CD601-02)
Lab Tek Chambered cover glass (Thermo Fisher Scientific, catalog number: 150682)
SMM 293-TII expression medium (Sino Biological, catalog number: M293TII)
Sf-900 II SFM medium (Thermo Fisher Scientific, catalog number: 10902096)
Polyethyleneimines (PEI) (Polysciences, catalog number: 23966-2)
Amylose beads (New England Biolabs, catalog number: E8035S)
LB broth powder (Sangon Biotech, catalog number: A507002)
Isopropyl-beta-D-thiogalactopyranoside (IPTG) solution (Sangon Biotech, catalog number: B541007)
Tris (Sigma-Aldrich, catalog number: 77-86-1)
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 329-98-6)
Acetic acid (Sangon Biotech, catalog number: A501931)
Ammonium acetate (Sangon Biotech, catalog number: A600032)
Phosphate buffered saline (PBS) (HyClone, catalog number:SH80255.01)
HEPES (Sigma-Aldrich, catalog number: 7365-45-9)
Imidazole (Sigma-Aldrich, catalog number: 288-32-4)
Glycerol (Sinopharm, catalog number: 56-81-5)
DTT (dithiothreitol) (Inaclo, catalog number: 0281)
Maltose (Sigma-Aldrich, catalog number: 69-79-4)
Rosetta (DE3) pLysS-competent E. coli cells (TIANGEN, catalog number: CB108-02)
Strep-Tactin resin (IBA Lifesciences, catalog number: 2-1201-002)
D-desthiobiotin (Sigma-Aldrich, catalog number: D1411)
HisPur(tm) Ni-NTA Resin (Thermo Fisher Scientific, catalog number: 88222)
MBP Trap HP column (GE Healthcare, catalog number: 28-9187-78)
TEV protease (Solarbio, catalog number: P2060)
Sodium chloride (NaCl) (Sangon Biotech, catalog number: A610476)
PIPES (Sangon Biotech, catalog number: A620434)
Potassium chloride (KCl) (Sangon Biotech, catalog number: A610440)
MgCl2 (Sangon Biotech, catalog number: B601193)
EGTA (MedChemExpress, catalog number: HY-D0861)
Ethylenediaminetetraacetic acid (EDTA) (MedChemExpress, catalog number: HY-Y0682)
Adenosine 5'-triphosphate (ATP) (MedChemExpress, catalog number: HY-B2176)
MOPS (MedChemExpress, catalog number: HY-D0859)
Triton X-100 (Solarbio, catalog number: T8200)
Protease inhibitor cocktails (MedChemExpress, catalog number: HY-K0012)
Glucose oxidase (Solarbio, catalog number: G8030)
Catalase (Solarbio, catalog number: C8070)
Bovine serum albumin (BSA) (Solarbio, catalog number: A8010)
Methyl cellulose (MedChemExpress, catalog number: HY-125861)
Sucrose (Solarbio, catalog number: S8271)
ATP-Mg (Sigma Aldrich, catalog number: A9187)
Buffer A (see Recipes)
Buffer B (see Recipes)
Buffer C (see Recipes)
Lysis buffer (see Recipes)
Basic buffer (see Recipes)
Loading buffer (see Recipes)
High-salt buffer (see Recipes)
Wash buffer (see Recipes)
TIRF buffer (see Recipes)
Elution buffer (see Recipes)
Storage buffer (see Recipes)
Equipment
Nickel column (Bio-Rad, catalog number: 12009287)
Mono Q 5/50 GL column (GE Healthcare, catalog number: 17-5166-01)
HiTrap Q HP (GE Healthcare, catalog number: 17-1154-01)
Superdex200 Increase 10/300 GL (GE Healthcare, catalog number: 28990944)
Ultrafiltration discs (Sigma-Aldrich, catalog number: PLGC07610)
Allegra X-14 centrifuge, type 45 Ti rotor manual, JS-4.750 swinging-bucket rotor and buckets, 4 × 50 mL, 9 × 15 mL adapters (Beckman Coulter, model: Allegra X14, catalog number: A99464)
Water bath at 37 °C (Thermo Fisher Scientific, model: TSCIP19)
Incubator at 37 °C with 5% CO2 (Thermo Fisher Scientific, model: HERAcell 150i)
Confocal fluorescence microscopy (NIKON, A1 HD25)
Nikon SIM/STORM (TIRF)
Field emission scanning electron microscopy (FE-SEM) (FEI Quanta 200)
Critical Point Dryer (Shianjia Biotechnology, model: SCD-350M)
Software
NIS-Elements software (Laboratory Imaging)
ImageJ
Procedure
Purification of Arp2/3 complex
Construction of expression vector: Sub-clone the full-length open reading frames of seven subunits of human Arp2/3-ArpC1a-ArpC5 complex into pCAG vectors. To facilitate protein purification, add two strep-tags to the N-termini of ArpC1a (Doolittle et al., 2013a and 2013b).
Cell transfection: Grow HEK293F cells in SMM 293-TII expression medium to a density of 1.5–1.8 million cells per milliliter. Transiently transfect the plasmids to HEK293F cells using PEI. Mix 0.25 mg of plasmids of each subunit of complex (1.75 mg in total) and 3 mg of PEI in 50 mL of fresh medium for 25 min, then add to 800 mL of culture. Culture the transfected cells at 37 °C supplemented with 5% CO2 in a shaker for 48–72 h.
Extraction and purification of target proteins:
Harvest cells by centrifugation and resuspend in ice-cold buffer A.
Solubilize cell membrane using 1% Triton X-100 for 1 h while rotating at 4 °C.
Centrifuge cell lysates at 20,000 × g for 1 h at 4 °C using a type 45 Ti rotor to remove cell debris.
Collect and load the supernatant onto Strep-Tactin resin.
Remove unbound proteins by extensive wash with buffer B.
Elute the bound proteins with buffer B supplemented with 10 mM D-desthiobiotin.
Further purify the target protein by Superdex200 Increase 10/300 GL column using buffer C.
Pool and concentrate peak fractions containing all seven subunits to 10 mg/mL.
Purification of PWCA
Construction of vector: Clone the PWCA domain of human WHAMM protein into a pCAG vector containing a N-terminal MBP-tag (Liu et al., 2017). The PWCA domain is expressed and purified from the HEK293F cell line.
When the culture reaches a density of 1.2–1.6 million cells per milliliter, transfect cells with 1 mg of plasmids using 2 mg of PEI.
Extraction and purification of target proteins:
After 48–72 h post transfection, harvest cells and lyse in ice-cold lysis buffer for 1 h while rotating at 4 °C.
Remove the insoluble material by centrifugation at 20,000 × g for 1 h at 4 °C.
Incubate the supernatant with amylose beads for 2 h while rotating at 4 °C.
Remove unbound proteins by extensive wash with the basic buffer and elute the bound proteins using the same buffer with 10 mM maltose.
Further purify the eluted proteins by anion exchange using a Mono Q 5/50 GL column.
Load the proteins onto the Mono Q column at 1 mL/min using the loading buffer as running buffer.
Use high-salt buffer for gradient elution.
Pool and load the fractions containing the PWCA domain onto a Superdex 75 10/300 GL column for final purification.
Purification of capping protein (CapZ)
Construction of vector: Clone the cDNA of capping protein (α1β2, Mus musculus) into a pET-3d vector containing a N-terminal 6×His-tag.
Transfer the expression vector into Rosetta (DE3) pLysS-competent E. coli cells via transformation for protein expression.
Extraction and purification of target protein:
Grow bacteria in LB medium at 37 °C and induce protein expression at OD600 0.6–1.0 with 0.5 mM IPTG for 3 h.
Collect cell pellets by centrifugation and resuspend in ice-cold lysis buffer.
Lyse cells by ultrasonication; then, remove cell debris by centrifugation at 20,000 × g using the Type 45 Ti rotor at 4 °C for 1 h.
Collect the supernatant and load it onto Ni-NTA resin.
Wash the resin with wash buffer.
Elute the bound proteins with elution buffer.
Further purify the eluted proteins by anion exchange using a Mono Q 5/50 GL column.
Load the proteins onto the Mono Q column at 1 mL/min using the loading buffer as running buffer.
Use high-salt buffer for gradient elution.
Pool and load the fractions containing capping protein onto a Superdex 75 10/300 GL column for final purification.
Purification of myosin 1D
Grow Sf9 cells in sf-900 II SFM medium to a density of 2 × 106 cells/mL and incubate with virus harboring full-length Myo1D construct containing a N-terminal 6×His-tag.
After 60 h, collect cells, lyse by freeze-thaw cycles, and centrifuge at 4 °C.
Remove insoluble material by centrifugation at 20,000 × g for 30 min.
Incubate supernatants on a roller at 4 °C for 1 h with 1 mL of Ni-NTA resin and transfer into a column.
Wash the columns with wash buffer.
Elute myosin 1D with elution buffer.
The protein concentration of myosin 1D is determined by OD280.
Concentrate the protein by ultrafiltration discs (10 KD).
Store the protein in storage buffer.
Determine the purity of protein by Coomassie blue–stained SDS-poly-acrylamide gels.
Expression and purification of Ub8
Grow bacteria transformed with 6×His-Ub8 construct in LB medium at 37 °C and induce at OD600 ~0.8 with 0.5 mM IPTG at 18 °C for 16 h.
Re-suspend cells in 50 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM phenylmethylsulphonyl fluoride (PMSF).
Transfer the cell suspension on ice and sonicate to disrupt the cells, followed by centrifugation at 47,850 × g for 1 h at 4 °C.
Isolate the proteins through a Ni-NTA column.
Further purify the proteins using a Hitrap Q HP column.
Acidify the solution of Ub8 using acetic acid.
Apply to a Hitrap SP HP column.
Elute with a linear gradient of NaCl (0–1 M) in 50 mM ammonium acetate pH 4.5.
Further purify Ub8 proteins by gel filtration. Equilibrate the gel filtration columns (Superdex 200 10/300) in 150 mM NaCl, 40 mM Tris-HCl (pH 7.4), 10% glycerol, and 1 mM DTT.
Flash-freeze Ub8 proteins and store at -80 °C.
Expression and purification of p62
Transfer the MBP-mCherry-p62 construct (His6-MBP-Tev-mCherry vector backbone) into BL21 (DE3) E. coli cells via transformation and spread to bacterial culture plate for overnight culture at 37 °C.
Pick up a single colony into a liquid LB medium (approximately 15 mL) and incubate at 37 °C in a bacterial shaker at 220 rpm for 14–18 h.
Inoculate 1% of the bacterial solution into 1 L liquid LB medium and expand the culture at 37 °C.
When OD600 reaches ~0.6, add 0.2 mM IPTG to induce protein expression at 16 °C overnight.
Collect bacteria at 1,800 × g for 15 min. Wash again with PBS.
Add 30 mL of lysis buffer, vortex, and shake to fully resuspend the bacteria.
Put the resuspended bacteria into a 50 mL glass beaker on ice.
Ultrasonic crush the bacteria for 10 min, at 60% intensity, sonication for 5 s, interval 3 s.
Centrifuge the sonication solution at 13,000 × g for 30 min at 4 °C.
Collect the supernatant and purify the proteins with MBP Trap HP column.
Use TEV protease to remove MBP tag from MBP-mCherry-p62 fusion protein (Raran-Kurussi et al., 2017). Add the equivalent amount of TEV protease to the standard reaction buffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, and 1 mM DTT) containing MBP-mCherry-p62 fusion protein and incubate overnight (4 °C) or 1–2 h (37 °C).
The resultant mCherry-p62 protein is used for in vitro phase separation assay.
In vitro reconstitution of p62 bodies on the Arp2/3-derived actin network (Figure 1)
Prepare actin monomers (1 μM, 50% Oregon-green labeled) as described in another protocol (Jiang and Huang, 2017). The growth of actin filaments can be observed under a Nikon microscope equipped with a 100× oil objective by the TIRF illumination.
To initiate branched actin polymerization, add 1 μM actin (50% Oregon-green labeled), 100 nM Arp2/3 complex, 600 nM CapZ, and 300 nM PWCA to a tube, then add TIRF buffer for a 50 μL mixed solution in total.
Carefully transfer 10 μL of mixed solution to the center of a Lab Tek Chambered cover glass.
In the focal plane of the chamber, robust branched actin polymerization can be observed by TIRF microscopy.
Add 10 μM Myo1D to the solution and continue TIRF imaging.
Add 0.5 μM mCherry-p62 and 3 μM Ub8 to the solution and continue TIRF imaging.
The reconstituted p62 body can also be examined by scanning electron microscopy. For the conventional fixation procedure, grow p62 bodies on coverslips, fix with 2.5% glutaraldehyde in PBS buffer for 2 h at room temperature, wash three times with PBS buffer, and then post-fix with 1% osmium for 20 min at room temperature. Then, dehydrate all samples with a graded series of ethanol (50%, 70%, 80%, 90%, 100%, and 100%) for 2 min each and dry with Critical Point Dryer using CO2. Coat the dried samples with an approximately 5 nm thick gold film by sputter coating before examination with focused ion beam scanning electron microscopy using a Helios detector at an acceleration voltage of 2.0 kV (Rong et al., 2012).
Figure 1. Schematic of the p62 body reconstitution assay. Molecules are not drawn to scale.
Data analysis
In this reconstitution assay, all components are derived from purified proteins. Oregon-green-labeled actin monomers can self-assemble into actin filaments (Figure 2, left panel). Addition of Arp2/3 complex, CapZ, and PWCA leads to the formation of branched actin network (Figure 2, right panel). Myo1D, as a motor protein, is not necessary for the branched actin network; however, its inclusion strengthens such branched actin structures. The scaffold proteins of p62 bodies (mCherry-p62 and Ub8) are then introduced on top of branched actin network to reconstitute cytoskeleton-associated p62 bodies, which can be observed by either TIRF microscopy or scanning electron microscopy (Figure 3 and Figure 4). Then, the NIS-Elements software is used for image measurement and co-location analysis. In such in vitro assay, phase separation of p62 bodies is greatly accelerated with lower concentration threshold of scaffold proteins in the presence of branched actin network than that using p62/Ub8 alone (Feng et al., 2022). Use ImageJ to process immunofluorescence images (rotating, cropping, and adjusting brightness and contrast when necessary).
Figure 2. Total internal reflection fluorescence (TIRF) images showing the actin polymerization. Oregon-green-labeled actin filaments (left panel) and reconstituted branched actin network (right panel). Scale bar = 10 μm.
Figure 3. In vitro–reconstituted p62 bodies on branched actin network. Scale bar = 5 µm.
Figure 4. Morphology of reconstituted p62 bodies observed by FE-SEM. Field emission scanning electron microscopy (FE-SEM) image showing the structures of four reconstituted p62 bodies on the Arp2/3-derived actin network. Scale bar = 1 µm.
Notes
When mixing the components for branched actin polymerization, it is advised to firstly co-incubate PWCA and Arp2/3 complex for 5 min before mixing with actin and CapZ. Otherwise, the efficiency of branched actin polymerization will be decreased.
It is recommended to ultracentrifuge the actin monomers to remove the precipitate before use.
Recipes
Buffer A
20 mM HEPES pH 7.4
40 mM NaCl
1 mM DTT
Store at 4 °C, use within two weeks
Buffer B
20 mM PIPES pH 6.8
150 mM KCl
2 mM MgCl2
5 mM EGTA
1mM EDTA
0.5 mM DTT
0.2 mM ATP
5% glycerol
Store at 4 °C, use within two weeks
Buffer C
20 mM MOPS pH 7.0
100 mM KCl
2 mM MgCl2
5 mM EGTA
1 mM EDTA
0.5 mM DTT
0.2 mM ATP
5% glycerol
Store at 4 °C, use within two weeks
Lysis buffer
20 mM Tris pH 8.0
150 mM NaCl
1 mM DTT
1 mM EDTA
5% glycerol
1% Triton X-100
Protease inhibitor cocktails
Store at 4 °C, use within two weeks
Basic buffer
20 mM Tris pH 8.0
150 mM NaCl
1 mM DTT
1 mM EDTA
Store at 4 °C, use within two weeks
Loading buffer
20 mM Tris pH 8.0
80 mM NaCl
1 mM DTT
1 mM EDTA
Store at -20 °C
High-salt buffer
20 mM Tris pH 8.0
500 mM NaCl
1 mM DTT
1 mM EDTA
Store at 4 °C, use within two weeks
Wash buffer
20 mM HEPES pH 7.4
500 mM NaCl
1 mM DTT
5% glycerol
30 mM imidazole
Store at 4 °C, use within two weeks
TIRF buffer
1 mM MgCl2
50 mM KCl
1 mM EGTA
10 mM imidazole pH 8.0
0.2 mM ATP
100 mM DTT
100 μg/mL glucose oxidase
20 μg/mL catalase
0.2% BSA
0.5% methyl cellulose
Store at 4 °C, use within two weeks
Elution buffer
50 mM Tris pH 7.5
250 mM imidazole
300 mM NaCl
0.2 mM EGTA
Store at 4 °C, use within two weeks
Storage buffer
50 mM HEPES PH7.4
300 mM NaCl
50 μM ATP-Mg
10% sucrose
Store at 4 °C, use within two weeks
Acknowledgments
We thank Drs. Jiangfeng Shen, Xiaoyu Fu, Wanqing Du, Wenkang Zhao, Xuezhao Feng, Mingrui Ding, Jinpei Zhang and Daxiao Sun for their helpful efforts and suggestions on optimizing this protocol. This work was supported by the Ministry of Science and Technology of China (2017YFA0506300), and the National Natural Science Foundation of China (31771536 and 31860316).
Competing interests
The authors declare no competing interests.
References
Banani, S. F., Lee, H. O., Hyman, A. A. and Rosen, M. K. (2017). Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18(5): 285-298.
Campellone, K. G., Webb, N. J., Znameroski, E. A. and Welch, M. D. (2008). WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport. Cell 134(1): 148-161.
Doolittle, L. K., Rosen, M. K. and Padrick, S. B. (2013a). Purification of native Arp2/3 complex from bovine thymus. Methods Mol Biol 1046: 231-250.
Doolittle, L. K., Rosen, M. K. and Padrick, S. B. (2013b). Purification of Arp2/3 complex from Saccharomyces cerevisiae. Methods Mol Biol 1046: 251-271.
Feng, X., Du, W., Ding, M., Zhao, W., Xirefu, X., Ma, M., Zhuang, Y., Fu, X., Shen, J., Zhang, J., et al. (2022). Myosin 1D and the branched actin network control the condensation of p62 bodies. Cell Res 32(7): 659-669.
Jiang, Y. and Huang, S. (2017). Direct Visualization and Quantification of the Actin Nucleation andElongation Events in vitro by TIRF Microscopy. Bio Protoc 7(5): e2146.
Liu, T., Dai, A., Cao, Y., Zhang, R., Dong, M. Q. and Wang, H. W. (2017). Structural Insights of WHAMM's Interaction with Microtubules by Cryo-EM. J Mol Biol 429(9): 1352-1363.
Raran-Kurussi, S., Cherry, S., Zhang, D. and Waugh, D. S. (2017). Removal of Affinity Tags with TEV Protease. Methods Mol Biol 1586: 221-230.
Rong, Y., Liu, M., Ma, L., Du, W., Zhang, H., Tian, Y., Cao, Z., Li, Y., Ren, H., Zhang, C., Li, L., Chen, S., Xi, J. and Yu, L. (2012). Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation. Nat Cell Biol 14(9): 924-934.
Sun, D., Wu, R., Zheng, J., Li, P. and Yu, L. (2018). Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation. Cell Res 28(4): 405-415.
Wong, E. and Cuervo, A. M. (2010). Autophagy gone awry in neurodegenerative diseases. Nat Neurosci 13(7): 805-811.
Zaffagnini, G., Savova, A., Danieli, A., Romanov, J., Tremel, S., Ebner, M., Peterbauer, T., Sztacho, M., Trapannone, R., Tarafder, A. K., et al. (2018). p62 filaments capture and present ubiquitinated cargos for autophagy. EMBO J 37(5): e98308.
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Biochemistry > Protein > Self-assembly
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4,657 | https://bio-protocol.org/en/bpdetail?id=4657&type=0 | # Bio-Protocol Content
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Peer-reviewed
Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids
MC Man Chun Chiu
CL Cun Li
YY Yifei Yu
XL Xiaojuan Liu
JH Jingjing Huang
ZW Zhixin Wan
KY Kwok Yung Yuen
JZ Jie Zhou
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4657 Views: 1804
Reviewed by: Vivien Jane Coulson-ThomasSudhir Verma Anonymous reviewer(s)
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Cited by
Original Research Article:
The authors used this protocol in Cell Discovery Jun 2022
Abstract
A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing.
Graphical overview
Keywords: Lung organoids Airway organoids Alveolar organoids Long-term expansion Proximal differentiation Distal differentiation
Background
Recent advances in stem cell technology enable the generation of mini-organs or organs-in-a-dish, known as organoids. By definition, organoids are three-dimensional cultures derived from stem cells; they mimic the in vivo architecture and functionality of the corresponding tissue or organ (Lancaster and Knoblich, 2014; M. Li and Izpisua Belmonte, 2019; Schutgens and Clevers, 2020). Organoids have become a robust and innovative tool for modeling developmental biology, physiology, and pathology.
Organoids can be derived from either pluripotent stem cells or adult stem cells (ASC). When provided with appropriate niche factors, ASCs isolated from tissues can self-renew and self-organize into organ-like multicellular clusters composed of multiple tissue-specific cell types, which morphologically and functionally simulate the in vivo counterparts. The generation of the first ASC-derived organoid, the human intestinal organoid, was reported in 2009 (Sato et al., 2009 and 2011). Afterward, ASC-derived organoids were established for a variety of human organs and tissues, including the prostate (Chua et al., 2014; Karthaus et al., 2014), liver (Huch et al., 2013b;Hu et al., 2018), stomach (Wroblewski et al., 2015; Schlaermann et al., 2016), pancreas (Huch et al., 2013a), mammary gland (Sachs et al., 2018), and lung (Zhou et al., 2018; Sachs et al., 2019). These ASC-derived organoids retain the fundamental cellular, structural, and functional properties of the native organ and maintain genotypic and phenotypic stability during long-term culture.
The human respiratory tract is lined with two distinct types of epithelia: the airway and the alveolar epithelium. We established the long-term expandable ASC-derived human lung organoid from lung tissues in collaboration with Clevers’ lab (Zhou et al., 2018; Sachs et al., 2019). We further developed a proximal differentiation protocol and generated 3D and 2D airway organoids that morphologically and functionally phenocopy the airway epithelium to a near-physiological level (Zhou et al., 2018). A detailed protocol describing the derivation of lung organoids and the generation of differentiated airway organoids was published recently (C. Li et al., 2022). However, derivation of the alveolar epithelium, which consists of type 1 and type 2 alveolar epithelial cells (AT1 and AT2, respectively), from the lung organoids remained elusive.
We recently reported a bipotential lung organoid culture system that can generate both airway and alveolar epithelial cells (Chiu et al., 2022). Here, we provide a detailed protocol to generate 3D alveolar organoids from long-term expanding human lung organoids, the same source for generating airway organoids as previously reported. We also describe a protocol to generate optimized 2D airway organoids from lung organoids, which mimic the airway epithelium more favorably. Altogether, we established a bipotential lung organoid culture system that could enable bidirectional differentiation into alveolar organoids upon distal differentiation or airway organoids upon proximal differentiation. The lung organoids serve as a stable source for long-term expansion, while differentiated airway and alveolar organoids faithfully phenocopy the human airway and alveolar epithelium, respectively. These organoids are robust and physiologically active tools that are applicable to various experimental manipulations, to explore the biology and pathology of the human lungs.
Materials and Reagents
Nunc 15 and 50 mL conical sterile polypropylene centrifuge tubes (Thermo Scientific, catalog numbers: 339650, 339652)
Surgical scalpel blade No. 22 (Swann-Morton, catalog number: 0508)
100 mm TC-treated culture dish (Corning, catalog number: 430167)
T175 cell culture flask (Greiner Bio-One, catalog number: 661175)
Nunc non-treated 24-well plate (Thermo Scientific, catalog number: 144530)
Nunclon Sphera 24-well plate (Thermo Scientific, catalog number: 174930)
Costar 6.5 and 12 mm Transwell®, 0.4 μm pore polyester membrane inserts (Stem Cell Technologies, catalog numbers: 38024, 38023)
40 and 100 μm cell strainer (Falcon, catalog numbers: 352340, 352360)
Steritop threaded bottle top filter (0.22 μm) (Merck Millipore, catalog number: SCGPS01RE)
10/100/1,000 μL QSP low retention filtered pipette tips (Thermo Scientific, catalog numbers: TFLR102-10-Q, TFLR113-100-Q, TFLR1121000-Q)
5/10/15 mL Stripette serological pipettes (Corning, catalog numbers: 4487, 4488, 4489)
Pasteur pipettes length ca. 225 mm (Brand, catalog number: 747720)
5 mL round bottom polystyrene test tube (Falcon, catalog number: 352052)
β-BODIPYTM FL C12-HPC (Invitrogen, catalog number: D3792)
LysoTrackerTM red DND-99 (Invitrogen, catalog number: L7528)
Matrigel growth factor–reduced (GFR) basement membrane matrix, phenol red–free, LDEV-free (Corning, catalog number: 356231)
Advanced DMEM/F-12 (Gibco, catalog number: 12634010)
DMEM (Gibco, catalog number: 10569010)
ZeocinTM selection reagent (100 mg/mL) (Gibco, catalog number: R25005)
GeneticinTM selective antibiotic (G418 sulfate) (50 mg/mL) (Gibco, catalog number: 10131035)
HEPES (Gibco, catalog number: 15630056)
GlutaMAX supplement (Gibco, catalog number: 35050061)
Penicillin-Streptomycin (Gibco, catalog number: 15140122)
Recombinant human Rspondin1 (Stem Cell Technologies, catalog number: 78213; Peprotech, catalog number: 120-38)
Rspondin1 expressing 293T cell line (Sigma-Aldrich, catalog number: SCC111)
Recombinant human Noggin (Stem Cell Technologies, catalog number: 78060; Peprotech, catalog number: 120-10C)
B-27 supplement (50×), serum-free (Gibco, catalog number: 17504044)
N-Acetyl-L-cysteine (Sigma-Aldrich, catalog number: A9165)
Nicotinamide (Sigma-Aldrich, catalog number: N0636)
Y-27632 dihydrochloride (Tocris, catalog number: 1254)
A 83-01 (Tocris, catalog number: 2939)
SB 202190 (Sigma-Aldrich, catalog number: S7067)
Recombinant human KGF (FGF-7) (Peprotech, catalog number: 100-19)
Recombinant human FGF-10 (Peprotech, catalog number: 100-26)
Recombinant human heregulin β-1 (Peprotech, catalog number: 100-03)
Primocin (InvivoGen, catalog number: ant-pm-1)
Dexamethasone (Tocris, catalog number: 1126)
8-Bromo-cAMP, sodium salt (Tocris, catalog number: 1140)
IBMX (Tocris, catalog number: 2845)
CHIR 99021 (Tocris, catalog number: 4423)
Recombinant murine WNT3A (Peprotech, catalog number: 315-20)
L WNT3A cell line (ATCC, catalog number: CRL-2647)
PneumaCult-ALI medium (StemCell Technologies, catalog number: 05001)
Heparin solution (StemCell Technologies, catalog number: 7980)
Hydrocortisone stock solution (StemCell Technologies, catalog number: 7925)
DAPT (Tocris, catalog number: 2634)
PIPES (Sigma-Aldrich, catalog number: P1851)
Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C9407)
TrypLE select enzyme (10×), no phenol red (Gibco, catalog number: A1217701)
Phosphate buffered saline (PBS) (Gibco, catalog number: 10010023)
Fetal bovine serum (FBS), qualified, heat inactivated (Gibco, catalog number: 10082147)
Buffer EL erythrocyte lysis buffer (Qiagen, catalog number: 79217)
UltraPureTM 0.5 M EDTA, pH 8.0 (Invitrogen, catalog number: 15575020)
Formaldehyde solution [i.e., 37% paraformaldehyde (PFA)] (Sigma-Aldrich, catalog number: 252549)
Triton X-100 (Sigma-Aldrich, catalog number: X100)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)
DAPT (Sigma-Aldrich, catalog number: D9542)
Phalloidin - Atto 647N (Sigma-Aldrich, catalog number: 65906)
ProLongTM glass Antifade mountant (Invitrogen, catalog number: P36980)
Glutaraldehyde (purchased from electron microscope unit, HKU)
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331)
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465)
Basal medium (see Recipes)
Expansion medium (see Recipes)
Distal differentiation medium (DD medium) (see Recipes)
Proximal differentiation medium (PD medium) (see Recipes)
Rspondin1 conditioned medium (see Recipes)
Noggin conditioned medium (see Recipes)
WNT3A conditioned medium (see Recipes)
Equipment
SterilGARD e3 Class II Type A2 biosafety cabinet (Baker Co, catalog number: SG404-INT)
Forma Steri-Cycle i160 CO2 165 L incubator (Thermo Fisher Scientific, catalog number: 51030301)
New Brunswick Innova® 44/44R stackable incubator shaker (Eppendorf, catalog number: M1282-0002)
Centrifuge (Eppendorf, model: 5810R)
FE20-Kit FiveEasy benchtop pH meter (Mettler Toledo, model: FE20-KIT)
LightCycler 96 Instrument (Roche, catalog number: 381711)
Inverted routine microscope (Nikon, model: Eclipse TS100)
Confocal microscope (Zeiss, model: LSM 800)
Transmission electron microscope (FEI, model: Tecnai G2 20 S-TWIN)
Cell analyzer (BD Biosciences, model: FACSCanto II & LSRFortessa)
Midi Plus pipetting controllers (Sartorius, model: 710931)
Research Plus mechanical pipette (Eppendorf, catalog number: 3123000900)
Procedure
Derivation and expansion of lung organoids
Preparation
Prepare basal medium (Recipe 1) and keep it on ice or at 4 °C.
Prepare expansion medium (Recipe 2) and prewarm at 37 °C.
Thaw matrigel at 4 °C and keep it on ice.
Note: Matrigel will solidify at room temperature.
Prewarm a 24-well suspension culture plate in a standard cell culture incubator with 5% CO2 and humidified atmosphere at 37 °C.
Prepare glass Pasteur pipettes with a narrow opening (≤1 mm) by burning the tips with a flame, such as a Bunsen burner, to narrow the opening from ~1.5 to ≤1 mm. Cool down the Pasteur pipettes. Autoclave the Pasteur pipettes prior to use.
Note: The burning procedure also aims to smoothen the sharp edge of the glass pipette and reduce cell damage. Any fine tip Pasteur pipette with a narrow opening (≤1 mm) and smooth edge can be used instead.
Rinse the Pasteur pipettes with 2–3 mL of basal medium by pipetting up and down a few times to avoid excessive attachment of cells before using them to mechanically shear the organoids (i.e., steps A3e and B2e).
Derivation of lung organoids
Obtain freshly resected lung tissues from patients who underwent surgical operations due to various diseases. Transport the tissue in the cold basal medium (and preferably on ice at 4 °C) and process it as soon as possible.
Note: The lung tissues/biopsies should be resected from the distal lung region, should be composed of normal tissues adjacent to diseased tissues (with both bronchioles and alveoli), and should be ~0.5 cm in diameter.
Mince the tissue into small pieces (≤1 mm) with a sterile scalpel in a 100 mm cell culture dish. Wash the tissue pieces with 10 mL of cold basal medium and transfer them to a 15 mL centrifuge tube. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Resuspend the tissue pieces in 8 mL of cold basal medium supplemented with collagenase at a final concentration of 2 mg/mL. Digest the tissue pieces for 30–40 min at 37 °C in a shaking incubator at 120 rpm.
Shear the digested tissue pieces by pipetting up and down 20 times using a 10 mL serological pipette. Filter the cell suspension through a 100 μm cell strainer.
(Optional) Recover remaining tissue pieces from the cell strainer with cold basal medium and transfer them to a 15 mL centrifuge tube for a second round of mechanical shearing by pipetting and then filtering to increase the cell yield.
Add FBS to the flowthrough with a final concentration of 2% to terminate enzymatic digestion. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
(Optional) Resuspend the pellet in 2 mL of erythrocyte lysis buffer and incubate for 5 min at room temperature to remove the red blood cells.
Wash the cells with 10 mL of cold basal medium. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Resuspend the pellet in cold matrigel. Add 80–160 μL of matrigel for cells obtained from a lung tissue of size ~0.5 cm in diameter. Dispense 40 μL of the cell suspension to each well of a prewarmed 24-well suspension culture plate and incubate for 10–15 min at 37 °C in a standard cell culture CO2 incubator to let the matrigel droplet solidify.
Add 500 μL of expansion medium to each well and incubate the plate in a standard cell culture CO2 incubator. Replenish the medium every 2–3 days with caution not to disrupt the matrigel droplets. Observe the organoids and monitor their growth under a light microscope regularly (Figure 1).
Note: In the initial culture, there are non-epithelial cell types such as lung fibroblasts, which will gradually disappear after 2–3 passages.
Figure 1. Derivation and expansion of lung organoids. (A). Top, a schematic graph outlines the derivation and expansion of lung organoids (LO) in expansion medium (Exp medium). Bottom, photomicrographs show growing lung organoids derived from isolated lung cells on day 0, 5, 9, 13, and 19. Scale bar = 500 μm. (B). Lung organoids were applied to immunofluorescence staining to label P63+ basal cells, FOXJ1+/ACCTUB+ ciliated cells, MUC5AC+ goblet cells, CC10+ club cells, and SFTPB+ AT2 cells (green). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 20 μm.
Expansion of lung organoids
Passage and split the lung organoids every 2–3 weeks.
Disrupt the matrigel droplets by pipetting up and down the medium 10 times with a 1,000 μL pipette. Transfer the mixture to a 15 mL tube. Top up with cold basal medium to a total volume of 10 mL if several wells of organoids are harvested for passaging. Incubate the mixture on ice for 5 min. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Wash the organoids once with cold basal medium. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
(Optional) Resuspend the organoids in 1–2 mL of 10× TrypLE and incubate for 5 min at 37 °C to digest the organoids. Add FBS to the organoids with a final concentration of 2% to terminate enzymatic digestion (i.e., add 20 μL of FBS for 1 mL of 10× TrypLE).
Note: Trypsinization is preferred when the mechanical shearing approach is inadequate to shear the lung organoid into small pieces or when the size of the organoids is highly variable, particularly when subsequent experimentations require organoids of a relatively uniform size.
Shear the organoids into single cells and small cellular clusters by pipetting up and down 20–40 times with a Pasteur pipette.
Check the sheared organoids under a light microscope to ensure organoid fragments are small enough (e.g., less than 10 cells). Repeat the mechanical shearing if necessary.
Wash the cells with the cold basal medium. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Resuspend the pellet in an estimated volume of cold matrigel, which enables a split of the organoids with a ratio of 1:2 to 1:10 based on organoid density in the original culture and the methods used for passage. We normally apply a ratio of 1:2 to 1:4 when passaging with mechanical shearing and 1:6 to 1:10 when passaging with enzymatic digestion.
Note: The cell density should be approximately 2 × 104–4 × 104 cells in each 40 μL of matrigel.
Dispense 40 μL of the cell suspension to each well of a prewarmed 24-well suspension culture plate and incubate for 10–15 min at 37 °C in a standard cell culture CO2 incubator to let the matrigel droplet solidify.
Add 500 μL of expansion medium to each well and incubate the plate in a standard cell culture CO2 incubator. Replenish the medium every 2–3 days with caution not to disrupt the matrigel droplets.
Note: The lung organoids can be passaged every 2–3 weeks for approximately one year (i.e., 16–24 passages).
Distal differentiation of lung organoids to generate alveolar organoids
Preparation
Prepare distal differentiation medium (DD medium) (Recipe 3) and prewarm at 37 °C.
Distal differentiation in suspension culture
Culture lung organoids in the expansion medium for 12–14 days to prepare sufficient organoids for differentiation culture (lung organoids from step A3j).
Disrupt the matrigel droplets by pipetting up and down the medium 10 times with a 1,000 μL pipette. Transfer the mixture to a 15 mL tube. Top up with cold basal medium to a total volume of 10 mL if several wells of organoids are harvested for passaging. Incubate the mixture on ice for 5–10 min. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Note: Use cold basal medium (keep on ice) to wash the organoids and incubate them on ice to completely remove the matrigel. Residual matrigel may compromise the effect of subsequent suspension culture.
Wash the organoids with cold basal medium. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Resuspend the organoids in 1–2 mL of 10× TrypLE and incubate for 5 min at 37 °C to digest the organoids. Add FBS with a final concentration of 2% to terminate enzymatic digestion (i.e., add 20 μL of FBS for 1 mL of 10× TrypLE).
Shear the organoids into single cells by pipetting up and down 20–40 times using a Pasteur pipette.
Check under a light microscope to ensure the organoids are sheared into single cells. Repeat the mechanical shearing if necessary.
Wash the cells with 10 mL of cold basal medium. Filter the cell suspension through a 40 μm cell strainer. Centrifuge at 400 × g for 5 min at 4 °C. Discard the supernatant.
Resuspend the pellet in 1–10 mL of DD medium based on the estimated cell number. Count the number of cells with a hemocytometer under a microscope. Adjust the cell concentration to 2 × 105 per milliliter with DD medium.
Dispense 500 μL of cell suspension (1 × 105 cells) to each well of a Nunclon Sphera 24-well suspension culture plate.
Notes:
1) Approximately 2.4 × 106 cells are required for generating a plate of alveolar organoids in 24-well format.
2) Do not put too many cells in each well for suspension culture because cells may form aggregates if the density is too high, which may compromise alveolar differentiation.
3) Avoid scratching the bottom of the wells during pipetting, which may damage the super low cell attachment surface of the Nunclon Sphera culture plates.
Incubate the cells in a standard cell culture incubator for 10–14 days for maturation (Figure 2). Directly add 100 μL of DD medium to each well every other day without removing the old medium, since evaporation reduces the volume of the medium during incubation. Evenly distribute the cells by gentle shaking before putting the plate back into the incubator.
Notes:
1) Make sure to evenly distribute the cells; they may form aggregates if closely centered in the well, which may compromise alveolar differentiation.
2) Organoids with a lumen and thin wall are discernible under a microscope from day 3–4 after suspension cultured in DD medium. After 10 days of distal differentiation culture, the alveolar organoids are mature enough to simulate the native alveolar epithelium, which is applicable for subsequent experimental manipulation.
3) The thin and flat AT1 cells are delicate. Pipette gently to avoid disturbing the AT1 morphology during the differentiation culture and downstream procedures.
Figure 2. Generation of alveolar organoids. (A). Top, a schematic graph outlines the distal differentiation protocol to generate alveolar organoids (AlvO) in distal differentiation medium (DD medium). Exp medium = expansion medium. Bottom, photomicrographs show single cell suspension on day 0 and mature alveolar organoids on day 14. Scale bar = 100 μm. (B). Alveolar organoids were applied to immunofluorescence staining to label AQP5+ AT1 cells (red) and SFTPB+/HTII-280+ AT2 cells (green). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 20 μm.
Proximal differentiation of lung organoids to generate optimized 2D airway organoids
Preparation
Prepare proximal differentiation medium (PD medium) (Recipe 4, no PIPES, pH 7.4) and prewarm at 37 °C.
Prepare proximal differentiation medium (PD medium) (Recipe 4, with PIPES, pH 6.6) and prewarm at 37 °C.
Pre-incubate the Transwell inserts with basal medium overnight in a standard cell culture incubator. For a 24-well insert, add 200 and 500 μL of basal medium to the top and bottom chamber, respectively.
Proximal differentiation in Transwell inserts
Culture lung organoids in the expansion medium for 12–14 days after passaging to prepare sufficient cells for differentiation culture (lung organoids from step A3j).
Dissociate lung organoids into single cells according to steps B2b–B2g.
Resuspend the pellet in 1–4 mL of expansion medium based on the cell number. Count the number of cells with a hemocytometer. Adjust the cell concentration to 1.3 × 106 per milliliter with expansion medium.
Remove basal medium from the top and bottom chambers of the pre-incubated Transwell inserts. Seed 100 μL of cell suspension (1.3 × 105 cells) to the top chamber of 24-well inserts. Add 500 μL of expansion medium to the bottom chamber. Incubate the cells in a standard cell culture CO2 incubator for two days to let cells grow and attach.
Note: Approximately 3.12 × 106 cells are required for generating a plate of 2D airway organoids in 24-well Transwell inserts.
Replace the expansion medium with PD medium (pH 7.4) in both the top and bottom chambers. Incubate the cells in a standard cell culture CO2 incubator for two days to let cells reach confluence.
Note: The 2D organoids reach confluence after changing from expansion medium to PD medium (pH 7.4) for 2–3 days.
Replace the PD medium pH 7.4 with PD medium pH 6.6 in the top chamber, while keeping the PD medium pH 7.4 in the bottom chamber (i.e., pH 6.6/7.4 on top/bottom). Incubate the cells in a standard cell culture CO2 incubator for 10 days for maturation (Figure 3). Replenish the PD medium pH 6.6 in the top chamber and PD medium pH 7.4 in the bottom chamber every 2–3 days.
Note: Motile beating cilia are normally discernible in the organoids under a light microscope from day 7 after differentiation in PD medium. After 14 days of proximal differentiation culture, the 2D airway organoids are ready for experimental manipulation.
Figure 3. Generation of optimized 2D airway organoids. (A). A schematic graph outlines the proximal differentiation protocol to generate 2D airway organoids (AwO) in proximal differentiation medium (PD medium) at different pH. Exp medium = expansion medium. (B). 2D optimized airway organoids were applied to immunofluorescence staining to label ACCTUB+ ciliated cells (red). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 20 μm.
Characterization of organoids
Examine mRNA expression of cell type–specific genes or other cellular genes
RT-qPCR assay: add 350 μL of cell lysis buffer to harvest the organoids for RNA extraction, reverse transcription, and detection of cellular gene expression.
Note: Use the autologous undifferentiated lung organoids for comparison/control.
Assess protein expression of cell type–specific markers and other cellular proteins
Immunofluorescence staining and imaging
i. Fix the organoids with 1 mL of 4% PFA at room temperature for 60 min. Remove the PFA and wash the organoids once with 2% FBS/PBS.
ii. Permeabilize the organoids with 1 mL of 0.1% Triton X-100 at room temperature for 10 min. Remove the Triton X-100 and wash the organoids once with 2% FBS/PBS.
iii. Block the organoids with 1 mL of 3% BSA at room temperature for 60 min. Remove the BSA (no need to wash).
iv. Incubate the organoids with primary antibodies diluted in 2% FBS/PBS at 4 °C overnight. Remove the antibodies and wash them with 2% FBS/PBS five times.
Note: Refer to the product datasheet for the optimal dilution of the antibody.
v. Incubate the organoids with secondary antibodies diluted in 2% FBS/PBS at 4 °C overnight. Remove the antibodies and wash the organoids with 2% FBS/PBS five times.
Note: Avoid exposure to light after addition of fluorescent materials (i.e., secondary antibody).
vi. Counterstain the organoids with DAPI and Phalloidin diluted in 2% FBS/PBS at 4 °C overnight. Remove the dyes and wash the organoids once with 2% FBS/PBS.
vii. Mount the organoids on a glass slide with glass coverslip using Prolong glass antifade mountant and proceed to confocal imaging.
Note: The glass slide can be stored at 4 °C in the dark for ~1–2 weeks.
Flow cytometry analysis
i. Incubate the organoids in 1 mL of 10 mM EDTA at 37 °C for 30–60 min.
ii. Shear the organoids into single cells by pipetting up and down 20–40 times using a Pasteur pipette.
iii. Fix the cells with 1 mL of 4% PFA at room temperature for 15–30 min. Remove the PFA and wash the cells once with 2% FBS/PBS.
iv. Permeabilize the cells with 1 mL of 0.1% Triton X-100 at 4 °C for 5 min. Remove the Triton X-100 and wash the cells once with 2% FBS/PBS.
v. Incubate the cells with primary antibodies diluted in 2% FBS/PBS at 4 °C for 60 min. Remove the antibodies and wash the cells once with 2% FBS/PBS.
Note: Refer to the product datasheet for the optimal dilution of the antibody.
vi. Incubate the cells with secondary antibodies diluted in 2% FBS/PBS at 4 °C for 60 min. Remove the antibodies and wash the cells once with 2% FBS/PBS.
Note: Avoid exposure to light after addition of fluorescent materials (i.e., secondary antibody).
vii. Resuspend the cells in 2% FBS/PBS at a density of 1 × 106 cells per milliliter and transfer the cells to a 5 mL round bottom polystyrene flow tube. Proceed to flow cytometry.
Others
Electron microscopy (EM) imaging:
Fix the organoids with 1 mL of 2.5% glutaraldehyde overnight, followed by EM processing (sectioning and staining) for ultrastructural imaging.
Note: Please refer to the detailed protocol provided by the Electron Microscope Unit of HKU (https://emunit.hku.hk/documents/SamplePreparationTechnique.pdf).
Live cell staining and imaging to assess AT2 cell functionality in alveolar organoids:
i. Resuspend the alveolar organoids in 0.5 mL of DD medium supplemented with 1 μM of β-BODIPYTM FL C12-HPC. Incubate the organoids at 37 °C overnight in a standard cell culture incubator.
Note: Avoid exposure to light after the addition of fluorescent materials.
ii. Collect the alveolar organoids into a 15 mL centrifuge tube. Wash the organoids with 10 mL of basal medium. Centrifuge at 300 × g for 5 min. Discard the supernatant.
iii. Resuspend the organoids in 0.5 mL of DD medium supplemented with 100 nM of LysoTracker red. Incubate the organoids at room temperature for 30 min.
iv. Repeat the washing (step D3b.ii).
v. Mount the live organoids onto a glass slide and proceed to confocal imaging immediately.
Data analysis
For the related downstream characterization and analysis of organoids such as gene expression, immunofluorescence staining, flow cytometry, electron microscopy, and functional assessment, please refer to our publications (Zhou et al., 2018; Chiu et al., 2022).
Notes
All procedures were conducted in a biosafety cabinet.
The protocols were reproducible and have been applied to generate lung organoids and differentiated alveolar/airway organoids derived from multiple donors in our laboratory.
The organoids derived from different donors may have a certain degree of variation due to individual genotypic and phenotypic variation (e.g., genes, age, health status).
The lung organoids could be cryopreserved for long-term storage and biobanking.
The conditioned medium (Rspondin1, Noggin, and WNT3A) can be replaced by commercially available recombinant proteins.
The expansion and differentiation media (Recipes 2–4) remain stable for use for approximately 2–4 weeks if properly stored at 4 °C. Prepare fresh medium for optimal performance.
Recipes
Basal medium
Reagent Final concentration Amount
Advanced DMEM/F-12
HEPES (1 M)
GlutaMAX (200 mM)
Penicillin-Streptomycin (10,000 U/mL)
Total n/a
10 mM
2 mM
100 U/mL
n/a 500 mL
5 mL
5 mL
5 mL
515 mL
Expansion medium
Reagent Final concentration Amount
Basal medium
Rspondin1
Noggin
B-27 supplement (50×)
N-Acetyl-L-cysteine (500 mM)
Nicotinamide (1 M)
Y-27632 (50 mM)
A 83-01 (500 μM)
SB 202190 (10 mM)
FGF-7 (5 μg/mL)
FGF-10 (100 μg/mL)
Primocin (50 mg/mL)
Heregulin β-1* (10 μM)
Total n/a
10%
10%
1×
1.25 mM
10 mM
5 μM
500 nM
1 μM
5 ng/mL
20 ng/mL
100 μg/mL
5 nM
n/a Top up to 100 mL
10 mL
10 mL
2 mL
250 μL
1 mL
10 μL
100 μL
10 μL
100 μL
20 μL
200 μL
50 μL
100 mL
*Only required for initial derivation of organoids (first passage)
Distal differentiation medium (DD medium)
Reagent Final concentration Amount
Basal medium
Dexamethasone (10 μM)
8-Bromo-cAMP (100 mM)
IBMX (50 mM)
B-27 supplement (50×)
Primocin (50 mg/mL)
WNT3A*
CHIR99021* (10 mM)
Total n/a
50 nM
100 μM
100 μM
1×
100 μg/mL
50%
3 μM
n/a Top up to 100 mL
500 μL
100 μL
200 μL
2 mL
200 μL
50 mL
30 μL
100 mL
*Add either 50% WNT3A or 3 μM CHIR99021
Proximal differentiation medium (PD medium)
Reagent Final concentration Amount
PneumaCult-ALI Basal Medium
PneumaCult-ALI 10× Supplement
PneumaCult-ALI Maintenance Supplement (100×)
Heparin (2 mg/mL)
Hydrocortisone (96 μg/mL)
DAPT (10 mM)
Primocin (50 mg/mL)
PIPES*# (1 M)
Total n/a
1×
1×
4 μg/mL
480 ng/mL
10 μM
100 μg/mL
2 mM
n/a 450 mL
50 mL
5 mL
1 mL
2.5 mL
500 μL
1 mL
1 mL
510 mL
*Prepare two PD media, one with PIPES and one without
#For the PD medium with PIPES, adjust the pH to 6.6 with HCl and NaOH after addition of PIPES
Rspondin1 conditioned medium
Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin + 1.5 mL of zeocin selection reagent).
Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin).
Culture the Rspondin1 expressing 293T cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence.
Split the cells into six T175 flasks in growing medium and wait until they reach confluence.
Remove the growing medium and culture the cells in 50 mL basal medium (Recipe 1) for seven days.
Collect the medium (i.e., the Rspondin1 conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter.
Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.
Noggin conditioned medium
Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL penicillin-streptomycin + 5 mL of G418 sulfate).
Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin).
Culture the Noggin expressing HEK293 cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence.
Split the cells into six T175 flasks in growing medium and wait until they reach confluence.
Remove the growing medium and culture the cells in 50 mL of basal medium (Recipe 1) for seven days.
Collect the medium (i.e., the Noggin conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter.
Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.
WNT3A conditioned medium
Prepare selection medium (500 mL of DMEM + 60 mL of FBS + 5 mL penicillin-streptomycin + 625 μL of zeocin).
Prepare growing medium (500 mL of DMEM + 60 mL of FBS + 5 mL of penicillin-streptomycin).
Culture the Rspondin1 expressing 293T cells in a T175 flask in selection medium in a standard cell culture CO2 incubator and wait until they reach confluence.
Split the cells into six T175 flasks in growing medium and wait until they reach confluence.
Split the cells into thirty 100 mm cell culture dishes in growing medium (20 mL per dish) and culture the cells for seven days.
Collect the medium (i.e., the WNT3A conditioned medium). Centrifuge at 300 × g for 5 min to pellet the cells/cell debris and filter the medium through a 0.22 μm bottle top vacuum filter.
Aliquot the medium into small volumes (~50 mL) and store them at -80 °C.
Acknowledgments
We thank the Center for PanorOmic Sciences and Electron Microscope Unit, Li Ka Shing Faculty of Medicine, The University of Hong Kong, for assistance in confocal imaging, flow cytometry, and electron microscopy. This work was partly supported by funding from the Health and Medical Research Fund (HMRF, 17161272 and 19180392) of the Food and Health Bureau of the HKSAR government to J.Z.; General Research Fund (GRF, 17105420) and Collaborative Research Fund (CRF, C7042-21G) of the Research Grants Council of HKSAR government to J.Z.; Health@InnoHK, Innovation and Technology Commission, HKSAR Government to K.Y.Y.; and the donations of Hong Kong Sanatorium and Hospital, the Shaw Foundation Hong Kong, May Tam Mak Mei Yin, Richard Yu and Carol Yu, Lee Wan Keung Charity Foundation Limited, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Chan Yin Chuen Memorial Charitable Foundation, Perfect Shape Medical Limited, Kai Chong Tong, Foo Oi Foundation Limited to K.Y.Y.
This protocol was derived from our previous publications (Zhou et al., 2018; Chiu et al., 2022).
Competing interests
J.Z., K.Y.Y., M.C.C., and C.L. are listed as inventors on the patent of airway organoids (publication No: US-2021-0207081-A1). J.Z., M.C.C., C.L., and K.Y.Y. are inventors on a provisional patent of alveolar organoids (US application No: 63/238,485). The remaining authors declare no competing interests.
Ethics
This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364 and UW21-695). Informed consent was obtained from tissue donors for using their lung tissue for research purposes.
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4,658 | https://bio-protocol.org/en/bpdetail?id=4658&type=0 | # Bio-Protocol Content
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Peer-reviewed
Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
DM Divyanshu Mahajan
HT Hieng Chiong Tie
LL Lei Lu
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4658 Views: 431
Reviewed by: Ralph Thomas BoettcherSusanne ReinhardtTakashi Nishina
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Apr 2022
Abstract
The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging.
Graphical overview
Keywords: Golgi Golgi cisternae Imaging Airyscan microscopy Golgi localization
Background
The Golgi complex (Golgi) plays a central role in sorting and modifying secretory proteins and lipids (cargos) (Glick and Luini, 2011; Klumperman, 2011; Lu and Hong, 2014). In mammalian cells, the Golgi consists of a laterally linked network of Golgi stacks. As the structural unit of the Golgi, a Golgi stack comprises 4–7 flattened and densely packed membrane sacks called cisternae. The stacked cisternae of the Golgi can be further divided into three zones, namely the cis, medial, and trans-Golgi zones. At the trans-side of the Golgi stack, the trans-Golgi network localizes immediately outside the trans-Golgi zone and comprises tubular and vesicular membrane profiles. The secretory cargos synthesized in the ER reach the cis-Golgi and transit the medial before eventually exiting at the trans-side of the Golgi.
The structural unit of the Golgi, the Golgi stack, has an extremely diverse and complex morphology. It has an amorphous and twisted shape and many attached tubular and vesicular membrane profiles. The spatial resolution of the conventional light microscope is ~250 nm, which is insufficient to resolve the 200–400 nm thick Golgi stack. Therefore, under a fluorescence microscope, the mammalian Golgi appears as a perinuclear lump with almost unresolvable structural features. The difficulty in imaging the Golgi is also due to the dense and random orientation of the Golgi stacks. Hence, it is critical to perform multicolor imaging to identify different zones of the same Golgi stack. Although possessing a spatial resolution of 40–100 nm, super-resolution microscopic techniques such as stimulated emission depletion microscopy and single-molecule localization microscopy have difficulty resolving the Golgi's cisternal organizations, since it is very challenging for them to perform multicolor imaging (Schermelleh et al., 2019). In the past, conventional and super-resolution light microscopy have had only limited success in imaging the Golgi cisternae with primarily qualitative data, mostly revealing the relative distributions of no more than three Golgi proteins (Dejgaard et al., 2007; Bottanelli et al., 2016; Tie et al., 2016; Zhang et al., 2020; Hao et al., 2021).
Nocodazole treatment scatters the densely aggregated Golgi as disk-shaped individual structural units with relatively uniform morphological appearances. Extensive data have demonstrated that nocodazole-induced Golgi ministacks (hereafter Golgi ministacks) reliably represent the native Golgi stack (Rogalski et al., 1984; Van De Moortele et al., 1993; Cole et al., 1996; Trucco et al., 2004; Tie et al., 2018). Therefore, Golgi ministacks are a valid Golgi model. We developed a quantitative method called GLIM (Golgi localization by imaging the center of fluorescence mass) (Tie et al., 2016 and 2017). GLIM can achieve ~30 nm spatial resolution along the cis-trans Golgi axis. However, it only provides a numerical value denoting the axial localization but does not generate a pictorial representation of the cisternal distribution of Golgi proteins. By averaging en face views of Golgi ministacks, we later developed an en face averaging method to study the lateral organization of Golgi proteins (Tie et al., 2018). Recently, we have further advanced the averaging approach and introduced the side-averaging method to visualize the cisternal organization of Golgi ministacks directly (Tie et al., 2022). Averaging the Golgi ministack images can effectively enhance the common structural features and suppress the morphological variations or noises among individual Golgi ministacks. Side-averaging provides a systematic and quantitative imaging approach to reveal the axial and lateral cisternal distributions of Golgi proteins. Here, we present a detailed side-averaging protocol to image the intra-Golgi distribution of four Golgi proteins: giantin, GalT-mCherry, GM130, and GFP-OSBP.
Materials and Reagents
Cell culture materials and reagents
Φ 12 mm glass coverslip (no. 1.5) (Menzel, catalog number: CB00120RAC)
24-well cell culture plates (TC-treated and F-bottom) (Corning® Costar®, catalog number: 3524)
T25 cell culture flask (Nunc®, catalog number: 156367)
15 mL centrifuge tube (Greiner, catalog number: 188271)
0.45 µm syringe filter with Supor® membrane (25 mm, sterile) (Pall, catalog number: 4614)
Glass slide (3 × 1 inch) (Biomedia, catalog number: BMH.880103)
Kimwipe® (Kimtech, catalog number: 34155)
Dulbecco's modified Eagle's medium (DMEM) (Capricon, catalog number: DMEM-HPA-P50)
0.25% Trypsin-EDTA (Gibco, catalog number: 25200072)
Fetal bovine serum (FBS) (GE, Hyclone, catalog number: SV30160.03HI)
Opti-MEM (Invitrogen, catalog number: 31985070)
Lipofectamine 2000 (Invitrogen, catalog number: 116688-019)
Chemicals
Nocodazole (Merck, catalog number: 487928)
Dimethyl sulfoxide (DMSO) (Merck, catalog number: D8418-50ML)
Paraformaldehyde (Merck, catalog number: 1.04005.1000)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)
Saponin (Sigma-Aldrich, catalog number: 47036)
Glycerol (Sigma-Aldrich, catalog number: G5516-1L)
Mowiol 4-88 (MW ~31,000) (Merck, catalog number: 81381-50G)
1,4-Diazabicyclo [2.2.2] octane (DABCO) (Merck, catalog number: D27802-25G)
Tris base (Promega, catalog number: H5135)
Sodium chloride (NaCl) (Merck, catalog number: 1.06404.5000)
Potassium chloride (KCl) (Merck, catalog number: 1.04936.1000)
Sodium phosphate dibasic (Na2HPO4) (Merck, catalog number: S9763)
Potassium phosphate monobasic (KH2PO4) (Merck, catalog number: P0662)
Hydrochloric acid (HCl) (Merck, catalog number: 1.00317.2500)
NH4Cl (Merck, catalog number: 254134)
Colorless nail polish
GalT-mCherry mammalian expression plasmid DNA (Tie et al., 2016)
GFP-OSBP mammalian expression plasmid DNA (Addgene: pLJM1-FLAG-GFP-OSBP #134659)
Primary antibodies
GM130 mouse monoclonal antibody (BD Biosciences, catalog number: 610823; dilution 1:500)
Giantin rabbit polyclonal antibody (BioLegend, catalog number: 924302; dilution 1:1,000)
Secondary antibodies
Goat anti-mouse IgG conjugated with Alexa Fluor (AF) 647 (Invitrogen, catalog number: A21235; dilution 1:500)
Goat anti-rabbit IgG conjugated with AF488 (Invitrogen, catalog number: A11008; dilution 1:500)
Goat anti-rabbit IgG conjugated with AF647 (Invitrogen, catalog number: A32733; dilution 1:500)
Solutions
33 mM nocodazole stock solution (see Recipes)
Phosphate buffered saline (PBS) (see Recipes)
1 M Tris pH 8.0 (see Recipes)
4% paraformaldehyde fixative (see Recipes)
100 mM NH4Cl (see Recipes)
10% saponin (see Recipes)
Antibody dilution buffer (see Recipes)
Mowiol mounting medium (see Recipes)
Equipment
A pair of sharp forceps
Water bath
Airyscan microscope: Zeiss LSM980 system equipped with a motorized stage, a temperature-controlled environment chamber, 63×/1.46 Oil Plan-Apochromat objective, and Zeiss Airyscan 2 detector module. GFP or AF488, mCherry, and AF647 are excited by 488, 561, and 639 nm diode laser lights, respectively. The emission lights from GFP or AF488, mCherry, and AF647 are collected by BP495-560, BP570-630, and LP655 filters.
37 °C cell culture incubator with 5% CO2 (Nuaire, catalog number: NU5510E)
Benchtop centrifuge (Eppendorf, catalog number: EPPE5401000.064)
Software
Fiji (NIH, https://fiji.sc/)
Macros (Supplementary Files 1–6): P1-Rotate_Resize_Normalize, P2-Resize_Add_Line, P3-Reflection_Average, ROI_to_image, Side average_Axial line intensity profile, and Side average_Lateral line intensity profile.
Zeiss Zen software
Microsoft Excel
Microsoft Word
Procedure
In this protocol, HeLa cells grown on two coverslips are prepared to study the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP by side-averaging. For coverslip #1, cells transiently expressing GalT-mCherry are immunostained for giantin and GM130 with fluorophores AF488 and AF647, respectively. For coverslip #2, cells transiently co-expressing GFP-OSBP and GalT-mCherry are immunostained for giantin with fluorophore AF647. Cells are imaged under a Zeiss Airyscan microscope.
Prepare glass coverslip for cell culture
Add 200 µL of DMEM to two wells in a 24-well plate: well #1 and #2.
Wash two pieces of coverslip in 70% ethanol. Then, wipe them with Kimwipe® tissue and place them into the two DMEM-containing wells. Hence, the two pieces of coverslip are referred to as coverslip #1 and #2.
Keep the 24-well plate in the 37 °C cell culture incubator until use.
Seed HeLa cells onto the glass coverslip
Maintain HeLa cells in a T25 flask with DMEM supplemented with 10% FBS (complete medium) in the 37 °C cell culture incubator. Cells are sub-cultured at ~80% confluency.
Aspirate the culture medium from the T25 flask and add 1 mL of 0.25% Trypsin-EDTA. Return the flask to the 37 °C cell culture incubator for 2 min.
Add 3 mL of complete medium to the T25 flask, pipette several times to detach the cells, and collect the cells in a 15 mL centrifuge tube.
Pellet cells by centrifugation at 500 × g for 2 min in a benchtop centrifuge.
Aspirate the supernatant and resuspend the cells in 5 mL of complete medium by pipetting.
Aspirate the DMEM medium in wells #1 and #2. Add 0.4 mL of cell suspension (~1 × 105 cells) to the two wells and incubate in the 37 °C cell incubator.
Cells are ready for transfection when they are ~80% confluent and well spread on the glass coverslip.
Transfection to express GalT-mCherry and GFP-OSBP
Change well #1 and #2 to 500 µL of fresh complete medium.
Add 500 ng GalT-mCherry plasmid DNA to 50 µL of Opti-MEM. Add 1 µL of lipofectamine 2000 to 50 µL of Opti-MEM in a separate tube. After 5 min incubation at room temperature, mix the content of the two tubes and incubate the resulting mixture at room temperature for 20 min. Add the final mix to well #1.
Note: Other transfection reagents should also be suitable to transfect HeLa cells.
Add GalT-mCherry and GFP-OSBP plasmid DNA, 250 ng each, to 50 µL of Opti-MEM. Add 1 µL of lipofectamine 2000 to 50 µL of Opti-MEM in a separate tube. After 5 min incubation at room temperature, mix the content of the two tubes and incubate the resulting mixture at room temperature for 20 min. Add the final mix to well #2.
Change well #1 and #2 to the fresh complete medium after 6 h incubation in the 37 °C cell culture incubator.
Nocodazole treatment to induce Golgi ministacks
Dilute the nocodazole stock solution 1,000-fold by pipetting 1 µL of stock solution into 1 mL of complete medium. The working concentration of nocodazole is 33 µM. Prewarm the nocodazole-containing complete medium to 37 °C.
Aspirate the medium from wells #1 and #2 and add 500 µL of prewarmed nocodazole-containing complete medium.
Incubate the 24-well plate for 3 h in the 37 °C cell culture incubator.
Immunofluorescence labeling
Note: Avoid intense ambient light to prevent fluorophores from photobleaching.
Fixation
Aspirate the nocodazole-containing complete medium from wells #1 and #2. Next, rinse cells with 0.5 mL of PBS twice and add 0.5 mL of 4% paraformaldehyde fixative solution. After 20 min incubation at room temperature, wash the wells twice with 0.5 mL of PBS, with 100 mM NH4Cl, and again with 0.5 mL of PBS.
Immunofluorescence labeling
Dilute primary antibodies in the antibody dilution buffer according to their recommended dilution factors. Prepare two types of primary antibody mixtures. Mixture #1 contains giantin and GM130 primary antibodies, while mixture #2 has giantin primary antibody only.
Invert the lid of the 24-well plate. Add 10 µL of mixture #1 and #2 onto the inverse side of the lid, corresponding to wells #1 and #2, respectively. Using a pair of sharp forceps, lift the coverslips #1 and #2, cell-side down, and place them onto the corresponding drops of mixture #1 and #2 on the lid. Put the lid into a Ziplock bag with a piece of wet Kimwipe® tissue inside. Seal the Ziplock bag and allow the cells to incubate with the primary antibody at room temperature for 1 h. Transfer the two pieces of glass coverslip, cell-side up, and place them onto the corresponding wells. Wash the glass coverslip with 0.5 mL of PBS three times.
Wash the inverse side of the 24-well plate lid extensively with water. Next, wipe dry the inverse side of the lid with Kimwipe® tissue. Re-use the inverse side of the lid for secondary antibody incubation.
Repeat the above steps described in step E2b) for the fluorescence-conjugated secondary antibodies. Use goat anti-rabbit AF488 and goat anti-mouse AF647 for coverslip #1 and goat anti-rabbit AF647 for coverslip #2.
Mounting
Add two drops of Mowiol mounting medium, 10 µL each, onto a piece of glass slide. Transfer coverslip #1 and #2 from the 24-well plate onto the two drops of Mowiol mounting medium, cell-side down. Dry glass coverslip at room temperature for 1 h. Seal the edge of the glass coverslip with colorless nail polish. The glass slide is then sealed in a Ziplock bag and stored at -20 °C until imaging.
Note: Other mounting media should work too. However, we prefer Mowiol mounting medium as it solidifies, which makes it easier to handle the coverslips.
Imaging
Acquire cell images (16-bit) under the 63× objective using the SR mode in the Zeiss Airyscan microscope. Select cells with good signals and well-spread morphologies. Collect multiple three-channel image stacks with a z-step of 170 nm and pixel size of 45 nm. Image seven cells with one cell per image. Process raw Airyscan images using Zeiss Zen software.
Data analysis
Side-averaging analysis
Side-averaging requires three-channel images, among which two channels are reserved for giantin and GalT-mCherry. Giantin is a cisternal rim-localized marker to identify a Golgi ministack's spatial orientation. It appears as a ring and double-punctum in the en face and side-view of a Golgi ministack, respectively (Tie et al., 2018). On the other hand, the trans-Golgi marker GalT-mCherry denotes the trans-side of side-views. The testing Golgi protein, GM130 or GFP-OSBP, in this case, is imaged in the third channel.
Install Macros. There are six Microsoft Word docx files, Supplementary Files 1-6, each containing code for one macro. Open Supplementary File 1 in Microsoft Word. Copy and paste the text content (the macro code) to Fiji's macro editor (Plugins > New > Macro). Next, save the macro using the provided macro name in the second line of the text as an .ijm file. Install the macro (Plugins > Macros > Install) and select the saved .ijm file. Finally, repeat the above steps for Supplementary Files 2-6 to install the rest of the macros. The following six macros, corresponding to Supplementary Files 1-6, respectively, should be found under Plugins > Macros: P1-Rotate_Resize_Normalize, P2-Resize_Add_Line, P3-Reflection_Average, ROI_to_image, Side average_Axial line intensity profile, and Side average_Lateral line intensity profile.
Open the processed Airyscan image in Fiji and select the giantin channel. Average three consecutive z-sections around the best focus plane (Image > Stacks > Z project). Next, split the three-channel image into three single images (Image > Color > Split Channels). Finally, merge them again as a new three-channel image so that GalT-mCherry is Channel 1 (C1) or red, giantin is Channel 2 (C2) or artificially green, and GM130 or GFP-OSBP is Channel 3 (C3) or artificially blue. Save the new three-channel image.
Open the ROI Manager (Analyze > Tools > ROI Manager). Manually draw three regions in the background using the Freehand selections drawing tool while pressing the Shift key. The background regions should be within the cytoplasm and free of Golgi ministacks (Figure 1A). Combine the three background regions as one region of interest (ROI) and add it to the ROI Manager by pressing t or clicking Add of the ROI Manager. Save the background ROI to a sub-folder (RoiSet-background) for later revision. Set measurement parameters: Standard deviation and mean (Analyze > Set Measurements > check “Standard deviation” and “Mean gray value”).
NOTE: Combining three background ROIs would sample more regions of the background.
Select giantin or C2 as the active channel. Next, measure the standard deviation and mean of the background ROI (Analyze > Measure). The values are displayed in the Results windows.
Copy the standard deviation and mean gray values to Excel and calculate the threshold value as mean + 3SD. Subtract the threshold value from C2 (Process > Math > Subtract). When a dialog window pops up with the message Process all 2 images?, click No to subtract the background from the current channel only.
Repeat steps 4 and 5 by selecting C1 and C3 as active channels.
Save the background-subtracted three-channel image with the suffix “BGST” (background subtracted) for later revision. Then, remove all ROIs in the ROI Manager by clicking Clear in the ROI Manager.
Visually inspect giantin image and identify Golgi ministack side-views, characterized by giantin double-punctum (Figure 1B). Select the Rectangle icon (rectangle drawing tool) from the Fiji toolbar. Manually draw a small square in any part of the image while monitoring the status bar to ensure w = 40 pixel and h = 40 pixel. Move the resulting 40 × 40 pixel square to box a double-punctum. Then, add the ROI to the ROI Manager by pressing t (Figure 1C). Move the 40 × 40 pixel square to box other double-punctum and repeat the above steps. Exhaust the image to select all double-punctum (Figure 1B). Save all ROIs to a sub-folder (RoiSet-side-view) for later revision.
Convert ROIs to individual three-channel images (hereafter referred to as cropped images) (Figure 1C). The names of cropped images have a sequential number (1, 2, 3, etc.) as their suffixes. The conversion can be performed either manually (Ctrl + Shift + D or Image > Duplicate) or automatically by launching the macro “ROI_to_image” (Plugins > Macros > ROI_to_image).
Figure 1. Selection of side-views of Golgi ministacks. (A) Drawing and selecting the background ROI. In Step (1), three ROIs are manually drawn while holding the Shift key. In Step (2), three ROIs are added to the ROI Manager as one background ROI by pressing t. (B) Golgi ministacks are labeled by GalT-mCherry (red), giantin (green), and GM130 (blue). Side-views are boxed with square ROIs with 40 × 40 pixels (yellow boxes). Note that the lines of yellow boxes are thickened to increase their visibility. Below are images showing a typical en face and side-view Golgi ministacks. (C) ROIs are added to the ROI Manager. (D) Side-view ROIs are converted to individual three-channel images (cropped images) (40 × 40 pixel). (E) Manually flip or rotate the cropped images so that the GalT-mCherry signal is above the giantin double-punctum.
Manually flip or rotate all cropped images (Image > Transform: flip or rotate) so that GalT-mCherry signal, which usually appears as a lump, is above the giantin double-punctum. Hence, the trans-side of the Golgi ministack is positioned up.
Ensure the background pixels of all cropped images are 0 in all three channels (Figure 2). To that end, first rescale intensities of the three-channel image to 0 and 1 (Image > Adjust > Brightness&Contrast > Set Minimum displayed value to 0, Maximum displayed value to 1, and check Propagate to the other 2 channels of this image) to display all non-zero pixels as the maximum brightness (Figure 2A). Next, manually draw a ROI around non-zero pixels in the background region (Figure 2B) and remove them or make them zero (Edit > Clear or Delete) (Figure 2C). Alternatively, estimate the background intensity value and subtract it from each channel. This step requires significant effort and is critical for high-quality side-averaging.
NOTE: “Clear” affects all channels, while “Delete” affects only the active channel.
Figure 2. Manually delete or clear background objects in a cropped image. (A) Rescale the intensity of the cropped image to 0 and 1 to reveal the background object. (B) Manually draw an ROI around the background object. (C) Remove the background object.
Clear all ROIs in the ROI Manager by clicking Clear.
In a cropped image, select giantin or C2 as the active channel. Then, rescale intensities of the three-channel image to 0 and 1 as described in step 11.
Manually draw two ROIs to mark the two giantin puncta (left and right) and add the ROIs to the ROI Manager (Figure 3).
Figure 3. Manually mark the two giantin puncta in a cropped image. (A) In the giantin or C2 channel, rescale the intensity of the cropped image to 0 and 1. (B) Manually draw two ROIs corresponding to the two giantin puncta. (C) Add the two ROIs to the ROI Manager.
Click Deselect in the ROI Manager and launch the macro P1-Rotate_Resize_Normalize (Plugins > Macros > P1-Rotate_Resize_Normalize). Upon prompt, choose a directory to export the processed channel images. The macro splits and processes the three-channel cropped image into three single-channel images. Then, it saves them in the specified directory with prefixes “C1,” “C2,” or “C3,” indicating the respective channel or Golgi markers.
Repeat steps 12–15 for all cropped images.
Import processed single-channel images from step 15 as C1, C2, and C3 image stacks (File > Import > Image Sequence > in "Filename contains", key in “C1,” “C2,” or “C3”).
Acquire the side-averaged image by averaging C1, C2, or C3 image stacks (Image > Stacks > Z-Projection). Then, save the three side-averaged images (Figure 4, left column).
Resize the side-averaged image by launching the macro P2-Resize_Add_Line (Plugins > Macros > P2-Resize_Add_Line) (Figure 4, middle two columns). The macro tool resizes the side-averaged image isotropic with a pixel size of 5.6 nm so that the resulting image represents the ministack of the mean population size (Tie et al., 2022). Two images are saved with the following prefixes: “resized1” and “resized2.” “resized2” image differs from “resized1” image in that the former has a horizontal white line to indicate the y position of the center of fluorescence mass (ym). File names and folder paths are displayed in the Log window.
Figure 4. Cropped images are processed by the macro P1-Rotate_Resize_Normalize and subsequently averaged. The resulting averaged images of GFP-OSBP, giantin, GalT-mCherry, and GM130 (left column) are further processed by macros P2-Resize_Add_Line (middle two columns) and then by P3-Refelection_Average (right two columns).
Process the “resized1” or “resized2” image using the macro P3-Refelection_Average to generate a reflection-averaged image (Plugins > Macros > P3-Refelection_Average) (Figure 4, right two columns). The operation assumes that the side-averaged Golgi ministack image should be symmetrical about the y-axis. Images are saved with "mirror" appended at the beginning of their file names. File names and folder paths are displayed in the Log window. The reflection averaged “resized1” and “resized2” images are suitable for constructing merged composite images showing the cisternal organization of multiple Golgi markers.
Generate a composite image with side-averaged Golgi proteins, including giantin, GalT-mCherry, GM130, and GFP-OSBP. Open “resized2” images and merge them by artificially assigning GM130 as blue, giantin as green, GalT-mCherry as red, and OSBP-EGFP as cyan (Image > Color > Merge Channels) (Figure 5). The composite image displays the cisternal organization of a Golgi ministack.
Figure 5. A composite side-view image showing the relative cisternal distribution of, from the cis- to trans-side of the Golgi, GM130, giantin, GalT-mCherry, and GFP-OSBP. Reflection-averaged “resized2” images of GM130, giantin, GalT-mCherry, and GFP-OSBP are used to construct the composite image.
Data plotting
Process “resized1” image using the macro, Side average_Axial line intensity profile, to generate the axial line intensity profile (Plugins > Macros > Side average_Axial line intensity profile) (Figure 6). Two windows are displayed. The Plot of results window shows the axial line intensity profile. x = 0 corresponds to the position of the center of the fluorescence mass, ym. The Results window shows the numerical values of x (pixels) and normalized intensity.
By side-averaging, we can calculate the localization quotient (LQ) of GFP-OSBP, , using the below equation:
where yGFP–OSBP is the ym of GFP-OSBP, yGM130 is the ym of GM130, and yGalT–mCherry is the ym of GalT-mCherry. Similarly, we can also calculate LQside of giantin. The LQside values of OSBP-EGFP and giantin are measured to be 1.72 (n = 15) and 0.54 (n = 16), consistent with our GLIM data, 1.74 ± 0.1 (n = 59) and 0.57 ± 0.05 (n = 103), respectively. The axial size of the Golgi ministack, defined as yGalT–mCherry – yGM130, is measured to be 293 ± 15 nm (n = 16), which agrees with the mean axial size of a population, 274 ± 5 nm (n = 204) (Tie et al., 2022).
Process “resized1” image using the macro, Side average_Lateral line intensity profile, to generate the lateral line intensity profile (Plugins > Macros > Side average_Lateral line intensity profile) (Figure 6). Lateral line intensity profiles of rim-localized Golgi proteins, such as giantin, show two peaks. On the other hand, those of interior-localized Golgi proteins, such as Golgi enzymes including GalT-mCherry, show a single broad peak. The lateral size of the Golgi ministack, defined as the distance between the two half-maximum points of the outer slopes in the lateral line intensity profile of giantin, is 723 ± 17 nm (n = 16), which agrees with the mean lateral size of a population, 700 ± 10 nm (n = 204) (Tie et al., 2022).
Figure 6. The axial and lateral line intensity profiles of “resized1” images of GM130, giantin, GalT-mCherry, and GFP-OSBP. The “resized1” images are processed by macros Side average_Axial line intensity profile and Side average_Lateral line intensity profile.
Recipes
33 mM nocodazole stock solution
Add 1 mL of DMSO to a vial containing 10 mg of nocodazole powder and vortex. Aliquot and store the stock solution at -20 °C. Centrifuge the nocodazole stock solution to remove insoluble residues before use.
PBS
Dissolve 0.2 g of KCl, 8.0 g of NaCl, 0.245 g of KH2PO4, and 1.44 g of Na2HPO4 in 800 mL of water. Adjust the pH to 7.4 and top up the volume to 1 L with water. Store at room temperature until use.
1 M Tris pH 8.0
Dissolve 121.1 g of Tris base in 800 mL of water. Adjust the pH to 8.0 using 1 M HCl and top up the volume to 1 L with water. Store at room temperature until use.
4% paraformaldehyde fixative
Dissolve 4.0 g of paraformaldehyde powder in 100 mL of hot PBS. Filter the solution through a 0.45 µm syringe filter with Supor® membrane. Aliquot and store the solution at -20 °C.
CAUTION: Use proper personal protective equipment while working with paraformaldehyde powder, as it is carcinogenic and causes skin irritation.
100 mM NH4Cl
Dissolve 14.86 g of NH4Cl powder in 1 L of water. Store at room temperature until use.
10% saponin
Dissolve 1.0 g of saponin powder in 10 mL of water, aliquot, and store at -20 °C until use.
Antibody dilution buffer
Dissolve 2.0 g of BSA in 94 mL of PBS and add 5 mL of FBS and 1 mL of 10% saponin. The solution is filtered through a 0.45 µm syringe filter with Supor® membrane, aliquoted, and stored at -20 °C.
Mowiol mounting medium
Dissolve 1.2 g of Mowiol 4-88, 0.36 g of DABCO, 3.0 g of glycerol, and 1.2 mL of 1 M Tris pH 8.0 in 7.8 mL of water. Dissolve the mixture by vortexing with incubation in a 60 °C water bath. Aliquot and store the solution at -20 °C until use.
Acknowledgments
This project is supported by the Ministry of Education, Singapore, under its Tier 2 MOE-T2EP30221-0001 and Tier 1 RG 25/22. This protocol's original research has been published (Tie et al., 2022). We thank R. Zoncu for sharing the plasmid DNA, pLJM1-FLAG-GFP-OSBP.
Competing interests
The authors declare no conflicts of interest or competing interests.
References
Bottanelli, F., Kromann, E. B., Allgeyer, E. S., Erdmann, R. S., Wood Baguley, S., Sirinakis, G., Schepartz, A., Baddeley, D., Toomre, D. K., Rothman, J. E. et al. (2016). Two-colour live-cell nanoscale imaging of intracellular targets. Nat Commun 7: 10778.
Cole, N. B., Sciaky, N., Marotta, A., Song, J. and Lippincott-Schwartz, J. (1996). Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol Biol Cell 7(4): 631-50.
Dejgaard, S. Y., Murshid, A., Dee, K. M. and Presley, J. F. (2007). Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins. J Histochem Cytochem 55(7): 709-719.
Glick, B. S. and Luini, A. (2011). Models for Golgi traffic: a critical assessment. Cold Spring Harb Perspect Biol 3(11): a005215.
Hao, X., Allgeyer, E. S., Lee, D. R., Antonello, J., Watters, K., Gerdes, J. A., Schroeder, L. K., Bottanelli, F., Zhao, J., Kidd, P., et al. (2021). Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution. Nat Methods 18(6): 688-693.
Klumperman, J. (2011). Architecture of the mammalian Golgi. Cold Spring Harb Perspect Biol 3:a005181.
Lu, L. and Hong, W. (2014). From endosomes to the trans-Golgi network. Semin Cell Dev Biol 31: 30-39.
Rogalski, A. A., Bergmann, J. E. and Singer, S. J. (1984). Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane. J Cell Biol 99(3): 1101-1109.
Schermelleh, L., A. Ferrand, T. Huser, C. Eggeling, M. Sauer, O. Biehlmaier, and G.P.C. Drummen. (2019). Super-resolution microscopy demystified. Nat Cell Biol 21:72-84.
Tie, H. C., Chen, B., Sun, X., Cheng, L. and Lu, L. (2017). Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass. J Vis Exp(126): 55996.
Tie, H. C., Ludwig, A., Sandin, S. and Lu, L. (2018). The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack. Elife 7: e41301.
Tie, H. C., Mahajan, D., Chen, B., Cheng, L., VanDongen, A. M. and Lu, L. (2016). A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes. Mol Biol Cell 27(5): 848-861.
Tie, H. C., Mahajan, D. and Lu, L. (2022). Visualizing intra-Golgi localization and transport by side-averaging Golgi ministacks. J Cell Biol 221(6): e202109114.
Trucco, A., Polishchuk, R. S., Martella, O., Di Pentima, A., Fusella, A., Di Giandomenico, D., San Pietro, E., Beznoussenko, G. V., Polishchuk, E. V., Baldassarre, M., et al. (2004). Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments. Nat Cell Biol 6(11): 1071-1081.
Van De Moortele, S., Picart, R., Tixier-Vidal, A. and Tougard, C. (1993). Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells. Eur J Cell Biol 60(2): 217-227.
Zhang, Y., Schroeder, L. K., Lessard, M. D., Kidd, P., Chung, J., Song, Y., Benedetti, L., Li, Y., Ries, J., Grimm, J. B., et al. (2020). Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging. Nat Methods 17(2): 225-231.
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4,659 | https://bio-protocol.org/en/bpdetail?id=4659&type=0 | # Bio-Protocol Content
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Peer-reviewed
Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry
Lilli T. E. Bay
TS Trond Stokke
RS Randi G. Syljuåsen
Helga B. Landsverk
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4659 Views: 951
Reviewed by: Gal HaimovichJosé M. Dias Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nucleic Acids Research Aug 2022
Abstract
RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription.
Graphical overview
Keywords: RNA polymerase II RNAPII Transcription Flow cytometry Cell cycle Extraction Barcoding Multiplexing
Background
RNA polymerase II (RNAPII) plays an essential role in protein production by transcribing DNA into mRNA. In addition, it participates in the detection, signaling, and repair after DNA damage (Ljungman and Lane, 2004; Lagerwerf et al., 2011; Lans et al., 2019; Landsverk et al., 2021), and thus also plays a key role in the DNA damage response. Furthermore, RNAPII is essential for cell cycle progression, and is itself regulated by the cell cycle (Enserink and Chymkowitch, 2022). As DNA damage greatly affects the cell cycle by activation of cell cycle checkpoints (Hauge et al., 2021), measuring RNAPII levels on chromatin in individual cell cycle phases is highly relevant both in the absence and presence of DNA damage. Previously, cell cycle effects on RNAPII have been studied, for instance by microscopy (Chan et al., 2012; Teves et al., 2018) and/or chromatin immunoprecipitation/fractionation followed by sequencing (Liang et al., 2015, Wang et al., 2021) or western blotting (Akoulitchev and Reinberg, 1998; Palozola et al., 2017). However, although sequencing or chromatin fractionation techniques can give high resolution sequence information and/or quantitative data, they have so far been based on cell lysates made from a large number of pooled cells. In order to investigate cell cycle effects, these techniques require cell synchronization, which can induce replication stress or changes to transcription. Microscopy offers single-cell resolution and does not require synchronization but is limited in the number of cells analyzed. To overcome these obstacles, we recently described a new method to study RNAPII chromatin binding in single cells through the cell cycle (Bay et al., 2022). This flow cytometry method allows for the determination of cell cycle phases without the need to synchronize cells. Also, thousands of cells are measured per sample per experiment, ensuring accurate quantitative readouts. Using this method on cells treated with the transcriptional inhibitor 5,6-Dichloro-1-beta-Ribo-furanosyl Benzimidazole (DRB), which arrests RNAPII in promoter proximal regions, and the proteasome inhibitor MG132, we showed that promoter proximal RNAPII is degraded under unperturbed conditions, and that this is further enhanced after UV treatment (Bay et al., 2022). In line with our results, degradation of promoter proximal RNAPII after UV treatment was also observed in an independent study by live-cell imaging, chromatin immunoprecipitation followed by sequencing, and western blotting of chromatin fractions (Steurer et al., 2022). Furthermore, using our method, we could observe cell cycle differences in the chromatin loading of RNAPII after UV treatment (Bay et al., 2022). Here, we present the detailed protocol for this method.
A first critical step in the protocol is the cell extraction step, where non-chromatin-bound factors are removed by treatment with mild detergent Triton X-100 (0.5%) prior to fixation (Figure 1). This allows measurements of only chromatin-bound RNAPII. The use of flow cytometry to measure chromatin-bound factors after detergent treatment was first described in the early 90s for the study of chromatin-bound DNA polymerase α (Stokke et al., 1991). Similar assays using extraction prior to fixation have also been used to measure chromatin-binding of other proteins involved in transcription, replication, and repair by flow cytometry (Stokke et al., 1993; Forment et al., 2012; Forment and Jackson, 2015) or immunofluorescence microscopy (Syljuåsen et al., 2005; Britton et al., 2013). Notably, this is the first time such a protocol has been described for RNAPII by flow cytometry. Triton X-100 treatment is performed in a sucrose-containing buffer, similar to pre-extraction/cytoskeletal buffers commonly used in immunofluorescence microscopy (Britton et al., 2013; Kilgas et al., 2021). However, as RNAPII is known to be released from chromatin in mitosis (Parsons and Spencer, 1997), NaCl concentrations were optimized based on the expected low chromatin binding of RNAPII in mitotic cells (Bay et al., 2022). Moreover, as RNAPII is a target for proteasome-mediated degradation (Wilson et al., 2013), particular care should be taken to avoid degradation during cell processing. In this protocol, degradation is prevented by the addition of proteasome inhibitors during extraction, by performing the extraction on ice, and by fixing the cells rapidly after extraction.
Another key feature of the method is multiplexing, i.e., measurement of several different parameters simultaneously in single cells. One advantage of multiplexing is that it allows the incorporation of barcoding into the assay. In this protocol, barcoding is performed by labeling non-treated control cells with an amine-reactive Alexa 647, which binds to intracellular proteins (Krutzik and Nolan, 2006). The resulting Alexa 647–labeled cells are added to all the individual samples prior to staining with antibodies or the EdU Click-iT reaction and can be separated from the sample cells during analysis. The barcoded cells thus provide an internal standard for normalization of the RNAPII signals in the individual samples. This greatly facilitates quantification and accuracy of the assay and enables all the samples to be compared with each other. Multiplexing further permits analysis of RNAPII chromatin levels in specific phases of the cell cycle, by analyzing RNAPII chromatin levels only in cells with a certain DNA and EdU content (Bay et al., 2022).
Choice of antibodies is also critical in this method, as every new antibody used requires validation. The antibodies described here have been validated by us and others (Heidemann et al., 2013; Steurer et al., 2018; Bay et al., 2022), and recognize the N-terminal domain of RNAPII and the serine 5– (pRNAPII S5) and serine 2–phosphorylated (pRNAPII S2) forms of RNAPII. pRNAPII S5 is mostly associated with promoter-proximal regions, and global levels can therefore indicate degree of promoter-proximal paused RNAPII (Bay et al., 2022). pRNAPII S2 can be used as a marker for productive elongation (Bay et al., 2022). Finally, the use of EdU to label replicating cells is an important step to distinguish between G1 and early S phase, as well as between late S and G2. EdU is a nucleotide analogue, which is incorporated into the DNA molecule upon DNA replication. As their DNA content is highly similar, G1 vs. early S phase and G2 vs. late S-phase cells are not distinguishable by measurements of DNA content alone. By including EdU labeling, cells with similar DNA content can be separated in G1 vs. early S or G2 vs. late S based on the presence or absence of EdU incorporation, which marks the replicating (S phase) cells. However, this step may be omitted if such finely separated cell cycle determination is not required. If EdU is omitted, determination of cell cycle phase can be done by DNA content alone to obtain G1, S, and G2 fractions [Figure 2 and Dale Rein et al. (2015)].
Figure 1. Overview of method
Materials and Reagents
Cell culture and harvest
Serological pipettes, polystyrene
Sterile conical-bottom tubes, 15 mL
Human cervical cancer HeLa Kyoto cells (but other cell types may be used)
Penicillin-Streptomycin (P/S) (Thermo Fisher, catalog number: 15140122)
Fetal bovine serum (FBS) (Biowest, catalog number: S181B-050)
Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMAX supplement, pyruvate (Thermo Fisher, catalog number: 31966047)
Culture dishes, 6 cm (VWR, catalog number: 353004)
EdU (1 μM) (Thermo Fisher, catalog number: A10044)
Trypsin 0.25% with EDTA, 100 mL (Life Technologies, catalog number: 25200-056)
Phosphate-buffered saline (10× PBS) (Thermo Fisher, catalog number: 70011-051)
10% formalin solution, neutral-buffered ready-made solution (Sigma-Aldrich, catalog number: HT5011)
Extraction
HEPES (pH 7.9) (Sigma-Aldrich, catalog number: H3784)
MgCl2 (Sigma-Aldrich, catalog number: 208337)
NaCl (Sigma-Aldrich, catalog number: S9888)
Sucrose (Sigma-Aldrich, catalog number: S0389)
Triton X-100 (Sigma-Aldrich, catalog number: T9284-100ML)
PhosSTOP phosphatase inhibitors (Merck, catalog number: 4906837001)
cOmplete protease inhibitor cocktail EDTA-free (Merck, catalog number: 5892791001)
MG132 (Sigma-Aldrich, catalog number: M7449)
ddH2O Milli-Q water
Chromatin extraction buffer (10 mL) (see Recipes)
Staining and preparation of cells for flow cytometry analysis
BD Falcon 5 mL polystyrene with cell strainer cap (VWR, catalog number: 734-0001)
Aluminum foil
Alexa Fluor 647 NHS Ester (Succinimidyl Ester) (Thermo Fisher, catalog number: A20006)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540)
Phosphate-buffered saline (10× PBS) (Thermo Fisher, catalog number: 70011-051)
Fetal bovine serum (FBS) (Biowest, catalog number: S181B-050)
Anti-RNAPII (D8L4Y) (Cell Signaling Technology, catalog number: 14958S)
Anti-pRNAPII S5 (3E8) (Sigma-Aldrich, catalog number: 04-1572)
Anti-pRNAPII S2 (3E10) (Sigma-Aldrich, catalog number: 04-1571)
Donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor 488 (Thermo Fisher, catalog number: A-21206)
Donkey anti-rat IgG H+L Alexa Fluor 488 pre-adsorbed (Abcam, catalog number: ab150153)
Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit (Thermo Fisher, catalog number: C10646)
Bovine serum albumin (BSA) heat-shock fraction, protease free, essentially globulin free, pH 7, ≥98% (Sigma-Aldrich, catalog number: A3059-100G)
Na2HPO4 (Sigma-Aldrich, catalog number: S3264)
KH2PO4 (Sigma-Aldrich, catalog number: P5655)
KCl (Sigma-Aldrich, catalog number: P3911)
NaCl (Sigma-Aldrich, catalog number: S9888)
Ethylenediaminetetraacetic acid (EDTA, pH 7.5)
Igepal CA-630 (Sigma-Aldrich, catalog number: I8896-50ML)
Skim milk powder (Millipore, catalog number: 70166-500G)
DNA-stain Hoechst 33258 (624 μg/mL) (Sigma-Aldrich, catalog number: B1155-25MG)
LX Alexa Fluor 647 NHS Ester (barcoding) (see Recipes)
Flow buffer (1 L) (see Recipes)
Detergent buffer (see Recipes)
Click-iT permeabilization and wash reagent (see Recipes)
Click-iT reaction cocktail (see Recipes)
Equipment
Sterile cell culture hood
37 °C cell culture incubator with CO2 control
Centrifuge (Hettich Rotina 380R Centrifuge, rotor 1754, radius 167 mm)
LSRII flow cytometer (BD Biosciences), Quartz cuvette flow cell, instrument settings can be found in Table 1.
Table 1. LSRII flow cytometer instrument settings
Parameter Laser Band-Pass (nm) Longpass Dichroic filter (nm) Detector
Alexa Fluor 488 488 nm–100 mW 525/30 505 PMT B
SSC 488 nm–100 mW 488/10 - PMT C
FSC 488 nm–100 mW 488/10 - FSC Diode–Silicon photodiode
Hoechst 405 nm–100 mW 450/50 - PMT G
Alexa Fluor 647 640 nm–40 mW 670/14 - PMT C
Alexa Fluor 594 561 nm–45 mW 610/20 600 PMT D
Software
DiVa software is used when obtaining flow cytometry data
FlowJo V10_5 software is used for analyzing the flow cytometry data
Procedure
Preparations: Prepare the chromatin extraction buffer (see Recipes below); 100 μL is required per sample. As one tablet of cOmplete EDTA-free protease inhibitor cocktail and one tablet of PhosSTOP phosphatase inhibitors are needed per 10 mL solution, adjust with +10 mL if more than 10 mL solution is required. Add MG132 to a final concentration of 20 µM (add 2 µL per milliliter of 10 mM stock). Mix thoroughly by vortexing and place on rotator at 4 °C until use. The chromatin extraction buffer should be prepared fresh before each experiment.
Cultivate human cervical cancer HeLa Kyoto cells in DMEM supplemented with 10% FBS and 1% P/S. For 1.0 × 106–1.5 × 106 cells at time of harvest, plate 6.0 × 105 cells in 6 cm culture dishes, approximately 24 h prior to harvest. In addition to dishes intended for samples and the non-treated control cells, include additional dishes intended for use for barcoding and secondary antibody controls. (Approximately 3 × 105 barcoded cells are needed per sample, and 1.0 × 106–1.5 × 106 cells are needed for the secondary controls at time of harvest.)
Note: The protocol may be adapted for other cell types, e.g., we have used RPE and MRC5 cells.
Prepare 1 μM EdU in DMEM supplemented with 10% FBS and 1% P/S at 37 °C and mix well. Remove cell culture media from the culture dishes and replace with 4 mL per dish of the EdU-containing DMEM, 1 h prior to further treatment or harvest in the absence of additional treatment.
Notes:
1) Samples intended as barcoding controls and controls for secondary antibodies are not labeled with EdU and are therefore left untreated in this protocol. However, they can be EdU-positive if desired.
2) This step may be omitted if cell cycle phase determination by DNA stain alone is sufficient.
Harvest cells:
Remove culture medium and wash the cell layer once with 500 µL of trypsin solution or PBS at room temperature (RT).
Remove trypsin or PBS solution, add 500 µL of fresh trypsin solution, and transfer the dishes to a 37 °C cell culture incubator. After approximately 1 min, check if cells have started to detach by gently tapping the side of the dish. If the cells still adhere to the surface, place them back in the incubator until detachment is observed.
When most cells have detached, resuspend the cells in 4 mL of DMEM and transfer the cell suspension of each dish to separate and labeled 15 mL sterile conical-bottom tubes.
Isolate cell pellets by centrifugation at 634 × g for 5 min and remove supernatant.
Note: If timing is important, e.g., after DNA damage, add cold medium to the cells after they have detached, keep the 15 mL sterile conical-bottom tubes containing the cells on ice, and spin the cells at 4 °C to stop enzymatic reactions as much as possible prior to extraction and fixation.
Extract the cells: to prepare, place the chromatin extraction buffer on ice (in a Styrofoam box with ice) and the 10% formalin solution at RT.
Add 100 μL of ice-cold chromatin extraction buffer to each cell pellet, mix by pipetting, and immediately place the tube on ice. Start the timer at 5 min after the first sample and then continue with remaining samples. Time is of essence, so work efficiently. If the incubation period exceeds 5 min, then adjust for all samples to obtain equal extraction time.
When time is up, add 900 μL of formalin to the first sample, transfer to RT, and continue with the other samples in the same pace and order as before. Start the timer at 10 min after the last sample has been mixed with formalin and placed at RT.
Isolate cell pellets by centrifugation at 634 × g for 5 min at RT.
Resuspend the cells in 500 µL of PBS and continue with staining.
Notes:
1) The extraction is a critical step. Fixation must occur rapidly after extraction, or degradation of RNAPII may occur. Within the time window described here, the time prior to fixation is likely more critical to keep constant than the fixation time itself.
2) Samples intended as barcoding cells and secondary antibody controls should also be extracted prior to fixation.
3) When the cells have been fixed and are in PBS, they can be left overnight at 4 °C prior to barcoding and staining.
4) Prior to barcoding, use PBS without FBS to resuspend the cells, as FBS competes with cellular proteins for binding to Alexa Fluor 647 NHS Ester. However, wash steps with PBS without FBS should be avoided as they lead to loss of cells.
Stain cells for barcoding:
Combine cells from tubes containing cells intended for barcoding from step 4d into one tube. (Approximately 3 × 105 barcoded cells should be prepared per sample.)
Add 12.5 μL of LX Alexa Fluor 647 NHS Ester (see Recipes below) per 500 μL of PBS. Mix by inverting the tube and incubate in the absence of light at RT for 30 min.
Quench barcoding by adding 5 mL of PBS with 5% FBS, mix well by inverting the tube, and leave in the dark at RT for 5 min.
Spin down barcoded cells by centrifugation at 1127 × g for 5 min at RT.
Remove supernatant by suction and resuspend pellet containing barcoded cells with 6 mL of PBS with 5% FBS.
Add 5 mL of PBS with 2% FBS to tubes containing samples, secondary antibody controls, and non-treated control cells from step 4d.
Distribute the barcoded cells from step 5e equally between all tubes containing samples, secondary antibody controls, and non-treated control cells from step 5f (e.g., for 10 tubes, distribute 6 mL of barcoded cell solution/10 = 0.6 mL of barcoded cell suspension per tube).
Stain samples with primary and secondary antibodies
Spin down cells in tubes, now also containing barcoding cells, by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.
Note: The second spin is to ensure the staining is as equal as possible between the samples. If this step is omitted, remaining supernatant adhered to the sides of the 15 mL tube may fall into the staining solution and affect the final staining volume, and thus antibody concentrations during staining.
Resuspend pellet in 50 μL of detergent buffer (see Recipes) and leave at RT for 5 min.
Add 50 μL of detergent buffer with primary antibody: rabbit RNAPII NTD 1:50 (i.e., final dilution 1:100), rat pRNAPII S5 1:50 (i.e., final dilution 1:100), or rat pRNAPII S2 1:50 (i.e., final dilution 1:100). Incubate in the absence of light at RT for 1 h.
Notes:
1) The tube with cells intended for use as secondary antibody control is resuspended in 100 μL of detergent buffer without primary antibody.
2) If EdU staining is included, each sample can be stained with one antibody only.
Add 5 mL of PBS with 2% FBS to each tube.
Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.
Add 100 μL of detergent buffer with secondary antibody: anti-rabbit Alexa Fluor 488 1:500 or anti-rat Alexa Fluor 488 1:500. Incubate in the absence of light at RT for 30 min.
Label EdU with the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit. Prepare by thawing the reaction buffer additive (10×), fluorescent dye azide 594, and copper protectant.
Add 5 mL of PBS with 2% FBS to each tube.
Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.
Resuspend cell pellet in 100 μL of 1× Click-iT permeabilization and wash reagent (see Recipes) and leave for 15 min at RT.
Prepare Click-iT reaction cocktail (see Recipes). This should be used within 15 min of preparation.
Add 500 μL of Click-iT reaction cocktail and incubate in the absence of light at RT for 30 min.
Add 5 mL of PBS with 2% FBS to each tube.
Spin down cells by centrifugation at 1127 × g for 5 min at RT. Remove most of the supernatant by suction (until approximately 100 μL is left), spin down again, and remove rest of the supernatant with a 200 μL pipette.
Label DNA with Hoechst 33258.
Resuspend cells in 500 μL of flow buffer (see Recipes) containing 2.4 μL/mL Hoechst 33258. Incubate in the absence of light at RT for 15 min.
Store in the absence of light at 4 °C overnight or until running flow cytometry.
Data analysis
Acquire data on the flow cytometer
In the flow cytometer acquisition software (DiVA), select the parameters shown in Table 2.
Table 2. Flow cytometry acquisition parameters
Parameter Scale
Side scatter (SSC) linear
Forward scatter (FSC) linear
Hoechst-W (width) linear
Hoechst-A (area) linear
Alexa Fluor 647 log
Alexa Fluor 594 log
Alexa Fluor 488 log
Open the graphs shown in Table 3.
Table 3. Flow cytometry acquisition graphs
Output x-axis y-axis
Size and granularity of cells FSC SSC
Single cells Hoechst-W Hoechst-A
Barcoding Alexa Fluor 594 Alexa Fluor 647
DNA profile Hoechst-A histogram
DNA vs. RNAPII/pRNAPII S5/pRNAPII S2 Hoechst-A Alexa Fluor 488
EdU profile Hoechst-A Alexa Fluor 594
Adjust voltages for FSC vs. SSC such that the majority of the cell population is within the graph window.
Adjust voltage for Hoechst such that the first peak of the histogram is located at 50 K (corresponds to G1 phase).
Gate for single cells based on the distribution of cells in the Hoechst-W vs. Hoechst-A plot (see Figure 2A). While the Hoechst-A will increase during the cell cycle as the DNA content doubles in size, the Hoechst-W (corresponding to the time it takes for the cell to traverse the laser beam) will not increase as much. Doublets (e.g., two G1 cells clumped together) may have equal Hoechst-A signal as G2 cells and can contaminate the G2 cell population. Bu using a narrow Hoechst-W gate, doublets are removed from analysis, as they will have a larger Hoechst-W than single cells (because they take longer time to traverse the laser beam).
Separate barcoded cells (ctrl, shown in orange) from sample (shown in black) based on Alexa Fluor 647 signal and EdU incorporation (Alexa Fluor 594) (Figure 2B). Note that there is bleed-through from high EdU signal into the Alexa Fluor 647 channel, as a subset of the sample population with high Alexa Fluor 594 staining also show high Alexa Fluor 647 intensity. Nevertheless, the barcoded cells form a distinct population that can be separated from the sample cells. Importantly, there is no bleed-through from the EdU (Alexa Fluor 594 signal) into the RNAPII signal (Alexa Fluor 488) (Figure 3).
Gate for cell cycle phases based on EdU incorporation vs. DNA content (Figure 2Ci): G1 cells are negative for EdU and have G1 DNA content. Early S-phase cells have DNA content close to G1 but are EdU positive. Mid S have an intermediary DNA content and are EdU positive. Late S phase have similar DNA content as G2 cells but are EdU positive. G2 cells are EdU negative and have G2 DNA content. Mitotic cells are observed as the small population below the G2 cells and are not included in the analysis of G2 cells. Alternatively, gate for cell cycle phases by DNA content (Figure 2Cii): a strict G1 gate based on DNA content is performed by selecting only the cells to the left of the summit of the G1 peak. This strict gate will minimize contamination of S-phase cells. Likewise, G2 cells are selected to the right of the summit of the G2 peak to minimize contamination of S-phase cells. The S-phase gate should be located between the G1 and G2 peaks. Choosing a location well separated from these will minimize contamination of G1 and G2 cells. The cell cycle gates should be as equal as possible in all the samples during analysis.
Determine median RNAPII levels in individual cell cycle phases.
Raw data output files are generated as .fcs files.
Acquire 20,000 cells of the population identified as sample after separating the sample from the barcoded cells (Figure 2B). If the number of cells is limiting, the minimum number of cells acquired should be 5,000. Use either slow or medium speed when acquiring data on the flow cytometer. Keep the speed constant for all samples.
Figure 2. Gating strategy of flow cytometry data. A. Adjust Hoechst voltage so that the G1 peak summit is located at 50 K and gate for single cells as shown. B. Separate the barcoded cells from the sample cells by Alexa Fluor 647 vs. Alexa Fluor 594 signal as shown. C. Gate for cell cycle phases in the sample as indicated based on EdU incorporation and DNA content (Hoechst-A) as shown (i) or DNA content alone (ii) (see text for more details). D. Analyze RNAPII levels in the entire sample, and in individual cell cycle phases, by obtaining the median levels from the cell cycle phase populations defined in C. The cells in the different cell cycle phases from C are shown in the RNAPII scatter plots in D.
Figure 3. No bleed-through from Alexa Fluor 594-A (EdU) into Alexa Fluor 488-A (RNAPII) measurements. Scatter plots showing A) Alexa Fluor 594 (EdU) and B) Alexa Fluor 488 (RNAPII) intensities vs. Hoechst-A in sample and barcoded cells. Sample cells were identical to the barcoded cells, with the exception of EdU incorporation (performed on sample cells only) and Alexa Fluor 647 barcoding (performed on barcoded cells only). Sample cells show high Alexa Fluor 594 intensity in S phase compared to barcoded cells, which were not treated with EdU. However, intensities of the sample cells overlap completely with the intensities of the barcoded cells in the Alexa Fluor 488 scatter plot, showing that there is no bleed-through from Alexa Fluor 594 into the Alexa Fluor 488 measurements using the conditions described in this protocol.
Analysis of the flow cytometry data
Import .fsc files into a workspace on the flow cytometer software FlowJo V10_5 or other versions of the software.
Use the gating strategy depicted in Figure 2.
RNAPII profiles can be viewed in Layout Editor and median RNAPII levels can be determined in Table Editor.
RNAPII level in the sample is determined by subtracting the background fluorescence (using the median level of Alexa Fluor 488 staining in the whole secondary antibody control sample) and by normalizing to the median level of Alexa Fluor 488 staining in the whole barcoded cell sample (Equation 1, G1 levels are shown as an example of cell cycle phase). Also include non-treated cells as one of the samples. This should be used to measure changes after treatment (Equation 2, G1 levels are shown as an example).
Recipes
Chromatin extraction buffer (10 mL)
Reagent Stock Final concentration Volume
HEPES, pH 7.9 1 M 20 mM 200 μL
MgCl2 0.1 M 1.5 mM 150 μL
NaCl 5 M 140 mM 280 μL
Sucrose 2.5 M 300 mM 1,200 μL
Triton X-100 100% 0.5% 50 μL
ddH2O Milli-Q water Adjust with water up to total 10 mL
Prior to use, add one tablet of cOmplete EDTA-free protease inhibitor cocktail and one tablet of PhosSTOP phosphatase inhibitors per 10 mL of solution. Add 2 μL of 10 mM MG132 per milliliter of buffer for a final concentration of 20 μM. The extraction buffer should be prepared fresh the day of extraction and kept at 4 °C or on ice prior to use.
LX Alexa Fluor 647 NHS Ester (barcoding)
Add 2 μL of Alexa Fluor 647 NHS Ester (5 mg/mL) stock in 553.5 μL of DMSO; the resulting solution is named working solution (18 μg/mL). Dilute the working solution further by combining 200 µL of the working solution with 160 µL of DMSO. The final solution is called the LX solution (10 µg/mL). Once diluted, the working solution and the LX solution can be stored at -80 °C and are stable for several weeks.
Flow buffer (1 L)
Reagent Stock Final concentration Volume
Na2HPO4 0.25 M 6.5 mM 26 mL
KH2PO4 1 M 1.5 mM 1.5 mL
KCl 1 M 2.7 mM 2.7 mL
NaCl 5 M 137 mM 27.4 mL
EDTA, pH 7.5 0.5 M 0.5 mM 1 mL
ddH2O Milli-Q water Adjust with water up to total 1 L
Add 100 μL of Igepal CA-630 100% to 100 mL of flow buffer to obtain a final concentration of 0.1%.
Detergent buffer
Flow buffer + 4% skim milk powder. For 4 mL of flow buffer, add 160 mg of skim milk powder.
Click-iT permeabilization and wash reagent
Reagent Volume
PBS 4.5 mL
Component E 500 μL
BSA 1%
Component E is from the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit.
Click-iT reaction cocktail
The copper protectant, fluorescent dye picolyl azide, and 10× reaction buffer additive are obtained from the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay kit. Dilute 10× reaction buffer additive 1:10 in ddH2O Milli-Q water.
Reagent Volume (for one reaction/sample)
PBS 438 μL
Copper protectant (Component F) 10 μL
Fluorescent dye picolyl azide 2.5 μL
Reaction buffer additive 1× 50 μL
Total reaction volume 500 μL
Adjust volumes according to number of samples in the experiment.
Acknowledgments
The method described here is further developed from Landsverk et al. (2020), Håland et al. (2015) and Stokke et al. (1993). We would like to thank the flow cytometry core facility at the Radium Hospital, Oslo University Hospital, for assistance and use of the flow cytometers. This work was funded by the Norwegian Research Council (275918) and Nansenfondet og de dermed forbundne fond.
Competing interests
The authors declare no competing interests.
References
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Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation
YY Yongil Yang
YS Yuanhua Shao
TC Timothy A. Chaffin
AA Amir H. Ahkami
EB Eduardo Blumwald
CJ C. Neal Stewart Jr.
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4660 Views: 862
Reviewed by: Pooja Verma Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Plant Biotechnology Journal Jul 2021
Abstract
Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters.
Graphical overview
Keywords: Poplar Mesophyll protoplasts Synthetic promoter Fluorescence Image analysis
Background
Transient transformation assays using plant protoplasts are effectively used for rapid evaluation of the subcellular localization of interesting proteins and how promoters regulate transcription, a lengthy process in stably engineered plants. In many cases, protoplasts are suitable proxies for the plant tissues used to isolate protoplasts—from physiological and molecular events to those in a whole organism. Therefore, protoplasts may be useful to investigate hormone responses, metabolomic processes, and cell responses against abiotic or biotic cues with minimal effort. Protoplasts provide a versatile plant cell system for screening many DNA fragments, which can be broadly applied in synthetic biology techniques. Especially, protoplast transformation can be effectively applied to determine synthetic promoter function using assays of induced marker genes (Cai et al., 2020; Jores et al., 2021; Yang et al., 2021). Furthermore, recent successes in rapid screening CRISPR/Cas9 constructs for gene edition after protoplast transformation show another application for protoplasts in synthetic biology (Lin et al., 2018; Toda et al., 2019; Rather et al., 2022). Additionally, with the recent appearance of practical methods for single cell–based -omics research using protoplasts (Chen et al., 2021; Farmer et al., 2021; Liu et al., 2021; Seyfferth et al., 2021; Liu et al., 2022; Mo and Jiao, 2022), the potential applications are continuing to grow in advanced research fields.
To date, plant protoplast transformation has been achieved in various herbaceous species as well as woody plants, since the initial methods were devised using Arabidopsis leaves (Yoo et al., 2007). Among them, poplar has been an attractive application for accelerating research in biofuel and bioproducts. Poplar protoplast isolation has been established using leaf mesophyll and stem tissues (Guo et al., 2012; Lin et al., 2014; Chen et al., 2021). A practical application for determining valuable stress-responsive synthetic promoters was successfully applied with poplar mesophyll protoplast transformation followed by fluorescence image analysis (Yang et al., 2021). The present protocol introduces the isolation of poplar mesophyll protoplasts to be applied for synthetic promoter assays by image analysis through fluorescence detection. This protocol uses less cell wall digestion enzyme and yields higher protoplast recovery compared to a regular poplar mesophyll protoplast preparation. Furthermore, a simpler and more convenient method using fluorescence measurement is utilized for the synthetic promoter screening.
Materials and Reagents
INRA 717-1B4 (Populus tremula × Populus alba) hybrid poplar
Glass plate
Single edge blades (Personna, catalog number: 94-120-71)
100 mm diameter and 20 mm deep Petri dishes (Fisher Scientific, catalog number: FB087511Z)
Magenta GA-7 plant culture box (Fisher Scientific, catalog number: 50-255-176)
BasixTM polystyrene serological pipette 10 mL (Thermo Scientific, catalog number: 14-955-234)
50 mL tube (Thermo Scientific, catalog number: 339650)
0.45 µm filter (Millipore, catalog number: SLHV004SL)
Round bottom disposable glass culture tube (10 mL) (Fisher Scientific, catalog number: 14-961-27)
96-well black/clear bottom plate (Thermo Scientific, catalog number: 165305)
40 µm sterile cell strainers (Fisher Scientific, catalog number: 08-771-1)
Murashige and Skoog (MS) basal salts with macronutrients and micronutrients (Caisson Labs, catalog number: MSP01)
MS vitamin solution (Caisson Labs, catalog number: MLV01)
MES hydrate (Thermo Scientific, catalog number: H5672.36)
KOH (Fisher Scientific, catalog number: M1064621000)
Sucrose (Fisher Scientific, catalog number: AA36508A1)
Activated charcoal (Sigma-Aldrich, catalog number: C9157)
GelzanTM CM (Gelrite®, Sigma-Aldrich, catalog number: G1910)
Indole-3-butyric acid (IBA) (PhytoTech Labs, catalog number: 1358)
Cellulase OnozukaTM R-10 (Yakurut Co., Cellulase OnozukaTM R-10)
MacerozymeTM R-10 (Yakurut Co., MacerozymeTM R-10)
D-mannitol (Sigma-Aldrich, catalog number: M4125)
KCl (Sigma-Aldrich, catalog number: P3911)
CaCl2 (Sigma-Aldrich, catalog number: C4901)
NaCl (Fisher Scientific, catalog number: BP358)
MgCl2 (Fisher Scientific, catalog number: AC223211000)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153)
Polyethylene glycol M.W. 6000 (PEG) (Sigma-Aldrich, catalog number: 81260)
Deionized-distilled water (ddH2O)
Rooting media (see Recipes)
Cell wall degradation enzyme mixture (see Recipes)
W5 solution (see Recipes)
MMg solution (see Recipes)
40% PEG solution (see Recipes)
WI solution (see Recipes)
Equipment
Growth chamber (Percival, model: CU-36L4)
Hausser ScientificTM 3200 hemacytometer (Fisher Scientific, catalog number: 02-671-54)
Micropipette sets 0.5–10 µL, 10–100 μL, and 100–1,000 µL (Corning, catalog numbers: 4071, 4073, 4075)
Micropipette tips 0.1–10 µL, 5–300 µL, and 100–1,000 µL (Fisher Scientific, catalog numbers: 02-707-454, 02-707-447, 02-707-408)
Water bath (Fisher Scientific, model: IsoTEMP 105)
Vacuum chamber (BVV, model: GV3G)
Orbital shaker (Lab-Line, model: 3520)
Centrifuge (Thermo Scientific, model: SorvallTM RC 6+) with swinging buckets (SH3000)
EVOS M7000 imaging system (Invitrogen, model: AMF7000)
Software
ImageJ (National Institutes of Health, imagej.nih.gov)
Procedure
Plant growth
Propagate a mini plant of 717-1B4 hybrid poplar in solidified rooting media (see Recipes) in a Magenta GA-7 plant culture box.
Grow the plant for approximately 1–2 months in a growth chamber controlled with 16:8 h light/dark conditions at 23 °C with 150 µmol/m2 s irradiance.
Preparation of cell wall degradation enzyme mixture
Prepare the cell wall degradation enzyme mixture as described (see Recipes).
Heat the solution at 55 °C for 10 min to degrade possible protease activity in buffer. Store the solution on ice.
Isolation of mesophyll protoplast from healthy leaves of propagated poplar
Collect the fully expanded leaves (>1 cm in width and >2 cm in length of leaf blade) from rooted poplar clones growing in Magenta boxes (Figure 2a).
Slice ten fully expanded leaves (expected recovery of ~7 × 106 cells) into 2 mm wide cut strips with a sharp single-edge blade on a glass plate.
Submerge leaf strips into 10 mL of cell wall degradation enzyme mixture in a deep 100 mm Petri dish.
Infiltrate the enzyme solution into the leave strips by vacuuming the solution in a vacuum chamber under ~40 mmHg pressure for 30 min. Turn off the vacuum but keep pressure for another 2.5 h.
Release the vacuum slowly. Lightly shake the mixture using orbital shaker (~80 rpm) for 10 min. Meanwhile, precool a 50 mL tube on ice.
Add 10 mL of precooled W5 solution (see Recipes) to the cell wall degradation enzyme–leaf strips mixture. Mix well by gentle hand shaking.
Put the 40 µm cell strainer on the precooled 50 mL tube on ice. Pass the mixture through the cell strainer to filter out cell debris from protoplast mixture. The green flowthrough contains the mesophyll protoplasts.
Remove the cell strainer and close the lid of the 50 mL tube. Spin down the cell by centrifugation at 100 × g for 3 min at 4 °C. Remove supernatant (~19.8 mL) by pipetting and then leave the green pellet with remaining solution (~0.2 mL).
Add 1 mL of W5 solution and resuspend the pellet. Place the tube on ice for 30 min for cell recovery.
Meanwhile, count the protoplasts using a hemacytometer under a microscope. Drop 10 µL of cell solution on hemacytometer and then cover the slide glass. Estimate cell count using EVOS imaging system.
Spin down the cell resuspension by centrifugation at 100 × g for 3 min. After completely removing W5 solution, add enough MMg solution (see Recipes) based on estimated cell count to bring the final concentration to 1 × 103 cells/µL.
PEG-mediated protoplast transformation
Dilute the plasmid with ddH2O to a final concentration of 1 µg/µL of pure plasmid DNA.
Add 100 µL of protoplast solution (1 × 105 cells) to a glass culture tube at room temperature. Add 20 µL of plasmid (20 µg) directly to protoplast without any loss in pipetting. Lightly tap the plasmid–protoplast mixture several times to mix.
Add 120 µL of 40% PEG solution (see Recipes) into 120 µL of plasmid–protoplast mixture. Slowly pour PEG solution at the surface of the protoplast solution to prevent pre-mixing of PEG with the plasmid–protoplast. The PEG will submerge smoothly below the plasmid–protoplast mixture, and clear separation between PEG and the plasmid–protoplast mixture will be observed. As soon as PEG is added to all mixtures, tap lightly several times until the mixture is green throughout. The final concentration of PEG is 20% in the mixture.
Incubate the mixture for 15 min at room temperature. Stop incubation by adding 500 µL of W5 solution to the transformed protoplast mixture. Note that adding at least two volumes of W5 solution (480 µL) is required for enough dilution of PEG.
Spin down the transformed protoplast by centrifugation at 100 × g for 3 min at 4 °C. Completely remove supernatant without any contact to the protoplast pellet.
Resuspend the protoplast pellet with 150 µL of WI solution (see Recipes) and gentle tapping.
Transfer protoplast–WI suspension into a 96-well optical microplate. Cover the lid and then incubate the plate at room temperature in the dark.
Fluorescence induction and image analysis
Note that fluorescence can be detected in the protoplast cells after 12 h up to one week depending on fluorescence species and experimental purpose. For the synthetic promoter screening, 48 h incubation is recommended.
To observe synthetic promoter activity in transformed protoplast, we measure fluorescence by scanning the 96-well plate using the EVOS 7000 image system. Put the incubated 96-well plate on plate holder of Evo image analyzer.
Set the reading mode to 96-well plate. Select the filter set for green fluorescence protein (GFP), red fluorescence protein (RFP), and brightfield images.
Take pictures under a 10× objective lens setting with optimal parameters. Calculate the transformation efficiency and promoter activity with these images.
To count fluorescent-expressed protoplast from images, use automatic options integrated in ImageJ software. Convert a color image to black/white format by going to Edit/Options/Conversions to scale when converting in menu bar. Then, turn image type to 16 bit grey scale by selecting Image option.
Click the Image/Adjust/Threshold for cells with the desired threshold parameter. Use this threshold setting for all images. Then, analyze particle by Analyze/Analyze particles option in the menu. Collect the count and particle information for further analysis. Three repeated transformations per plasmid were performed for statistical analysis.
Figure 1. Protocol for poplar protoplast isolation followed by synthetic promoter gene construct transformation into prepared protoplast
Data analysis
Poplar mesophyll protoplast transformation
The cell wall–digested poplar mesophyll protoplast is relatively smaller than those from Arabidopsis leaf and somatic cell–cultured soybean and potato (Figure 2b). Estimated protoplast cell counts were in a range of 6 × 106–7 × 106 cells in three diverse extractions (data not shown). Figure 2c shows a representative summary of different fluorescence images in protoplast transformed with a plasmid containing RFP driven by the 2× short 35S Cauliflower mosaic virus (CaMV) promoter, and GFP expressed by a designed synthetic promoter after 48 h incubation at room temperature (Figure 2c). The RFP and GFP fluorescence can be clearly distinguished overlaying the individual protoplasts. Transformation efficiency generally reaches up to 70%, which is determined by RFP-expressed cell counts over total protoplast cell counts with 20 µg of plasmid.
Figure 2. Representative images of isolated poplar mesophyll protoplast and fluorescent expression after transient plasmid transformation. a) Poplar clone in rooting media used for leaf section to isolate mesophyll protoplasts. The plants were grown up to two months to collect leaf blades suitable for protoplast isolation. b) Image of protoplasts isolated by 3 h after vacuum infiltration in cellulase enzyme solution. c) Images of GFP, RFP, and brightfield were taken separately after 48 h incubation following PEG-mediated transformation of 20 µg of plasmid containing 35S-driven RFP and synthetic promoter-driven GFP. Merged image was generated by EVOS M7000 imaging system based on both fluorescence and brightfield images overlayed on a single frame. Bars display 100 µm.
Synthetic promoter analysis by fluorescence detection using image analyzer
A total of 19 different plasmids were generated by replacing the promoter region with individual synthetic promoters (Figure 3a; the exact synthetic promoters’ sequences are not listed due to conflict of interest). The green GFP represents stress inducibility of each synthetic promoter against osmotic stress caused by incubation in 0.5 M mannitol (Yang et al., 2021). A 30% lower amount of cellulase and 2 h longer duration of vacuum infiltration were used here to isolate protoplasts, compared to the original established procedure for poplar mesophyll protoplast isolation (Guo et al., 2014). The fluorescent proteins were screened after 48 h incubation at room temperature. The images were collected as pairs of RFP and GFP expression from the same transformed protoplasts (Figure 3b). We observed RFP expression in whole-transformed protoplasts, confirming that transformation of current plasmid sets into protoplast were performed successfully following the present protocol (Figure 3b). In screening synthetic promoter activity, various ranges of the protoplasts expressing GFP were observed, meaning that we can distinguish different synthetic promoter-activated GFP expression by osmotic stress treatment. The ratio of GFP-expressed protoplast count over RFP-expressed protoplast count determined synthetic promoter activity for 19 synthetic promoters (Figure 3c).
Figure 3. Comparison images for screening synthetic promoters by fluorescent protein induction driven by osmotic stress–inducible synthetic promoters. a) Plasmid construct layout used for protoplast transformation. Promoter area was replaced with a synthetic promoter from each of 19 promoters. These were adopted from our lab and randomly labeled from A to S. b) Fluorescence images to screen poplar mesophyll protoplasts containing plasmid construct of different synthetic promoters driving GFP and 35S driving RFP. The RFP was used as an internal control to determine transformation yield. GFP inducibility was determined by 48 h incubation in 0.5 M mannitol solution. Bars display 100 µm. c) Plot of fluorescent expression ratio based on cell counts of protoplast of GFP-expression driven by synthetic promoter over RFP–control expression, showing reliable plasmid transformation into protoplasts. Protoplasts expressing both fluorescent proteins were counted by ImageJ as described in part E of the procedure section. Bars display the average of cell count ratio of GFP-expressed protoplast over RFP-expressed. Error bars show the standard deviation of ratio percentage from three replicates of protoplast transformation with each plasmid.
Recipes
Rooting media
Dissolve 4.43 g of MS basal salts with macronutrients and micronutrients, 1× MS vitamin solution, 0.5 g of MES hydrate, 30 g of sucrose, 5 g of activated charcoal, and 1.5 g of GelzanTM CM in 1 L ddH2O.
Adjust pH to 5.7 with 1 M KOH.
Autoclave the solution.
After the media temperature drops, add 100 µL of IBA (1 mg/mL of stock solution) to the media.
Pour 50 mL of media to each Magenta GA-7 box and let solidify completely.
Cell wall degradation enzyme mixture
Dissolve 0.2 g of cellulase OnozukaTM R-10 and 0.08 g of MacerozymeTM R-10 in the buffer solution including 1 mL of 0.2 M MES hydrate (pH 5.7), 5 mL of 0.8 M D-mannitol, and 100 µL of 2 M KCl.
Incubate the dissolved solution at 55 °C for 10 min. Then, cool on ice for at least 5 min.
Add 100 µL of 1 M CaCl2 and 100 µL of 10% BSA.
Sterilize the enzyme solution through a 0.45 µm filter, optionally.
W5 solution
Mix 2 mL of 0.2 M MES (pH 5.7), 6.16 mL of 5 M NaCl, 25 mL of 1 M CaCl2, and 500 µL of 2 M KCl with 166.34 mL of ddH2O.
MMg solution
Mix 5 mL of 0.8 M D-mannitol, 150 µL of MgCl2, and 200 µL of 0.2 M MES hydrate (pH 5.7) with 4.65 mL of ddH2O.
40% PEG solution
Prepare 40% PEG solution just before transformation.
Dissolve 4 g of PEG in the mixture including 2.5 mL of 0.8 M D-mannitol, 1 mL of 1 M of CaCl2, and 3 mL of ddH2O. If PEG is dissolved completely, the solution volume will have a total volume of 10 mL.
WI solution
Mix 6.25 mL of 0.8 M D-mannitol, 200 µL of 0.2 M MES (pH 7.5), 100 µL of 2 M KCl, and 3.45 mL of ddH2O.
Acknowledgments
This work was supported by funding from the Biological and Environmental Research in the U.S. Department of Energy Office of Science (DE-SC0018347). The protoplast isolation method in this protocol was modified from Guo et al. (2012). The plasmid constructs were adopted from unpublished work performed by the authors. The authors thank the technical staff and students in our laboratories for their support and contribution to the work.
Competing interests
C. Neal Stewart Jr. is an inventor on synthetic promoter and biotechnology intellectual property that is assigned to the University of Tennessee Research Foundation.
References
Cai, Y. M., Kallam, K., Tidd, H., Gendarini, G., Salzman, A. and Patron, N. J. (2020). Rational design of minimal synthetic promoters for plants. Nucleic Acids Res 48(21): 11845-11856.
Chen, Y., Tong, S., Jiang, Y., Ai, F., Feng, Y., Zhang, J., Gong, J., Qin, J., Zhang, Y., Zhu, Y., Liu, J. and Ma, T. (2021). Transcriptional landscape of highly lignified poplar stems at single-cell resolution. Genome Biol 22(1): 319.
Farmer, A., Thibivilliers, S., Ryu, K. H., Schiefelbein, J. and Libault, M. (2021). Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level. Mol Plant 14(3): 372-383.
Guo, J., Morrell-Falvey, J. L., Labbe, J. L., Muchero, W., Kalluri, U. C., Tuskan, G. A. and Chen, J. G. (2012). Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS One 7(9): e44908.
Jores, T., Tonnies, J., Wrightsman, T., Buckler, E. S., Cuperus, J. T., Fields, S. and Queitsch, C. (2021). Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. Nat Plants 7(6): 842-855.
Lin, C. S., Hsu, C. T., Yang, L. H., Lee, L. Y., Fu, J. Y., Cheng, Q. W., Wu, F. H., Hsiao, H. C., Zhang, Y., Zhang, R., et al. (2018). Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16(7): 1295-1310.
Lin, Y. C., Li, W., Chen, H., Li, Q., Sun, Y. H., Shi, R., Lin, C. Y., Wang, J. P., Chen, H. C., Chuang, L., et al. (2014). A simple improved-throughput xylem protoplast system for studying wood formation. Nat Protoc 9(9): 2194-2205.
Liu, H., Hu, D., Du, P., Wang, L., Liang, X., Li, H., Lu, Q., Li, S., Liu, H., Chen, X., et al. (2021). Single-cell RNA-seq describes the transcriptome landscape and identifies critical transcription factors in the leaf blade of the allotetraploid peanut (Arachis hypogaea L.). Plant Biotechnol J 19(11): 2261-2276.
Liu, Q., Li, P., Cheng, S., Zhao, Z., Liu, Y., Wei, Y., Lu, Q., Han, J., Cai, X., Zhou, Z., et al. (2022). Protoplast Dissociation and Transcriptome Analysis Provides Insights to Salt Stress Response in Cotton. Int J Mol Sci 23(5): 2845.
Mo, Y. and Jiao, Y. (2022). Advances and applications of single-cell omics technologies in plant research. Plant J 110(6): 1551-1563.
Rather, G. A., Ayzenshtat, D., Teper-Bamnolker, P., Kumar, M., Forotan, Z., Eshel, D. and Bocobza, S. (2022). Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato. Planta 256(1): 14.
Seyfferth, C., Renema, J., Wendrich, J. R., Eekhout, T., Seurinck, R., Vandamme, N., Blob, B., Saeys, Y., Helariutta, Y., Birnbaum, K. D. et al. (2021). Advances and Opportunities in Single-Cell Transcriptomics for Plant Research. Annu Rev Plant Biol 72: 847-866.
Toda, E., Koiso, N., Takebayashi, A., Ichikawa, M., Kiba, T., Osakabe, K., Osakabe, Y., Sakakibara, H., Kato, N. and Okamoto, T. (2019). An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. Nat Plants 5(4): 363-368.
Yang, Y., Lee, J. H., Poindexter, M. R., Shao, Y., Liu, W., Lenaghan, S. C., Ahkami, A. H., Blumwald, E. and Stewart, C. N., Jr. (2021). Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements. Plant Biotechnol J 19(7): 1354-1369.
Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.
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4,661 | https://bio-protocol.org/en/bpdetail?id=4661&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety
NT Ngoc Tung Tran *
ML Mikhail Lebedin *
ED Eric Danner *
RK Ralf Kühn
KR Klaus Rajewsky
VC Van Trung Chu
(*contributed equally to this work)
Published: Vol 13, Iss 8, Apr 20, 2023
DOI: 10.21769/BioProtoc.4661 Views: 674
Reviewed by: David PaulDr. Amit K. Tripathi Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Advances Jun 2022
Abstract
The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38–68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy.
Graphical overview
Keywords: Cas9D10A nickase PAM-out sgRNAs Spacer distance of 200-350 bp Gene repair approach AAV6 Gene correction efficiency Unintended on-target mutations Off-target mutations Human hematopoietic stem and progenitor cells (HSPCs)
Background
In the CRISPR/Cas9 system, a single-guide RNA (sgRNA)-directed Cas9 nuclease introduces double-strand breaks (DSBs) at the target region. DSBs are predominantly repaired by the non-homologous end joining (NHEJ) pathway, causing micro-insertions or deletions (indels or unwanted on-target mutations). If a DNA donor template with 5′ and 3′ homology arms (HAs) is provided, the homology-directed repair (HDR) pathway is activated to precisely replace the mutated DNA sequence (Cong et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Chu et al., 2015). Although the CRISPR/Cas9 system succeeded in repairing mutations, its undesired on-target mutations and potential off-target activities are still a major concern (Cradick et al., 2013; Frock et al., 2015; Fu et al., 2013; Pattanayak et al., 2013; Tsai et al., 2015). The target specificity of CRISPR/Cas9 has been increased by using truncated sgRNAs or extended sgRNAs with two additional G nucleotides at the 5′ end (Cho et al., 2014; Fu et al., 2014; Tsai et al., 2015), sgRNAs with high specificity (Akcakaya et al., 2018), or high-fidelity SpCas9 mutants (Kleinstiver et al., 2016; Slaymaker et al., 2016; Vakulskas et al., 2018). A nickase, a mutated version of Cas9, can be generated by introducing a D10A or H840A mutation to RuvC (E. coli protein that is an endonuclease) or HNH (histidine-asparagine-histidine motif) nuclease domain, respectively (Jinek et al., 2012; Nishimasu et al., 2014). A sgRNA-guided Cas9 nickase introduces a nick at the target sequence, generating a single-stranded break that is perfectly repaired by the nonmutagenic base excision repair pathway (Dianov and Hubscher, 2013). As a result, Cas9 nickase creates less indels than the Cas9 nuclease. In combination with a pair of PAM-out (facing away from each other) sgRNAs at a short distance of 38–68 bp, the Cas9D10A nickase generates offset double nicks that convert into site-specific DSBs. This approach led to efficient HDR- and NHEJ-mediated on-target events, while reducing off-target effects by 50–1,000-fold (Ran et al., 2013; Mali et al., 2013a). The limitation of this approach is that DSBs are still induced at the target sequence, leading to frequent indels and nonsense mutations in the target gene (Ran et al., 2013; Miyaoka et al., 2016).
Recently, we described a spacer-nick system that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a long distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor template delivery, the spacer-nick-based gene repair approach preserves efficient gene correction and minimizes adverse effects, such as unintended NHEJ-mediated on-target mutations and unwanted off-target genetic alterations in human hematopoietic stem and progenitor cells (HSPCs) (Tran et al., 2022). Additionally, we detailed modified GUIDE-seq and linear amplification high-throughput genome-wide translocation sequencing (LAM-HTGTS) methods to quantify genome-wide off-target mutations and capture all possible gene editing outcomes induced by sgRNA-guided nucleases in human HSPCs. The GUIDE-seq method is based on the integration of a blunt-end double-stranded (ds) oligodeoxynucleotide (dsODN) into the nuclease-induced DSBs and requires a large cell number (Tsai et al., 2015). To overcome this limitation and simplify this method to wet lab workflow, we modified it using Tn5 transposase to tagment genomic DNA and insert universal sequencing adapters into both ends of tagmented DNA fragments, allowing for an amplification of dsODN tags with specific primers (termed Tn5-mediated GUIDE-seq). Moreover, we modified the original HTGTS method previously described (Hu et al., 2016). This modification is based on linear amplification of primers outside of HAs and leads to a 5′- and 3′-based sequencing of the target region, allowing for a quantification of indels and deletion, AAV integrations, inversions, HDR, and translocations. Overall, we provide detailed step-by-step protocols for spacer-nick-mediated gene repair and off-target assessment in human HSPCs.
Materials and Reagents
Pipette filter tips [Sarstedt, catalog numbers: 70.3010.275 (10 μL), 70.3030.265 (20 μL), 70.3030.375 (100 μL), 70.3030.110 (200 μL), 70.3060.275 (1,000 μL)]
1.5 mL tubes (Eppendorf, catalog number: 0030 120.086)
2.0 mL tubes (Eppendorf, catalog number: 0030 120.094)
200 μL PCR plates (Sarstedt, catalog number: 72.1980.202)
200 μL PCR tubes (Neolab, catalog number: 7-5207)
Agarose (Biozym, catalog number: 840004)
100 bp DNA ladder (Thermo Fisher, catalog number: 5628019)
GeneRuler 1 kb Plus DNA ladder (Thermo Fisher, catalog number: SM1331)
AMPure XP beads (Beckman Coulter, catalog number: A63881)
DynabeadsTM MyONETM Streptavidin C1 (Thermo Fisher, catalog number: 65001)
Nuclease-free water (Sigma, catalog number: 3098)
Ethanol (Carl Roth, catalog number: P075.1)
Tris (Carl Roth, catalog number: 5429.2)
HCl (Carl Roth, catalog number: 9277.2)
NaOH (Carl Roth, catalog number: P031.2)
EDTA·2H2O (Carl Roth, catalog number: 8043.3)
NaCl (Carl Roth, catalog number: 3957.4)
PEG 8000 (Sigma, catalog number: 89510-250G-F)
Human CD34+ microbead kit (Miltenyi Biotec, catalog number: 130-046-702)
Ficoll-Paque Plus (GE Healthcare, catalog number: 17144002)
Serum-free StemSpanTM SFEM II medium (Stemcell, catalog number: 09655)
Human SCF (PeproTech, catalog number: 300-07)
Human TPO (PeproTech, catalog number: 300-18)
Human FLT3L (PeproTech, catalog number: 300-19)
Human IL-6 (PeproTech, catalog number: 200-06)
UM171 (Stemcell, catalog number: 72912)
StemRegenin 1 (SR1) (Stemcell, catalog number: 72342)
AAV6 system (Cell Biolabs, catalog number: VPK-410-SER6)
Fastdigest NotI (Thermo Fisher, catalog number: FD0593)
CrRNAs (IDT)
Alt-R® CRISPR-Cas9 tracrRNA (IDT, catalog number: 1072534)
Alt-R® S.p. Cas9D10A nickase V3 (IDT, catalog number: 1081063)
P3 primary cell 4D-NucleofectorTM X kit L (Lonza, catalog number: V4XP-3012)
Dead cell removal kit (Miltenyi Biotec, catalog number: 130-090-101)
KOD hot-start DNA polymerase (Merck Millipore, catalog number: 71085)
NucleoSpin Gel and PCR Cleanup (Macherey-Nagel, catalog number: 740609.50)
NucleoBond Xtra Maxi kit (Macherey-Nagel, catalog number: 740414.50)
GenFind V3 reagent kit (Beckman Coulter, catalog number: C34880)
Illumina Tagment DNA TDE1 Enzyme and Buffer kits (Illumina, catalog number: 20034197)
Zymo DNA clean and concentrator-5 (Zymo research, catalog number: D4003)
PrimeSTAR GXL polymerase (Takara, catalog number: R050B)
QubitTM dsDNA HS assay kit (Thermo Fisher, catalog number: Q32851)
High sensitivity D1000 ScreenTape assay (Agilent, catalog number: 5067)
QIAquick Gel Extraction kit (Qiagen, catalog number: 28704)
T4 DNA ligase (NEB, catalog number: M0202)
Hexaamminecobalt(III) chloride (HexCo) (Sigma, catalog number: 481521-25G)
Serum-free freezing medium BAMBANKER (Nippon Genetics, catalog number: 5802)
Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 20012027)
Platinum Taq polymerase (Thermo Fisher, catalog number: 15966005)
Nextera XT index kit v2 set A (Illumina, catalog number: FC-131-2001)
5 M Tetramethylammonium chloride solution (TMAC) (Sigma, catalog number: T3411-500ML)
Gel Loading Dye, Orange (6×) (NEB, catalog number: B7022S)
Q5 High-fidelity DNA polymerase (NEB, catalog number: M0491S)
Wizard Genomic DNA purification kit (Promega, catalog number: A1120)
ProNex size-selective purification beads (Promega, catalog number: NG2001)
High sensitivity DNA BioAnalyzer kit (Agilent, catalog number: 5067-4626)
TOP10 bacteria (Invitrogen, catalog number: C404003)
LB agar, powder (Invitrogen, catalog number: 22700025)
Carbenicillin (Sigma, catalog number: C1613-1ML)
T4 DNA ligase (NEB, catalog number: M0202M)
P3 Primary Cell electroporation buffer (see Recipes)
Completed serum-free StemSpanTM SFEM II medium (see Recipes)
70% ethanol (see Recipes)
50% (w/v) PEG 8000 (see Recipes)
5 M NaCl (see Recipes)
2.5 N NaOH (see Recipes)
0.5 M EDTA (see Recipes)
1 M Tris-HCl (pH 7.4) (see Recipes)
1 M Tris-HCl (pH 8.0) (see Recipes)
1× TE buffer (pH 8.0) (see Recipes)
B&W buffer (see Recipes)
20 mM hexaamminecobalt(III) chloride (see Recipes)
50 mM bridge adapter (see Recipes)
Forward and reverse oligos of dsODN (IDT) (Table 1)
Primers (Eurofins) (Table 1)
Table 1. Oligos and primers
Name Sequence
dsODN-forward 5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
dsODN-reverse 5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′
I5-Nextera-3′-GSP (-) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTTAATTGAGTTGTCATATGTTAATAACGGT
I5-Nextera-5′-GSP (+) TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATACCGTTATTAACATATGACAACTCAATTAA
I7-Nextera-reverse GTCTCGTGGGCTCGGAGATGTGTATAAG
Bridge-lower GCGACTATAGGGCACGCGTGGNNNNNN[AmC3]
Bridge-upper [Phos]CCACGCGTGCCCTATAGTCGC[AmC3]
U2-C-Adapter GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNGACTATAGGGCACGCGTGG
U1-C-GeneSpecific TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNN[GeneSpecific]
FC1-i5-U1 (N501) AATGATACGGCGACCACCGAGATCTACAC tagatcgc TCGTCGGCAGCGTC
FC2-i7-U2 (N701) CAAGCAGAAGACGGCATACGAGAT tcgcctta GTCTCGTGGGCTCGG
Equipment
96-well magnet stand (Thermo Fisher, catalog number: AM10027)
6-well magnet stand (Cytiva, catalog number: 28948964)
Tissue culture 6-well plates (Sarstedt, catalog number: 83.3920)
Tissue culture 12-well plates (Sarstedt, catalog number: 83.3921)
Freezing vials (Sarstedt, catalog number: 72.380.992)
16-well nucleocuvette strip (Lonza, catalog number: V4XP-3032)
Lonza 4D-Nucleofector (Lonza, catalog number: AAF-1003B)
Pipettes (Gilson)
Multichannel pipettes (Eppendorf)
PCR thermocycler (Eppendorf, catalog number: 6331000017)
NanoDrop (Thermo Fisher)
Thermomixer (Eppendorf)
Vortexer (Scientific Industries)
Centrifuge (Eppendorf)
Tissue culture incubator set at 37 °C, 5% CO2 (Binder)
TapeStation (Agilent, catalog number: G2991BA)
Qubit 2.0 fluorometer (Invitrogen, catalog number: Q32866)
ChemiDoc XRS gel imaging system (Bio-Rad)
Standard UV transilluminator (Cleaver Scientific)
TC20 automated cell counter (Bio-Rad, catalog number: 1450102)
Tube roller (Starlab, catalog number: N2400-7010)
2100 BioAnalyzer instrument (Agilent, catalog number: G2939BA)
Software
CrispRGold (the MDC, https://crisprgold.mdc-berlin.de)
R 4.0.3 programming language (R-project, https://www.r-project.org/)
R-studio integrated development environment (R-studio, https://www.rstudio.com/)
GelAnalyzer 19.1 gel densitometry software (GelAnalyzer, http://www.gelanalyzer.com/?i=1)
Procedure
Design spacer-nick sgRNAs
Note: For testing gene editing efficiencies of sgRNAs, you can select any available system that is suitable for your laboratory, such as T7EI (T7 endonuclease I) or Sanger sequencing following ICE analysis. Additionally, sgRNAs could be ordered as a pair of oligos, cloned into expression vectors, and tested in human cell lines such as HEK293T. You can also order synthetic sgRNAs, generate pre-assembled ribonucleoprotein (RNP) complexes of Cas9 nuclease and synthetic sgRNA, and perform in vitro T7EI assay on PCR amplicons.
Define 5′ and 3′ areas flanking mutation hotspots (Figure 1) with a spacer distance of 200–350 bp.
Figure 1. Design pools of spacer-nick sgRNAs. Scheme showing selected areas with a spacer distance of 200–350 bp, flanking a mutation. Pools of 5′ and 3′ sgRNAs with configurations of PAM sites facing outwards (PAM-out) are designed.
Design pools of sgRNAs on the selected areas with a PAM-out configuration using CrispRGold software. In combination with the Cas9D10A nickase, 5′ sgRNAs nick the plus strand, whereas 3′ sgRNAs nick the opposite strand of the dsDNA molecule.
Order designed sgRNAs, clone them into the suitable plasmids, and test gene editing efficiencies of these sgRNAs using T7EI assay or Sanger sequencing following ICE analysis.
Select several pairs of PAM-out sgRNAs (one 5′ sgRNA and one 3′ sgRNA) with equal and high gene editing efficiencies (>80%) for the spacer-nick gene repair approach.
Design DNA donor templates
Note: In order to quantify gene correction efficiencies, we insert a diagnostic restriction enzyme site by introducing silent mutations into correct sequences or develop correct integration PCR, which allows us to amplify repaired and non-repaired alleles. To clone DNA donor fragments into the AAV genome vector, we inserted NotI sites into both ends of DNA donor fragments together with six random nucleotides.
Design a DNA donor template carrying 5′ and 3′ HAs of at least 0.5–1.5 kb outside of each nick site (Figure 2).
In order to minimize a mini-homologous sequence (highlighted in green; Figure 2) between two nick sites, we modify this sequence by introducing silent mutations (in case of repairing mutations in coding exons) or by partially ablating this sequence (in case of inserting cDNA into loci).
Figure 2. Design the DNA donor template. Scheme showing configuration of the DNA donor template that carries 5′ and 3′ homology arms, flanking a modified sequence (marked in green) for gene correction and NotI sites at both ends for cloning into the pAAV vectors.
Add NotI restriction enzyme sites to both ends of the DNA donor template.
Order the dsDNA template from IDT.
Generation of spacer-nick RNPs
Note: We calculated the amounts of sgRNAs and Cas9D10A nickase for a reaction of 20 μL electroporation. For a reaction of 100 μL electroporation, scale up proportionally.
Order crRNAs, tracrRNA, and Cas9D10A nickase from IDT.
Generate sgRNA complex by mixing 20 μL of each crRNA (100 pmol) with 20 μL of tracrRNA (100 pmol) at a molarity ratio of 1:1 in a PCR tube.
Incubate the mixture at 95 °C for 5 min and ramp down at a rate of 1 °C/s to room temperature in the thermocycler.
Store sgRNA complexes at -20 °C up to six months or -80 °C up to one year.
Generate RNP complexes by mixing 2 μL of sgRNA (100 pmol) with 0.75 μL of Cas9D10A nickase (~50 pmol) at a molarity ratio of 2:1 in a PCR tube and incubate the mixture at 25 °C for 10 min in the thermocycler.
Note: Assembled RNPs should be freshly made before use.
Production and purification of AAV6 donor vectors
Note: We recommend using high-fidelity KOD hot-start DNA polymerase for PCR amplification of the DNA donor template. However, you can use any high-fidelity DNA polymerases that are available in your laboratory. Production and purification of AAV6 donor vectors are described in detail in our previous protocol (Tran et al., 2020).
Amplify the NotI-containing dsDNA donor template (step B4) with specific forward and reverse primers (carrying six random nucleotides, NotI site, and target-specific sequence) using any high-fidelity DNA polymerases.
Purify PCR products using NucleoSpin Gel and PCR Cleanup kit.
Digest PCR products with NotI restriction enzyme and load on 0.8% agarose gel.
Cut digested dsDNA band and purify it using any gel extraction kit.
Clone the purified dsDNA template into NotI-linearized pAAV vectors using T4 DNA ligase.
Transform the cloned pAAV vectors into competent TOP10 bacteria using heat-shock method.
Spread transformed bacteria on 10 cm LB agar dishes containing 50 μg/mL of carbenicillin and culture these dishes in a bacterial incubator at 30 °C overnight.
Pick bacterial colonies and inoculate these with 2 mL of liquid LB medium containing 50 μg/mL of carbenicillin at 30 °C overnight in a bacterial incubator with shaker.
Extract plasmids using any plasmid purification kit and confirm the insert by Sanger sequencing.
Produce large quantities of pAAV donor vectors using NucleoBond Xtra Maxi kit.
Transfect, purify, concentrate, and calculate copy number of AAV6 donor particles following our previous protocol (Tran et al., 2020).
Culture human HSPCs
Note: In order to achieve sufficient expansion of human CD34+ cells, we recommend that you culture ~4 × 105 HSPCs per 2 mL in a well of a 6-well plate and monitor the culture every day until these cells are used for electroporation. In case expanded cells are at high density, you should split them into new wells of a 6-well plate.
Isolate human CD34+ HSPCs from mobilized peripheral blood or bone marrow of healthy donors or patients using Ficoll-Paque Plus and follow human CD34+ microbead kit according to the manufacturer’s protocol.
Freeze isolated CD34+ HSPCs in serum-free freezing medium (BAMBANKER) at a density of 1 × 106 per milliliter and store in liquid nitrogen for long-term storage.
Thaw frozen vials and culture CD34+ HSPCs at a density of 4 × 105 per 2 mL of completed serum-free StemSpanTM SFEM II medium in a well of a 6-well plate.
Exchange half of the old medium with new medium 24 h post culture.
Seventy-two hours post culture, harvest expanded HSPCs and count cell number using TC20 cell counting system.
Transfer 2 × 105 HSPCs to a 1.5 mL Eppendorf tube and spin down at 300 × g for 5 min.
Remove supernatant, wash the cell pellet two times with room temperature PBS, and proceed to Section F or Section G.
RNP and dsODN electroporation for Tn5-mediated GUIDE-seq
Note: Prepare 1 mL of pre-warmed medium by adding 1 mL of completed serum-free StemSpanTM SFEM II medium to a well of a 12-well plate and place it into an incubator at 37 °C prior to electroporation. For long-term storage, annealed dsODN should be stored at -80 °C.
Order forward and reverse oligos of dsODN as previously described (Tsai et al., 2015) from IDT.
Anneal two oligos of dsODN by mixing 50 μL of each oligo (100 pmol), incubate at 95 °C for 5 min, and ramp down at a rate of 1 °C/s to room temperature in the thermocycler.
After the last wash with PBS in step E7, remove supernatant and resuspend the cell pellet with 20 μL of P3 Primary Cell electroporation buffer by pipetting up and down 10 times.
Add 2.75 μL of 5′ and 3′ assembled RNPs (step C5) and 0.5 μL (25 pmol) of the annealed dsODN to cell suspension and mix well by pipetting up and down five times.
Transfer the mixture to a well of a 16-well nucleocuvette strip.
Electroporate the cells using the DZ-100 program of Lonza 4D-Nucleofector.
Transfer the electroporated cells to pre-warmed serum-free StemSpanTM SFEM II medium.
Place the cell plate into an incubator at 37 °C and 5% CO2.
Change medium every 2–3 days.
At day 10 post electroporation, harvest the edited HSPCs by pipetting up and down five times, transfer cell suspension to a 15 mL Falcon tube, and spin down at 300 × g for 5 min.
Wash the cell pellet three times with room temperature PBS.
Deplete the dead cells by using dead cell removal kit and follow manufacturer’s specifications.
Isolate gDNA using GenFind V3 reagent kit according to the manufacturer’s protocol.
Measure concentration of gDNA using NanoDrop and dilute gDNA to 20 ng/μL.
Check quality of gDNA by loading 500 ng of gDNA on 0.8% agarose gel (Figure 3) and proceed to Section H.
Note: In order to achieve good results of Tn5-mediated DNA tagmentation, we recommend using GenFind V3 kit for gDNA isolation. This kit yields high quality of gDNA, and DNA fragment size is >20 kb (Figure 3). However, you can also use other gDNA isolation kits with equal quality that are available in your laboratory.
Figure 3. Quality control of the genomic DNA. 500 ng of gDNA of samples #1 and #2 are loaded on 0.8% agarose gel. GeneRuler 1 kb Plus DNA ladder is used as a marker. Optimal length of gDNA fragments is >20 kb.
RNP electroporation and AAV6 infection for LAM-HTGTS
Note: We normally infect the electroporated HSPCs with rAAV6 donor vectors at a MOI of 1 × 105 genome copy/cell. Higher MOI leads to cell death; we recommend carefully titrating your rAAV donor particles.
At the last wash with PBS in step E7, remove supernatant and resuspend 2 × 105 HSPCs with 20 μL of P3 Primary Cell electroporation buffer by pipetting up and down 10 times.
Add 2.75 μL of 5′ and 3′ spacer-nick RNPs (step C5) to cell suspension and mix well by pipetting up and down five times.
Transfer the mixture to a well of a 16-well nucleocuvette strip.
Electroporate the cells using the DZ-100 program of Lonza 4D-Nucleofector.
Transfer the electroporated cells to pre-warmed completed serum-free StemSpanTM SFEM II medium.
Place the plate into an incubator at 37 °C and 5% CO2.
Fifteen to thirty minutes later, add rAAV6 donor particles to the well containing the electroporated cells at a MOI of 1 × 105 genome copy/cell.
Change half the medium with new completed serum-free StemSpanTM SFEM II medium 6–8 h after AAV infection.
Change medium every 2–3 days.
At day 18 post infection, harvest the cells by pipetting up and down five times, transfer cell suspension to a 15 mL Falcon tube, and spin down at 300 × g for 5 min.
Wash the cell pellets three times with room temperature PBS.
Deplete dead cells by using dead cell removal kit following manufacturer’s specifications.
Isolate gDNA using Wizard Genomic DNA purification kit (see the note at step F15) and proceed to Section I.
Tn5 transposase-mediated GUIDE-seq
Note: Accurate concentration of gDNA is essential for reproducible Tn5-mediated tagmentation. We describe a Tn5-tagmented DNA protocol for exactly 100 ng of gDNA. If you want to conduct the protocol for more or less than 100 ng of gDNA, you should optimize these conditions. The Tn5-mediated DNA tagmentation should produce DNA fragments in the range of 300–1,500 bp. If DNA fragments are smaller or larger than this, you should reduce or increase the incubation time for the tagmentation, respectively. Importantly, you must carry out the Tn5-mediated DNA tagmentation at room temperature; do not use vortex for mixing. Primers used in this protocol are the same as described in Tran et al. (2022).
Add the following reagents stepwise to a well of an 8-well PCR strip as in Table 2:
Table 2. Tn5-mediated DNA tagmentation
Steps Reagents Volume (µL)
1 Nuclease-free water 3
2 gDNA (20 ng/µL) (Section F) 5
3 2× TD buffer 10
4 TDE1 (Tn5) enzyme 2
Total
20
Mix carefully by pipetting up and down 10 times, avoiding bubbles.
Close the PCR strip and centrifuge briefly at 280 × g for 30 s.
Incubate the PCR strip on the thermocycler at 55 °C for 7 min.
Transfer the PCR strip on ice.
Clean up Tn5-tagmented DNA fragments using Zymo DNA clean and concentrator-5 according to the manufacturer’s protocol.
Elute tagmented DNA fragments with 15 µL of nuclease-free water.
Check quality of tagmented DNA fragments by loading 3 µL of the elute on 1.2% agarose gel (see Figure 4) and proceed to step H9.
Figure 4. Verification of tagmented DNA fragments. Agarose gel analysis showing fragmentation of gDNA treated with (+) or without (-) Tn5 transposase. The Tn5-mediated tagmentation should produce DNA fragments in the range of ~0.3–1.5 kb (marked by white dash lines).
Prepare first PCR in a well of a 96-well plate according to Table 3.
Table 3. Amplifying dsODN-tagged amplicons
Reagents Volume (µL)
10× buffer (MgCl2 free) 3
MgCl2, 50 mM 1.2
dNTP mix, 10 mM 0.6
Platinum Taq polymerase (5 U/µL) 0.3
TMAC (0.5 M) 1.5
I5-Nextera-3′-GSP (-) or 5′-GSP (+)-forward (10 μM) 1.5
I7-Nextera-reverse (10 μM) 0.75
Tagmented DNA (step H7) 10
Nuclease-free water 11.15
Total 30
Spin the plate at 280 × g for 1 min.
Run the plate on the thermocycler with the touchdown program at the following conditions:
95 °C for 5 min.
15 cycles of [95 °C for 30 s, 70 °C (-1 °C/cycle) for 2 min, 72 °C for 30 s].
10 cycles of (95 °C for 30 s, 55 °C for 1 min, 72 °C for 30 s).
72 °C for 5 min.
4 °C hold.
Purify and clean up PCR products using AMPure XP beads (1.2×; 1.2 μL beads per 1 μL of sample) as follows:
Add 36 μL of AMPure XP beads to a PCR reaction.
Mix well by pipetting up and down 15 times using a 200 μL multichannel pipette.
Incubate the PCR plate at room temperature for 5 min.
Place the PCR plate on a 96-well magnetic stand for 5 min or until the solution is clear.
Remove the supernatant using the multichannel pipette.
Take the PCR plate out of the magnetic stand, add 200 μL of freshly made 70% ethanol to the beads, and mix by pipetting up and down.
Put the plate on the magnetic stand for 2 min and remove the supernatants.
Repeat steps f–g one more time.
Let the beads air dry for 3–5 min.
Take the plate out of the magnetic stand, add 18 μL of nuclease-free water, mix the beads, and incubate at room temperature for 2 min.
Put the plate on the magnetic stand for 5 min.
Transfer 15 μL of eluted DNA to a new well of a 96-well PCR plate and proceed to step H12.
Prepare second index PCR in a well of a 96-well plate according to Table 4.
Table 4. Indexing PCR fragments
Reagents Volume (µL)
Nuclease-free water 2.4
10× buffer for Platinum Taq Mg2+ free 3
50 mM Mg2+ 1.2
Platinum Taq (5 U/μL) 0.3
dNTP 10 mM 0.6
TMAC (0.5 M) 1.5
Nextera-I5 barcode-primer (10 μM) 3
Nextera-I7 barcode-primer (10 μM) 3
1st PCR product 15
Total 30
Run the plate on the thermocycler with touchdown program at the following conditions:
95 °C for 5 min.
15 cycles of [95 °C for 30 s, 70 °C (-1 °C/cycle) for 2 min, 72 °C for 30 s].
10 cycles of (95 °C for 30 s, 55 °C for 1 min, 72 °C for 30 s).
72 °C for 5 min.
4 °C hold.
Purify and clean up PCR products using AMPure XP beads (0.7×, see above).
Elute PCR products with 30 µL of 1× TE buffer (pH 8.0).
Verify size of PCR products by loading 3 µL of the eluate on a 1.5% agarose gel (Figure 5).
Figure 5. Verification of 5′ and 3′ enriched PCR products. Agarose gel analysis showing 5′ (left) and 3′ (right) enriched PCR products. Size range of PCR products is approximately 0.3–1.5 kb.
Measure DNA concentration using Qubit dsDNA HS high sensitivity kit following manufacturer’s specifications.
Calculate molar amount of each DNA library based on the size and concentration.
Pool equimolar amount of each DNA library into a 2 mL Eppendorf tube.
Clean and concentrate the pooled DNA library using AMPure XP beads (0.9×, see above).
Elute the pooled DNA library with 40 μL of nuclease-free water.
Save 2 μL of the elute for final quality control.
Mix 35 μL of the pooled DNA library with 7 μL of Gel Loading Dye, Orange (6×).
Load the mixture on two lanes (21 μL per lane) of 1.2% agarose gel.
Load 4 μL of 100 bp DNA ladder onto left and right lanes flanking the sample lanes.
Run the gel at 120 V for 30 min.
Under a standard UV transilluminator, cut out bands in a range of 350–1,000 bp (Figure 6).
Figure 6. Before and after size selection of the final library. Agarose gel images showing the DNA library before and after size selection. Size range (0.35–1.0 kb) of DNA fragments is excised and the DNA is extracted from the gel.
Extract DNA using QIAquick Gel Extraction kit following the manufacturer’s specifications.
Elute DNA with 100 μL of nuclease-free water.
Pool two lanes into a 2 mL Eppendorf tube.
Clean up and concentrate the final DNA library with AMPure XP beads (0.9×).
Elute DNA with 30 μL of 1× TE buffer (pH 8.0).
Measure DNA concentration of the final DNA library using Qubit dsDNA high sensitivity kit following manufacturer’s specifications.
Verify size range of the eluted DNA (step H22) and the final DNA library (step H32) using the D1000 ScreenTape kit (Figure 7).
Figure 7. TapeStation analysis for the final DNA library. The TapeStation analysis verifying length of the final DNA library. Size range of the DNA library is approximately 300–1,000 bp with a peak of 541 bp.
Calculate molar amount of the final DNA library.
Dilute the final DNA library to 4 nM with 1× TE buffer (pH 8.0).
Proceed to Illumina deep sequencing.
LAM-HTGTS
Note: Contamination of low molecular weight DNA fragments (<1 kb) will inhibit the LAM-HTGTS; therefore, it is crucial to remove any <1 kb fragments by cleaning gDNA up with ProNex size-selective purification beads. It is imperative to optimize the linear amplification PCR for it not to amplify any double-stranded products that will block the generation of the linear amplicons and the adapter ligation.
Ensure the high quality of gDNA by loading 50–100 ng on 0.8% agarose gel. If any <1 kb fragments are detected (Figure 8), purify the gDNA with 1.0× ProNex size-selective purification beads.
Figure 8. Quality control of the genomic DNA. Agarose gel image showing quality of gDNA before (UP) and after (PUR) ProNex bead purification. gDNA (UP: A and B) contains <1 kb fragments that are successfully removed by 1.0× ProNex beads purification (Pur: A and B).
Prepare linear amplification PCR using PrimeSTAR GXL DNA polymerase as in Table 5.
Table 5. Linear amplification PCR
Reagents Volume (µL)
Nuclease-free water 28.5
5× GXL buffer 10
dNTPs 2.5 mM each 4
Biotin-conjugated gene specific primer (1 µM) 2
Genomic DNA (30 ng/µL) (step G13) 5
GXL polymerase 0.5
Total 50
Mix the reaction by vortexing and spin down at 280 × g for 1 min.
Perform the amplification on the thermocycler using the following program:
98 °C for 5 min.
80 cycles of (98 °C for 30 s, 60 °C for 30 s, 68 °C 90 s).
68 °C for 2 min.
4 °C hold.
Note: Important for the first time, optional for the established reactions: check 5 μL of the reaction on 0.8% agarose gel to ensure there was no exponential off-target amplification.
Proceed with biotinylated PCR product capture through the following steps:
Transfer 2 μL of DynabeadsTM MyONETM Streptavidin C1 per each PCR reaction into a 1.5 mL Eppendorf tube and add 150 μL of B&W buffer to the tube.
Put the tube on the magnetic stand (6-well magnetic rack) for 5 min.
Remove the supernatant and add 150 μL of B&W buffer to the tube.
Take the tube out of the magnetic stand and resuspend the beads by pipetting up and down.
Put the beads on the magnetic stand for 5 min and remove the supernatant.
Resuspend the beads in 2 μL of nuclease-free water per each PCR reaction and mix the reactions as shown in Table 6.
Table 6. Capturing biotinylated PCR products
Reagents Volume (µL)
LAM-PCR product (step I4) 50
5 M NaCl 14
0.5 M EDTA 0.7
Washed StrepBeads 2
Nuclease-free water 3.3
Total 70
Incubate the reaction on the tube roller for 2–4 h.
Note: Four hours is recommended, but the reaction can be rolled overnight.
Capture the DNA–bead complexes on the magnetic stand.
Remove the supernatant and wash the beads with 150 μL of B&W buffer three times.
Wash the beads with 150 μL of nuclease-free water.
Resuspend the beads in 9 μL of nuclease-free water.
Proceed with on-bead ligation steps as follows:
Prepare and mix the reagents as in Table 7.
Table 7. On-bead ligation
Reagents Volume (µL)
DNA–Bead complexes (step I5k) 4.5
10× T4 DNA ligase buffer (NEB) 1
BridgeAdapter 50 mM (see Recipes) 0.5
T4 DNA ligase (NEB) 0.5
HexCo 20 mM 0.5
50% PEG8000 (see Recipes) 3
Total 10
Note: Add the DNA–bead complexes first to the reaction, before adding 50% PEG8000. Mix the reaction thoroughly by pipetting and vortexing.
Incubate the reactions in the thermocycler with the following program: 22 °C for 1 h, 16 °C for 1 h, 14 °C for 1 h, 10 °C for 1 h, and store at 4 °C.
Capture the DNA–bead complexes on the magnetic stand.
Remove the supernatant and wash the beads with 150 μL of B&W buffer three times.
Wash the beads with 150 μL of nuclease-free water.
Resuspend the beads in 20 μL of nuclease-free water.
Prepare and mix reagents for on-bead adapter PCR as in Table 8:
Table 8. Adapter PCR
Reagents Per reaction (µL)
Nuclease-free water 13.75
5× Q5 buffer 5
dNTPs 2
U1-C-Gene specific nested forward (10 μM) 1
U2-C-AdPrim (10 μM) 1
DNA–Bead complexes 2
Q5 polymerase 0.25
Total 25
Perform on-bead adapter PCR at the following conditions:
98 °C for 5 min.
30 cycles of (98 °C for 30 s, 60–65 °C 30 s, 72 °C 90 s).
72 °C for 2 min.
4 °C hold.
Load 5 μL of the reaction on 2% agarose gel along with the 100 bp ladder. The peak of the length distribution should be in the range of 300–500 bp (Figure 9).
Figure 9. Verification of on-bead adapter PCR products. Agarose gel analysis showing size range of on-bead adapter PCR products. The length distribution of on-bead adapter PCR products (A and B) should be in the range of 300–500 bp.
Purify PCR products with ProNex beads (1.2×) and proceed to Illumina library preparation.
Tag different samples with Illumina barcode sequences by mixing the reagents as in Table 9:
Table 9. Indexing PCR
Reagents Per reaction (µL)
Nuclease-free water 10.75
5× Q5 buffer 5
dNTPs 2
FC1-i5-U1 (10 μM) 1
FC2-i7-U2 (10 μM) 1
Purified PCR product from step I10 5
Q5 polymerase 0.25
Total 25
Perform indexing PCR at the following conditions:
98 °C for 5 min.
5–10 cycles of (98 °C for 30 s, 55 °C 30 s, 72 °C 90 s).
72 °C for 2 min.
4 °C hold.
Load 5 μL of the reaction on 2% agarose gel along with the 100 bp ladder. The peak of the length distribution should be around 300–500 bp (Figure 10).
Figure 10. Verification of indexed PCR products. Agarose gel analysis showing the length distribution of indexed PCR amplicons (samples: A to D) with a smear peaking at 200–1,000 bp. A negative control (NC) is used as a loading control.
Quantify the concentration of the products 500–700 bp long using densitometry in GelAnalyzer software.
Pool the samples so that the mass of the product per mass of gDNA used as an input (or number of the cells) is equal, to ensure a homogenous variant coverage.
Load 50–100 μL of the reaction on 0.8% agarose gel along with the 100 bp ladder and cut the 500–1,000 bp smear from the gel.
Extract the product from the agarose using QIAquick gel extraction kit. Measure the concentration on Nanodrop.
Ensure the proper size selection by loading the library diluted to 10 ng/μL on High Sensitivity D1000 ScreenTape or High Sensitivity dsDNA chip of BioAnalyzer (Figure 11).
Figure 11. BioAnalyzer track for the final library. The size distribution and molarity of the final DNA library are verified by the high sensitivity BioAnalyzer dsDNA chip. The length of the DNA library is in the range of 600–1,200 bp.
Calculate the molarity of the sample using ScreenTape or BioAnalyzer software and proceed with the library loading as described in the corresponding Illumina kit manufacturer’s manual.
Data analysis
For GUIDE-seq experiments, three independent experiments with two replicates each were performed. For LAM-HTGTS experiments, 3–6 independent experiments with two replicates each were performed. The pipelines for analyzing the GUIDE-seq and LAM-HTGTS were based on previous publications (Giannoukos et al., 2018; Danner et al., 2021). Detailed analysis of GUIDE-seq and LAM-HTGTS has been described in the Methods section and the supplementary figure S9 of the original paper (Tran et al., 2022). The Jupiter notebooks, conda environment, and scripts are available on https://github.com/ericdanner/SpacerNick. Briefly, GUIDE-seq reads were checked for correct priming and the sequence of the dsODN was trimmed to adjacent genomic sequences that are globally mapped to human genome (hg38) using Bowtie2. Mapped reads that were aligned to regions within 5,000 bps of the off-predicted target sites were quantified. For LAM-HTGTS analysis, reads were checked for correct priming and then aligned to the AAV ITR sequence to quantify AAV integrations. The unaligned reads were end-to-end mapped to in silico–generated outcomes. Remaining unmapped reads were then trimmed to the Cas9 target sites and globally aligned to human genome (hg38). This analysis allows for quantification of AAV integrations, wild type (non-targeted), indel and deletion, inversion, HDR, and translocation events.
Recipes
P3 electroporation buffer
Freshly prepare 20 μL of P3 Primary Cell electroporation buffer by adding 3.6 μL of supplement to 16.4 μL of P3 NucleofectorTM solution (Lonza).
Mix the buffer by vortexing and briefly spin down at 280 × g for 1 min.
Store at room temperature until use.
Serum-free StemSpanTM SFEM II medium
Serum-free StemSpanTM SFEM II medium (Stemcell) supplemented with human SCF (100 ng/mL), human TPO (100 ng/mL), human FLT3L (100 ng/mL), human IL-6 (100 ng/mL), 35 nM UM171, and 0.75 mM SR1.
Store at 4 °C for one week.
70% ethanol
Freshly mix 30% (v/v) with 70% (v/v) ethanol.
50% (wt/vol) PEG 8000
Dissolve 1 g of PEG 8000 in 2 mL of H2O and mix the solution with a thermomixer at 56 °C.
Filter the solution through a 0.22 μm filter, prepare aliquots, and store them at -80 °C.
5 M NaCl
Dissolve 292.2 g of NaCl in 800 mL of H2O and adjust the total volume to 1 L.
Autoclave and store the solution at room temperature.
2.5 N NaOH
Dissolve 10 g of NaOH in 100 mL of H2O.
0.5 M EDTA
Dissolve 186.1 g of EDTA·2H2O in 800 mL of H2O.
Stir vigorously on a magnetic stirrer.
Adjust the pH to 8.0 using 2.5 N NaOH and fill up to 1 L.
Autoclave and store the solution at room temperature.
1 M Tris-HCl (pH 7.4)
Dissolve 121.14 g of Tris in 800 mL of H2O.
Stir vigorously on a magnetic stirrer.
Adjust the pH to 7.4 using HCl and fill up H2O to 1 L.
Autoclave and store the solution at room temperature.
1 M Tris-HCl (pH 8.0)
Dissolve 121.14 g of Tris base in 800 mL of H2O.
Stir vigorously on a magnetic stirrer.
Adjust the pH to 8.0 using HCl and fill up H2O to 1 L.
Autoclave and store the solution at room temperature.
1× TE buffer (pH 8.0)
10 mM Tris-HCl (pH 8.0)
1 mM EDTA (pH 8.0)
Store the solution at room temperature
B&W buffer
1 M NaCl
5 mM Tris-HCl (pH 7.4)
1 mM EDTA (pH 8.0)
Filter and store the solution at room temperature
20 mM hexaamminecobalt(III) chloride (HexCo)
Dissolve 5.3 g of HexCo in 1 L of H2O to prepare a 20 mM working solution. Store the solution at room temperature.
50 mM bridge adapter
Anneal two oligonucleotides [sequences are identical to Hu et al. (2016)] by heating the 400 mM (total) mixture in 25 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.5 mM EDTA at 98 °C and ramp down at a rate of 1 °C/min to room temperature using the thermocycler. Dilute the mixture to 50 mM in nuclease-free water, aliquot, and store at -20 °C.
Acknowledgments
We thank H.P. Rahn, J. Pempe, C. Salomon, and C. Kocks (Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany) for excellent technical support. This protocol is derived from the original research paper (Tran et al., 2022; DOI: 10.1126/sciadv.abm9106).
Funding: This protocol was supported by the Berlin Institute of Health (BIH67000007, to V.T.C. and R.K.), the DFG (CH 1968/1-1, to V.T.C. and K.R.) and by the Helmholtz-Gemeinschaft, Zukunftsthema "Immunology and Inflammation" (ZT-0027, to K.R.).
Competing interests
K.R., V.T.C., N.T.T. and R.K. are inventors on a patent application related to this protocol filed by the Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC) (European Patent Application Nr. 21192657.1-1111, filed 23 August 2021). All other authors declare no competing interests.
Ethics
Human G-CSF-mobilized apheresis was derived from the Stem Cell Core Facility at Charite′ Hospital. The protocol has been reviewed and approved by the Institutional Review Board at Charite′ Hospital. Informed consent was obtained from all donors.
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Miyaoka, Y., Berman, J. R., Cooper, S. B., Mayerl, S. J., Chan, A. H., Zhang, B., Karlin-Neumann, G. A. and Conklin, B. R. (2016). Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep 6: 23549.
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F. and Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156(5): 935-949.
Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A. and Liu, D. R. (2013). High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.Nat Biotechnol 31(9): 839-843.
Ran, F. A., Hsu, P. D., Lin, C. Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y. and Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell 154(6): 1380-1389.
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X. and Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science 351(6268): 84-88.
Tran, N. T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kuhn, R., Rajewsky, K. and Chu, V. T. (2022). Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells.Sci Adv 8(22): eabm9106.
Tran, N. T., Trombke, J., Rajewsky, K. and Chu, V. T. (2020). Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells. STAR Protoc 1(1): 100028.
Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33(2): 187-197.
Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., et al. (2018). A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.Nat Med 24(8): 1216-1224.
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Biological Engineering > Biomedical engineering
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4,662 | https://bio-protocol.org/en/bpdetail?id=4662&type=0 | # Bio-Protocol Content
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E15.5 Mouse Embryo Micro-CT Using a Bruker Skyscan 1172 Micro-CT
EA Elena Astanina
SP Sara Petrillo
TG Tullio Genova
FM Federico Mussano
FB Federico Bussolino
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4662 Views: 472
Reviewed by: Alberto RissoneEVANGELOS THEODOROUAbhilash Padavannil
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Original Research Article:
The authors used this protocol in Nature Communications Sep 2022
Abstract
X-ray computed microtomography (µCT) is a powerful tool to reveal the 3D structure of tissues and organs. Compared with the traditional sectioning, staining, and microscopy image acquisition, it allows a better understanding of the morphology and a precise morphometric analysis. Here, we describe a method for 3D visualization and morphometric analysis by µCT scanning of the embryonic heart of iodine-stained E15.5 mouse embryos.
Keywords: Imaging X-ray computed tomography Micro-CT 3D visualization Mouse embryo Heart development
Background
Transgenic mouse models have been extensively used to study the roles of specific genes in heart development. Historically, to analyze morphological defects on heart formation in gain-of-function and loss-of-function models, the formalin-fixed paraffin-embedded embryos were sectioned and analyzed section-by-section. X-ray computed microtomography (µCT) is a powerful tool for visualizing the 3D morphology of the heart and performing morphometric analysis. µCT uses X-rays to create cross-sections of a physical object that can be used to recreate 3D models, reaching very high resolution (<1 µm). Here, we describe a method to apply this technique to biological samples. Our purpose was to study the cardiovascular system in the developing mouse embryo. This method overcomes the limits of traditional histology, such as the difficulty to obtain comparable sections, especially when working with very small and complex samples.
For µCT analysis of embryos, it is mandatory to increase the contrast of tissues. To this end, different methods and contrast agents are described in the literature, with small differences in protocols. The main agents available to this purpose and tested by the authors are iodine solution or phosphotungstic acid (PTA) (Wong et al., 2012 and 2013; Hsu et al., 2016; Handschuh and Glösmann, 2022). Osmium tetroxide is also used as a contrast agent, but because of its high toxicity it has not been tested by the authors. Both approaches of staining were previously used to study cardiac defects (Degenhardt et al., 2010; Lesciotto et al., 2020; Jamet et al., 2022). In our experience, the use of 2%–5% PTA solution requires methanol (3%–10%) to properly penetrate tissues. The concentration of PTA and methanol can be adjusted depending on the size of sample; for example, for small mouse embryos (E12.5), 3% of methanol should be sufficient; for E15.5 embryos, 5% methanol; and for large mouse embryos (E18.5), 10% of methanol should be necessary. We successfully used this method for the staining of E13.5 mouse embryos (Petrillo et al., 2018). Although the PTA method offers excellent contrast, it may cause artifacts due to sample shrinkage in embryos older than E13.5. The maximum size of the object suitable for the µCT is 50 mm; however, increasing sample size will reduce the resolution.
On the contrary, iodine-based staining offers good balance in terms of sample preservation and soft tissue contrast, especially for larger samples. In our experiment, in E15.5 embryos we used a 20% Lugol’s solution and 0.5% Tween-20. To preserve mineral structures, pH can be adjusted to 7.3.
Materials and Reagents
Mouse mating and embryo dissection
Petri dishes (100 mm) (BD Biosciences, catalog number: 351029 or similar)
35 mm cell culture dishes (BD Biosciences, catalog number: 352096 or similar)
Sterile disposable tubes (50 mL) (BD Biosciences, catalog number: 352070 or similar)
15 mL tubes
Mice
Pure ethanol (Sigma, catalog number: 493546)
Phosphate-buffered saline (PBS), without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 10010)
4% paraformaldehyde (PFA) (Santa Cruz Biotechnology, catalog number: sc-281692)
70% ethanol (see Recipes)
Micro-CT sample preparation
Lugol’s solution (Sigma, catalog number: 32922)
Tween-20 (Sigma, catalog number: P1379)
Staining solution with contrast agent (see Recipes)
Equipment
Blunt forceps (FST Standard Pattern forceps or similar)
Fine forceps (Dumont #5 or similar) and scissors (FST, catalog number: 14060-09 or similar)
X-Ray computed microtomography Skyscan 1172 (Bruker)
This instrument is a desktop µCT equipped with a fully distortion-corrected 11 MP X-ray camera (a 12-bit cooled CCD camera coupled to scintillator). The X-ray source is 20–100 kV, 10 W, with a <5 µm spot size. 0.8 µm is the highest resolution (only for small samples) and 25 µm is the lower resolution. The maximum object size is 50 mm. Skyscan 1172 is equipped with three filter positions (None, 0.5 mm Al, or Al+Cu).
Software
DataViewer 1.5.4.6 (Bruker)
NRecon 1.4.4 (Bruker)
CTVox 3.3.0 (Bruker)
Procedure
Mice mating
Keep mice on a 12:12 h light/dark cycle with unrestricted access to food and water. Use animals aged 2–4 months for mating.
For mating, put a male and two females in a cage after 6 pm.
Before 9 am on the next day, check for the formation of vaginal plug: lift a female by her tail, slightly dilate the vaginal opening with a blunt forceps, and check for a white mass.
Consider the day of plug detection as day E0.5. House the mated females separately from the male. Repeat mating procedure with non-mated females the following days.
Embryo dissection
Embryo dissection is performed as described in Zeeb et al. (2012).
At day E15.5, euthanize the pregnant female.
Place the mouse on a supine position and spray 70% ethanol on the abdomen.
Make a V-formed incision on the skin and abdominal cavity and lift the tissue to expose the internal organs.
Identify the V-shaped uterus with the string of embryos inside, cut at the proximal (cervical) and distal (ovaries) ends, and remove the uterus to a tube with ice-cold PBS.
Dissect the embryos in a dish with PBS on ice. First, cut the uterus into single embryos-containing pieces. Place each embryo in a 35 mm dish with PBS and remove the uterine tissues and yolk sac. Keep yolk sac membranes for genotyping if required.
Embryo fixation
Place single embryos in 15 mL tubes with 5 mL of 4% PFA and incubate at 4 °C for 24 h.
After fixation, keep the embryos in PBS at 4 °C. If needed, the samples can be kept at 4 °C for several days.
Micro-CT sample preparation
Prepare fresh staining solution of 20% Lugol’s solution + 0.5% Tween-20 pH 7.3.
Keep samples in staining solution in constant gentle agitation.
Change solution daily for 10 days.
Micro-CT acquisition
Perform acquisition at 80 kV using a 0.5 mm Al filter at a resolution of 3 µm, 0.4° of rotation step, 360° scan, and 4× frame averaging.
Micro-CT image reconstruction
Obtain image reconstruction using NRecon software (Bruker), with proper misalignment compensation, no smoothing, 5 ring artifacts reduction, and 30% beam-hardening correction.
Data analysis
Open dataset of the single embryo in DataViewer software (Figure 1).
Figure 1. A screenshot of DataViewer software. An embryo is visualized in virtual sections in three planes.
Select the plane by moving along the x- and y-axes. In our case, we were interested in performing the morphometric analysis of the embryo heart, in particular to measure the width of the ventricular walls and interventricular septum and the area of the pericardial cavity. The same plane, the best at representing the features we were interested in, was selected in all analyzed embryos.
Measure the features of interest by right-clicking to obtain the length in μm.
To perform a qualitative investigation, the dataset could be visualized in 3D by using CTVox software (Figure 2 and Video 1).
Figure 2. A screenshot of CTVox software. A E15.5 embryo is visualized in 3D slicing.
Video 1. Embryo
Recipes
70% ethanol
Obtained by mixing 70 mL of pure ethanol with 30 mL of water.
Staining solution with contrast agent
Staining solution with contrast agent is obtained by making a 20% Lugol’s solution with 0.5% Tween-20 and adjusting pH to 7.3.
Acknowledgments
This protocol was adapted from the previously published study (Astanina et al., 2022). This work was supported by AIRC – Associazione Italiana Per la Ricerca sul Cancro (grants 22910), Regione Piemonte (grant A1907A, Deflect), Fondazione CRT, Ministero dell’Università e della Ricerca (PRIN 2017, grant 2017237P5X), FPRC 5xmille 2016 MIUR (Biofilm), and ERA-Net Transcan-2 (grant TRS-2018-00000689) to F.B. and Ministero dell’Università.
Competing interests
The authors declare no conflict of interests.
Ethics
All animal procedures were approved by the ethics committee of the University of Turin and by the Italian Ministry of Health (protocol approval no. 864/2015‐PR).
References
Astanina, E., Doronzo, G., Corà, D., Neri, F., Oliviero, S., Genova, T., Mussano, F., Middonti, E., Vallariello, E., Cencioni, C., et al. (2022). The TFEB-TGIF1 axis regulates EMT in mouse epicardial cells. Nat Commun 13(1): 5191.
Degenhardt, K., Wright, A. C., Horng, D., Padmanabhan, A. and Epstein, J. A. (2010). Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ Cardiovasc Imaging 3(3): 314-322.
Handschuh, S. and Glösmann, M. (2022). Mouse embryo phenotyping using X-ray microCT. Front Cell Dev Biol 10: 949184.
Hsu, C. W., Wong, L., Rasmussen, T. L., Kalaga, S., McElwee, M. L., Keith, L. C., Bohat, R., Seavitt, J. R., Beaudet, A. L. and Dickinson, M. E. (2016). Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages. Dev Biol 419(2): 229-236.
Jamet, S., Ha, S., Ho, T. H., Houghtaling, S., Timms, A., Yu, K., Paquette, A., Maga, A. M., Greene, N. D. E. and Beier, D. R. (2022). The arginine methyltransferase Carm1 is necessary for heart development. G3 (Bethesda) 12(8).
Lesciotto, K. M., Motch Perrine, S. M., Kawasaki, M., Stecko, T., Ryan, T. M., Kawasaki, K. and Richtsmeier, J. T. (2020). Phosphotungstic acid-enhanced microCT: Optimized protocols for embryonic and early postnatal mice. Dev Dyn 249(4): 573-585.
Petrillo, S., Chiabrando, D., Genova, T., Fiorito, V., Ingoglia, G., Vinchi, F., Mussano, F., Carossa, S., Silengo, L., Altruda, F., et al. (2018). Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis. Cell Death Differ 25(3): 573-588.
Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P. and Henkelman, R. M. (2012). A novel 3D mouse embryo atlas based on micro-CT. Development 139(17): 3248-3256.
Wong, M. D., Spring, S. and Henkelman, R. M. (2013). Structural stabilization of tissue for embryo phenotyping using micro-CT with iodine staining. PLoS One 8(12): e84321.
Zeeb, M., Axnick, J., Planas-Paz, L., Hartmann, T., Strilic, B. and Lammert, E. (2012). Pharmacological manipulation of blood and lymphatic vascularization in ex vivo-cultured mouse embryos. Nat Protoc 7(11):1970-1982.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Tissue analysis > Tissue imaging
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4,663 | https://bio-protocol.org/en/bpdetail?id=4663&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Protocol for 3D Bioprinting Mesenchymal Stem Cell–derived Neural Tissues Using a Fibrin-based Bioink
MP Milena Restan Perez *
NM Nadia Z. Masri *
JW Jonathan Walters-Shumka *
SK Sarah Kahale
SW Stephanie Michelle Willerth
(*contributed equally to this work)
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4663 Views: 1397
Reviewed by: Oneil Girish BhalalaXinlei Li Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Biomolecules Aug 2021
Abstract
Three-dimensional bioprinting utilizes additive manufacturing processes that combine cells and a bioink to create living tissue models that mimic tissues found in vivo. Stem cells can regenerate and differentiate into specialized cell types, making them valuable for research concerning degenerative diseases and their potential treatments. 3D bioprinting stem cell–derived tissues have an advantage over other cell types because they can be expanded in large quantities and then differentiated to multiple cell types. Using patient-derived stem cells also enables a personalized medicine approach to the study of disease progression. In particular, mesenchymal stem cells (MSC) are an attractive cell type for bioprinting because they are easier to obtain from patients in comparison to pluripotent stem cells, and their robust characteristics make them desirable for bioprinting. Currently, both MSC bioprinting protocols and cell culturing protocols exist separately, but there is a lack of literature that combines the culturing of the cells with the bioprinting process. This protocol aims to bridge that gap by describing the bioprinting process in detail, starting with how to culture cells pre-printing, to 3D bioprinting the cells, and finally to the culturing process post-printing. Here, we outline the process of culturing MSCs to produce cells for 3D bioprinting. We also describe the process of preparing Axolotl Biosciences TissuePrint - High Viscosity (HV) and Low Viscosity (LV) bioink, the incorporation of MSCs to the bioink, setting up the BIO X and the Aspect RX1 bioprinters, and necessary computer-aided design (CAD) files. We also detail the differentiation of 2D and 3D cell cultures of MSC to dopaminergic neurons, including media preparation. We have also included the protocols for viability, immunocytochemistry, electrophysiology, and performing a dopamine enzyme-linked immunosorbent assay (ELISA), along with the statistical analysis.
Graphical overview
Keywords: 3D bioprinting Stem cells Mesenchymal stem cells Tissue engineering Dopaminergic neurons Bioink
Background
Parkinson’s disease (PD) occurs due to the depletion of dopaminergic neurons (DNs) in the substantia nigra pars compacta, which causes patients to lose their motor skills (Dauer and Przedborski, 2003). Three-dimensional bioprinting has become a sought-after technique to obtain models of such diseases, as they are low-cost and reproducible when compared to animal models, and enable a personalized medicine approach to modeling PD. 3D bioprinting deposits a cell-laden bioink in a layer-by-layer fashion using a computer-aided design (CAD) model to determine the structure with the goal of mimicking the extracellular matrix and 3D organization of cells as found in vivo (Maan et al., 2022). 3D bioprinting allows for a more accurate physiological representation of human tissues than those of 2D cell culture models. Thus, the printed models can be used as a tool for in vitro drug screening and can improve the translation between in vitro and in vivo studies (Hamadjida et al., 2019).
Bioprinting cell-laden bioinks requires large numbers of cells that must be able to withstand the stresses inherent to the bioprinting process. Mesenchymal stem cells (MSCs) derived from adipose tissue are an attractive cell type for tissue engineering as they can be obtained from patients in large numbers, while withstanding the shear stress of 3D bioprinting due to their robust characteristics (Tasnim et al., 2018). A 2011 study by Trzaska & Rameshwar differentiated bone marrow MSCs to DNs using 2D cell culture in 12 days (Trzaska and Rameshwar, 2011). In this paper, we modified the Trzaska & Rameshwar protocol by changing the sonic hedgehog to its agonist (purmorphamine) and adding both LDN-193189 (LDN) and SB431542 (SB). Bioprinted MSC-derived DN models could aid in drug discovery and enhance the chance of finding new treatments for neurological disorders like PD. In addition, bioprinted patient-derived MSC models can be used to study disease progression and to determine the most suitable treatment plan specific to each patient. Accordingly, 3D bioprinted tissue models play an important role in neural tissue engineering, especially with patient-derived stem cells that enable personalized medicine.
Human MSCs have previously been bioprinted for cartilage tissue engineering (Gao et al., 2017; Govindharaj et al., 2022). However, these articles lack detailed methodology of the bioprinting process required for other research groups to easily replicate the protocol and results. Our group has published methods describing how to make a fibrin-based bioink and how to print on the Aspect Biosystems RX1 bioprinter (Abelseth et al., 2019). However, this protocol uses stem cell–derived neural aggregates rather than a single-cell suspension, and the printing and culture parameters change depending on the cell type. Other protocols have been published on how to print with the CELLINK BIO X bioprinter, but do not explain the culture criteria and differentiation conditions for any specific cell line (Chrenek et al., 2022). Our protocol integrates several protocols into one by bridging the gap in current literature—describing how to bioprint on two different types of bioprinters, the Aspect RX1 and CELLINK BioX, how to culture MSCs before bioprinting, how to perform the preliminary cell differentiation experiments prior to bioprinting, and how to maintain and differentiate the 3D models after bioprinting. The procedures for immunocytochemistry, viability, electrophysiology, and ELISA are described in detail in this methods paper. This protocol serves as a foundation on how to bioprint other cell types such as human-induced pluripotent stem cells or a primary cell line. Although this protocol utilizes a cell-laden bioink, a cell-free scaffold can also be created using only bioink and seeding cells after the bioprinting process. Finally, this protocol describes bioprinting with an extrusion-based bioprinter, but other bioprinters could potentially be used, such as inkjet-based printers. In this case, preliminary tests without cells are required to determine the optimal printing parameters. Overall, this protocol aims to provide a standardized method for both the bioprinting of MSCs and the analysis of the bioprinted 3D tissue models.
Materials and Reagents
Human adipose–derived mesenchymal stem cells (HMSC-AD) (ScienCell, catalog number: 7510); storage: liquid nitrogen
Phosphate-buffered saline (PBS) (Fisher Scientific, catalog number: 10010031); storage: room temperature (RT)
Fibronectin (ScienCell, catalog number: 8248); storage: -80 °C. Aliquot the reagent into working volumes prior to storing
Mesenchymal stem cell growth supplement and basal medium (complete kit) (ScienCell, catalog number: 7501); storage: -20 °C, protect from light
Trypan blue (0.4%) (Thermo Fisher, catalog number: 15250061); storage: RT
Trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher, catalog number: 25200056); storage: -20 °C. Aliquot the reagent into working volumes
Fetal bovine serum (FBS) (Thermo Fisher, catalog number: 12483020); storage: aliquot into working volumes and store at -20°C
Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher, catalog number: 14040117); storage: RT
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301); storage: RT, protect from light
Poly-L-ornithine (PLO) (Sigma-Aldrich, catalog number: P4957); storage: 4 °C
Dulbecco’s modified Eagle medium (DMEM), high glucose, no glutamine (Sigma-Aldrich, 11-960-044); storage: 2–8 °C, protect from light
Laminin (Sigma-Aldrich, catalog number: L2020); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
Neurobasal media (Thermo Fisher, catalog number: 21103049); storage: 4 °C, protect from light
B-27 supplement (50×), serum free (Thermo Fisher, catalog number: 17504001); storage: -20 °C, protect from light. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
GlutaMAXTM supplement (Thermo Fisher, catalog number: 35050061); storage: RT, protect from light
Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
Purmorphamine (Sigma-Aldrich, catalog number: SML0868); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
Fibroblast growth factor 8 (FGF8) (R&D Systems, catalog number: 423-F8-025); storage: -80 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
LDN-193189 (STEMCELL Technologies, catalog number: 72147); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
SB431542 (STEMCELL Technologies, catalog number: 72232); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
Brain-derived neurotrophic factor (BDNF) (PeproTech, catalog number: 450-02); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
TissuePrint-HV Kit (Axolotl Bioscience); storage: -20 °C. Use for BIO X Printer
TissuePrint-LV Kit (Axolotl Bioscience); storage: -20 °C. Use on Aspect RX1 printer
TissuePrint Crosslinker (Axolotl Bioscience); storage: -20 °C. Use for both BIO X and Aspect RX1 printer
LIVE/DEAD Viability/Cytotoxicity kit (Thermo Fisher, catalog number: L3224); storage: -20 °C, protect from light
Formalin solution, neutral buffered, 10% (Sigma-Aldrich, catalog number: HT501128); storage: RT
Triton X-100 (Sigma-Aldrich, 9036-19-5); storage: RT
Normal goat serum (Abcam, catalog number: ab7481); storage: -20 °C. Aliquot the reagent into working volumes. Avoid thaw/freeze cycles
Primary antibody: anti-tyrosine hydroxylase (TH) antibody - neuronal marker (Abcam, catalog number: ab1120); storage: -20 °C. Aliquot reagent into working volumes. Avoid multiple freeze/thaw cycles
Primary antibody: anti-beta-III tubulin (TUJ1) antibody - neuron specific (R&D Systems, catalog number: MAB1195); storage: -20 °C. Aliquot reagent into working volumes. Avoid thaw/freeze cycles
Secondary antibody: goat anti-mouse (Alexa Fluor® 488) (Thermo Fisher, catalog number: D1306); storage: 4 °C, protect from light
Secondary antibody: goat anti-rabbit (Alexa Fluor® 568) (Thermo Fisher, catalog number: A11011); storage: 4 °C, protect from light
DAPI (Thermo Fisher, catalog number: D1306); storage: -20 °C (stock solution) or 4 °C (diluted solution). Avoid multiple freeze/thaw cycles
FLIPR Membrane Potential Assay Kit Blue (Molecular Devices, catalog number: R8042); storage: component A: RT; component B: 4 °C
Potassium chloride (KCl) (Caledon, catalog number: 5920170); storage: RT
Dopamine-specific enzyme-linked immunoassay (ELISA) kit (Abnova, catalog number: KA3838); storage: 4 °C
Cryovials (STEMCELL Technologies, catalog number: 200-0555)
T-75 flask, tissue culture treated
50 mL and/or 15 mL conical, sterile
Various sizes of pipette tips (1,000 μL, 200 μL, 20 μL), sterile and filtered
Kimwipes (Fisher Scientific, catalog number: 66662)
Corning® CoolCell® containers (Cole Palmer, catalog number: RK-04392-00); storage: RT/-80 °C
12-well cell culture plate (Sigma-Aldrich, catalog number: M8687)
LOP printhead (Aspect Biosystems, catalog number: 1256)
Mesh, square, 50 × 50 mm, pack of 10 (Aspect Biosystems, catalog number: 2067)
RX1 tubing, 74 cm (Aspect Biosystems, catalog number: 7779)
RX1 tubing, 54 cm (Aspect Biosystems, catalog number: 7777)
Empty cartridges without end and tip caps, 3 mL (CELLINK, catalog number: CSC010300502)
Sterile standard conical bioprinting nozzles, 22 G (CELLINK, catalog number: NZ4220005001)
Female/female luer lock adapter (CELLINK, catalog number: OH000000010)
5 cc luer lock syringe w/o needle (Terumo Medical Corporation, catalog number: SS-05L)
Various sizes of serological pipettes (25, 10, 5, and 1 mL)
Liquid nitrogen dewar
Control Group Cell Culture media (see Recipes)
Experimental Cell Culture media (see Recipes)
Complete Mesenchymal Stem Cell (MSC) medium (see Recipes)
Diluting PLO in PBS (see Recipes)
Diluting laminin in DMEM (see Recipes)
Calcein AM and Ethidium Homodimer-1 solution (see Recipes)
0.1% Triton-X solution (see Recipes)
5% NGS solution (see Recipes)
Primary Antibody solution (see Recipes)
Secondary Antibody solution (see Recipes)
300 nM DAPI solution (see Recipes)
70% ethanol solution (see Recipes)
Potassium chloride solution (see Recipes)
10% DMSO Freezing Media solution (see Recipes)
Equipment
BIO X printer (CELLINK, S-10001-001)
RX1 bioprinter (Aspect Biosystems, 46135)
DMI3000B microscope (Leica)
X-Cite Series 120Q Fluorescent light source (Excelitas Technologies, XI120-Q)
FormaTM Steri-CycleTM CO2 incubator (Thermo Fisher, 370)
Infinite M200 Pro Plate Reader (TECAN, 30050303)
Confocal Laser scanning microscope (FIPS-Zeiss) 10× and 20× magnification
Retiga 2000R digital CCD camera (QImaging)
Biosafety cabinet (BSC)
Water bath (VWR, 89032-299)
Lab ArmorTM beads (Thermo Fisher, A1254301)
Centrifuge 5810 R (Eppendorf, 5811000015)
-80 °C freezer
Pipette controller
DeNovix CellDrop FL (DeNovix CellDrop FL)
Software
Qcapture software 2.9.12
Excel (Microsoft)
ImageJ V1.52a
Prism 5 (GraphPad) statistical software
Procedure
Culturing MSCs for cell expansion
Please note that this protocol has been slightly adjusted from the ScienCell protocol for the adipose-derived MSCs used in this experiment. If you obtain MSCs from a different source, it is advised that you use the specific protocol for your MSCs when expanding your cells.
Coating T75 flask with fibronectin
Working in the BSC, add 5 mL of PBS and 150 μL of fibronectin to a 15 mL conical to make up a 30 µg/mL fibronectin solution. Mix and add to the T75 flask.
Gently move the flask forward and back and then side to side, to evenly coat the bottom of the plate, ensuring there are no dry spots.
Spray the exterior of the flask with 70% ethanol and place in the incubator at 37 °C with 5% CO2 overnight.
Seeding cells
Prepare the complete MSC medium and let it come to RT (refer to Note 15). Refer to Recipe 3.
Remove the fibronectin-coated plate from the incubator, spray the plate with 70% ethanol, and place in the BSC.
Aspirate the fibronectin solution and add 15 mL of complete MSC medium to the flask. Place the flask to the side.
Remove a frozen cryovial of HMSC-AD from the liquid nitrogen and place it in a 37 °C water bath without submerging the cap. Hold the cap and swirl the vial gently.
When a small ice crystal remains, spray the cryovial with ethanol and place it in the BSC.
Carefully remove the cap of the thawed cells without touching the interior threads.
Transfer the cells from the cryovial to the fibronectin-coated flask using a 1 mL pipette, drop by drop. Refer to Note 5.
Replace the cap on the flask. Move the plate forward and back and then side to side, to evenly distribute the cells.
Look at the cells under the microscope to ensure even distribution. Refer to Note 1.
Place the flask in the incubator at 37 °C and 5% CO2.
Do not disturb the plate for at least 16 h.
Change the media the next day and every 48 h thereafter, until the culture is approximately 50% confluent.
After culture is 50% confluent, perform daily media changes.
When culture is approximately 90% confluent, you can passage into a new plate/flask or freeze the cells.
Changing media
Warm MSC media to RT. Refer to Note 15.
Retrieve T75 flask with MSCs from the incubator.
Check the confluency and morphology of the cells under the microscope.
Sterilize the plate with 70% ethanol and place in the BSC.
Remove the media from the flask using a 25 mL serological pipette by tilting the flask so that the media accumulates at one corner and place the serological pipette in that corner. Be careful not to scrape the bottom of the flask or touch the pipette against the mouth of the flask.
Add 15 mL of complete MSC media.
Check under the microscope to ensure that the cells are still attached to the plate surface.
Place the flask in the incubator at 37 °C and 5% CO2.
Passaging cells
Warm complete MSC media, trypsin-EDTA, FBS, and DPBS to RT.
Retrieve the T75 flask with MSCs from the incubator.
Check the confluency and morphology of the cells under the microscope to confirm the MSCs are around 90% confluent.
Sterilize the plate with 70% ethanol and place in the BSC.
Aspirate the media from the plate using a 10 mL serological pipette.
Gently wash cells with 5 mL of DPBS by adding DPBS and removing it shortly after.
Add 5 mL of 0.25% trypsin-EDTA to the flask and incubate for approximately 1–2 min, or until the cells have round up, at 37 °C. Check cells mid-way through incubation to see if they are rounding or coming off the plate.
Spray the flask with 70% ethanol, then place in the BSC. Transfer the trypsin solution from the flask to a 50 mL conical containing equal parts of FBS to trypsin. If 5 mL of trypsin was added to the cells, add 5 mL of FBS to the conical.
Place the flask back in the incubator for one more minute without any solution.
At the end of the incubation, gently tap the flask to detach the cells. Check under the microscope to ensure the cells have all detached.
Add 5 mL of FBS to the flask and transfer the contents to the 50 mL conical.
Examine the flask under the microscope to ensure that there is less than 5% of cells in the flask.
An additional wash of the flask can be performed using 5 mL of DPBS to collect any remaining cells.
Centrifuge at 123× g for 5 min at RT.
Meanwhile, add 15 mL of complete MSC media to a prepared fibronectin-coated flask. Refer to step A1.
After centrifugation, aspirate the supernatant from the 50 mL conical. Be careful not to disturb the cell pellet.
Resuspend the cell pellet in 1 mL of complete MSC media and pipette up and down to ensure a homogenous cell suspension.
Count the cells using trypan blue and a cell counter or a hemocytometer (refer to step A5 for freezing protocol if you are not seeding the cells).
Plate cells at a density of 6,000 cells/cm2. Equation 1 can be used to calculate the quantity of cell suspension needed per well (or flask). A sample calculation for a T75 flask can be seen below. Refer to Note 6.
Add the calculated quantity of cell suspension to the flask containing 15 mL of media. Add the cells to the middle of the flask using a 1 mL pipette and move it back and forth, then side to side, to evenly distribute the cells. Refer to Note 1.
Check the cells under the microscope to ensure even distribution.
Place the flask in the incubator at 37 °C and 5% CO2.
Do not disturb the flask for at least 16 h.
Change the media the next day and every 48 hours thereafter, until the culture is approximately 50% confluent.
After culture is 50% confluent, perform daily media changes.
When culture is approximately 90% confluent, you can passage into a new plate or freeze the cells down.
Freezing cells
From step A4r, after performing the cell count, add approximately 5–10 mL of complete MSC media to the remaining cell suspension.
Centrifuge at 123× g for 5 min at RT.
Meanwhile, label the desired number of cryovials with the cell type, passage number, number of cells, date, and your initials. The number of cells per cryovial should be 1 million per 1 mL of 10% DMSO Freezing Media solution.
In a new conical, mix a 10% solution of DMSO in complete MSC media. Refer to Recipe 14.
After centrifugation, aspirate supernatant. Be careful not to disturb the cell pellet.
Add 1 mL of 10% DMSO diluted in complete MSC media per 1 million cells to the conical containing the cell pellet. Gently pipette up and down to resuspend the cells. This step must be done quickly, as cells could be adversely affected in DMSO at RT.
Add 1 mL of resuspended cells to each cryovial.
Transfer the cryovials to a Corning® CoolCell® containers and place in the -80 °C freezer for 24 h.
After 24 h, place cryovials in the liquid nitrogen dewar.
Differentiating MSCs to dopaminergic neurons on 2D cell culture plate
Prior to performing any bioprinting experiments, it is important to perform 2D cell culture experiments to determine if the MSCs will successfully differentiate to dopaminergic neurons and to optimize the cell density for differentiation. Please note that this protocol has been slightly adjusted from Trzaska and Rameshwar (Trzaska and Rameshwar, 2011).
Coating a 12-well plate with poly-L-Ornithine/Laminin
Dilute PLO in PBS to reach a final concentration of 15 μg/mL. Refer to Recipe 4.
Add 0.5 mL of PLO/PBS per well into the 12-well cell culture plate.
Gently move the plate black and forth and side to side to evenly coat the bottom of the plate, ensuring there are no dry spots in the wells.
Wrap in parafilm and incubate at RT for 2 h. Check the plates every 30 min to ensure that it does not evaporate. Alternatively, incubate the plate overnight at 2–8 °C.
After 2 h or the following day, aspirate the PLO using a 10 mL serological pipette.
Rinse each well with 1 mL/well of PBS.
Rinse twice with 0.5–1 mL/well of ice-cold DMEM. Refer to Note 2.
In the middle of the rinses, dilute the laminin solution in DMEM to reach a final concentration of 10 μg/mL. Refer to Notes 3 and 4. Refer to Recipe 5.
Add 0.5 mL of the diluted laminin solution per well.
Leave for 2 h in the incubator at 37 °C and 5% CO2. If the plate is not being used right away, place in the fridge at 4 °C for up to one week.
When ready to use, aspirate the laminin and seed the cells.
Seeding cells for dopaminergic differentiation
When the MSCs have become 90% confluent, passage them by following the step A4.
After performing the cell count, seed the cells onto the plate. A cell titration experiment should be performed seeding the cells at various densities to determine the optimal seeding density for your cell line. Recommendation: 1,200–25,000 cells/cm2. Refer to Notes 1, 6, and 7.
Culturing from day -1 to day 12
On day -1 (the day cells are seeded), add 1 mL of complete MSC media to each well to allow cells to adhere. Perform this for both control and experimental groups. A control group is added so that it is clear that the differentiation protocol worked. Refer to Note 7.
Place in the incubator overnight at 37 °C and 5% CO2.
On day 0, remove the complete MSC media and add the respective media to the control group and experimental group. Refer to Recipes 1 and 2.
Place the dish back in the incubator for nine days without any media changes. The cells can be checked under the microscope to monitor their progress.
On day 9, add 50 ng/mL of BDNF directly to the experimental group without performing any media changes. Do not add the BDNF to the control group.
Place the dish back in the incubator and incubate for an additional three days.
After 12 days, the cells can be analyzed using immunocytochemistry, live/dead assays, electrophysiology, and ELISA. Refer to Figure 1 for schematic of cell culture period.
Figure 1. Schematic of cell culture period for (A) control group and (B) experimental group for both 2D and 3D cultures
Preparing the bioink
Preparing TissuePrint - Low Viscosity (LV) and High Viscosity (HV) Bioink for the Aspect Biosystem bioprinter
Thaw component 1 (alginate) and component 3 (fibrin) at 4 °C up to one day before use and ensure they are both homogeneous prior to using.
Prior to mixing the bioink, passage the MSCs in accordance with step A4.
Once MSCs have been counted, centrifuge the cells at 123× g for 5 min. Refer to Note 8.
When centrifugation is completed, without disturbing the cell pellet, place the conical containing the MSCs in a conical rack and place in the incubator while the bioink components are being mixed. Do not remove the supernatant yet.
Thaw component 2 (genipin) at RT immediately before incorporating into the rest of the bioink.
Working in a sterile BSC, add component 2 to component 1 at RT. If component 2 starts to clump, slowly pipette up and down using a wide bore pipette to disperse the clump.
Remove the conical containing the MSCs from the incubator, disinfect the conical with 70% ethanol, and place in the BSC.
Carefully remove the supernatant without disturbing the cell pellet.
Add 1 mL of component 3 (at RT) to the cells and resuspend by pipetting up and down very gently. Harsh pipetting may cause cell death. Add the rest of component 3 to the cells and pipette up and down gently. The cell density per milliliter of bioink is dependent on the specific cell line used. Cell density titration experiments will need to be done to discover which density is best for each cell line. Typically, the cell density used is from 2 to 10 million cells per milliliter of bioink. Refer to Note 8.
Slowly add component 3 and MSC mixture into the mixture of component 1 and component 2 using a 1 mL pipette tip. Slowly mix the complete bioink solution by gently pipetting up and down. Avoid generating air bubbles. Refer to Note 9.
Preparing Crosslinker
Thaw component A and component B at 4 °C.
Add component B to component A using a serological pipette and pipette up and down to mix.
CAD design
Choosing a design for the Aspect Biosystems RX1 bioprinter
Select “New STL” and input parameters for the structure.
Select “Save STL” and name your STL file.
Select “Infill Editor” and under Material 1, choose a rectilinear infill pattern, with 40% infill, 0 perimeters, and a fiber diameter of 0.1 mm.
Select “Build Design” to convert the STL file into a Design file and name your file.
Choosing a design for the CELLINK BIO X bioprinter
Select “Bioprint” on the BIO X touchscreen.
Select the “3D models” tab and choose the dome model that has dimensions of 10 × 3 mm.
Select the “Surface” tab and select the 12-well plate and its vendor.
Select the “Printer” tab and choose “Tool 1” and the tool type, which should be the 3 mL Pneumatic syringe.
Select the “Layers” tab; for the “Grid” select rectilinear, and for the “Infill density” choose between 20%–25%. These parameters might need to be adjusted during the printing process depending on your printer.
Bioprinting
Bioprinting using the Aspect Biosystems RX1 bioprinter
The day before print day, autoclave all the equipment in Figure 2. Ensure that you have the necessary equipment for bioprinting, including the LOP printhead, RX1 tubing, and mesh sheets.
Refer to Video 1: Bioprinting with the RX1 Aspect Biosystem Bioprinter for a detailed explanation of how to print with this system.
Once the constructs have been bioprinted, use a sterile spatula to pick up the construct off the print stage, and use another spatula to carefully slide the construct off the first spatula onto the plate. The plate should be a PLO/Laminin-coated 12-well plate containing the appropriate media (one construct per well). Refer to Note 10.
Figure 2. Materials to be autoclaved for 3D bioprinting with the aspect. A) Conical holders (2×); B) 125 mL Nalgene bottles (2×); C) 125 mL Nalgene bottle printing lid and tubing fastener (2×); D) 15 mL bioink conical printing lid and tubing fastener; E) 500 mL squirt bottle; F) Funnel; G) 200 mL beaker (2×); H) 50 mL beaker; I) 20 mL beaker; J) Nalgene container; K) Printing stage; L) Plastic tweezers; M) Metal tweezers; N) Spatulas (2×).
Video 1. Bioprinting with the RX1 Aspect Biosystem
Bioprinting using the BIO X by CELLINK
The day before print day, autoclave all the equipment in Figure 3.
Refer to Video 2: Bioprinting with the CELLINK RX1 Bioprinter for a detailed explanation of how to print with this system. Please note that the bioink has been colored with green food dye for visualization purposes.
Once the constructs have been bioprinted, add 1 mL of the crosslinking solution to the well. Let this sit for 2–3 min and, using a sterile spatula, carefully transfer the 3D bioprinted constructs to a PLO/Laminin-coated 12-well plate containing the appropriate media (one construct per well). Refer to Note 10.
Figure 3. Materials needed for 3D bioprinting with the BioX. A) 12-well cell culture plate; B) Spatula; C) 5 cc luer lock syringe w/o needle; D) 3 mL cartridge; E) 22G, bioprinting nozzles and stopper; F) Female/female luer lock adapter.
Video 2. Bioprinting with the BioX Bioprinter
Culturing MSCs post-3D bioprinting
Coating plate with poly-L-ornithine/Laminin
Refer to Notes 1, 2, 3, 4, and 11.
Refer to protocol section B1 for steps on how to coat a plate with PLO/Laminin.
Culturing 3D bioprinted constructs
On day -1 (the day cells are 3D bioprinted), add 1 mL of complete MSC media to each well to allow cells to adjust to their new environment.
Place in the incubator overnight at 37 °C and 5% CO2.
On day 0, remove the complete MSC media and add 2 mL (for 12-well plate) of the respective media to the control group and experimental group. Refer to recipes 1 and 2. Ensure that the constructs are fully covered by the media—if a section of the construct is sticking out, add more media as needed.
Place the plate back in the incubator for nine days without any media changes. The cells can be monitored under a microscope to check their progress.
On day 9, add 50 ng/mL of BDNF directly to the experimental group without performing any media changes. Do not add the BDNF to the control group.
Place the plate back in the incubator and incubate for an additional three days.
After 12 days, the cells can be analyzed. Refer to Figure 1 for schematic of cell culture period.
Cell viability
The viability assay was performed using the LIVE/DEADTM Viability/Cytotoxicity kit on day 9 (before adding BDNF) and on day 12.
Remove the cells from the incubator and check under the microscope to see their morphology.
Aspirate media from each cell culture well.
Make a solution with 0.05% Calcein AM (component A) and 0.2% ethidium homodimer-1 (component B) in DPBS. Refer to Recipe 6. Determine beforehand how much complete solution you need according to how many wells you need to study. Typically, per well, 1 mL of complete solution is added. If you have 12 wells, make up a solution with a total volume of 12 mL. Make sure that the 3D construct is completely covered by the solution.
Wash once with 1–2 mL of DPBS.
Add the Calcein/ethidium solution to the wells to fully submerge the construct for 3D or the cells for 2D.
Incubate the cells at 37 °C with 5% CO2 for 30 min for 3D or 15 min for 2D.
Image the constructs with the Leica DMI3000B microscope with an X-Cite Series 120Q fluorescent light source using the Qcapture software 2.9.12.
Quantify viability by imaging one spot on the construct and taking 15 images in equally spaced Z-planes. At each Z-plane, take one image with the 488 nm laser to visualize live cells and one image with the 543 nm laser to visualize dead cells. Ensure when saving these images that you note if it is a live or dead image.
Create a Z-projection using ImageJ V1.52a software.
Count the number of live and dead cells using the ImageJ software to determine the viability throughout the construct. The live and dead cells are fluorescently labeled green and red, respectively, with the LIVE/DEADTM Viability/Cytotoxicity kit. Refer to the Data analysis section part A.
Immunocytochemistry
Immunocytochemistry can be performed on both 3D and 2D cultures on day 12 to visualize the early neuronal marker TUJ1 and dopamine-specific marker tyrosine hydroxylase (TH). Refer to Note 14.
Aspirate media from cell culture plate wells.
Wash the cells twice with PBS.
Fix the cells with 10% formalin solution and incubate at RT for 20 min for 3D culture or 10 min for 2D culture.
Aspirate formalin.
Wash cells twice with PBS, with 2 min incubations in between each wash, at RT.
Permeabilize the cells with 0.1% Triton-X and incubate at RT for 45 min for 3D culture or 10 min for 2D culture. Refer to Recipe 7.
Aspirate Triton-X solution.
Block cells with 5% NGS in PBS and incubate for 1–2 h at RT. Refer to Recipe 8.
Add two primary antibodies: anti-TH at 1:500 and anti-TUJ1 at 1:100 dilution in 5% NGS in PBS. Refer to Recipe 9.
Incubate at 4 °C overnight with shaking at 100 rpm.
Wash three times with PBS after incubation.
Add the secondary antibodies (goat anti-mouse Alexa Fluor® 488 and goat anti-rabbit Alexa Fluor® 568) at a 1:200 dilution in 5% NGS in PBS. Refer to Recipe 10.
Incubate for 2 h at RT with shaking at 100 rpm.
Wash the cells twice with PBS.
Stain the cells with 300 nM of DAPI diluted in PBS. Refer to Recipe 11.
Incubate for 5 min at RT.
Wash cells twice with PBS.
Image the cells using the FIPS-Zeiss Confocal Laser Scanning Microscope.
Following this, count the number of DAPI-positive, TUJ1-positive, and TH-positive cells using the ImageJ V1.52a software. Refer to the Data analysis section part B.
Electrophysiology
Turn off the lights in the BSC (the voltage-sensitive dye is light-sensitive).
Aspirate 1 mL of media and leave behind 1 mL of media. For the 3D constructs, be careful not to aspirate part of the construct.
Add 1 mL of FLIPR Membrane Potential Assay Kit Blue to the cells and 3D constructs so that the ratio between media and the FLIPR assay dye is 1:1.
Place the plate in the incubator for 45 minutes at 37 °C with 5% CO2.
Read the plate using a TECAN Infinite M200 Pro Plate Reader set to read fluorescent emission at 560 nm and take 25 reads from each well in a 5 × 5 grid.
After the initial readings, remove 1.56 mL of the solution in the well and add KCl at a concentration of 56 mM in distilled water. Refer to Recipe 13.
Incubate the 3D constructs or 2D cell cultures for 30 min at 37 °C with 5% CO2.
After incubation, read the plates using the TECAN Infinite M200 Pro Plate Reader set to read fluorescent emission at 560 nm and take 25 reads from each well in a 5 × 5 grid.
Refer to Note 12 and the Data analysis section part C.
Dopamine enzyme-linked immunosorbent assay (ELISA)
Collect the culture media from 3D or 2D cultures for both control and experimental groups on day 12. Collect the media from three different constructs, so that you have three replicates.
Analyze the concentration of dopamine released using a dopamine-specific ELISA kit by following the manufacturer’s instructions. Refer to Note 13.
Read the output absorbance using the TECAN Infinite M200 Pro Plate Reader set to 450 nm with a 635 nm reference wavelength, or as described in your chosen dopamine ELISA kit.
Refer to Data analysis section part D.
Data analysis
Viability
Open the live and dead images on ImageJ and create a Z project at one spot of the construct. Ensure that cells are not hiding behind other cells when doing the Z projection. If this occurs, perform the steps below on each image without performing a Z stack.
Count the live and dead cells separately by following the steps below:
Process > Subtract background > 12 pixels > Ok > Image > Adjust > Threshold > B&W > Adjust slider so only the cells are dark and background is white > Apply > Process > Binary > Fill Holes > Process > Binary > Convert to Mask > Process > Binary > Watershed > Analyze > Analyze Particles > 120-infinity (adjust accordingly) > Show > Outlines > Exclude on Edges > Ok.
Calculate the cell viability at each section of the construct using Equation 2.
Perform these calculations for three different constructs.
Immunocytochemistry
Open the images on ImageJ and apply a different color for each antibody.
Create a Z stack and save these files.
Create a composite image for visualization purposes.
Count the number of DAPI-positive, TUJ1-positive, and TH-positive cells using ImageJ.
Perform a two-tailed Student’s t-test, with a confidence level of 95% (p < 0.05). Perform this to determine if there is a statistically significant difference between the number of cells expressing neural cell markers to the number of nucleated cells between the control and experimental group. Perform this test on Prism 5 (GraphPad) statistical software.
Electrophysiology
To calculate the membrane potential, use equation 3 below, where R is the gas constant (8.314 J/Kmol), T is the temperature in Kelvin, F is Faraday’s constant in As/mol, z is the apparent charge of the external dye (assumed to be -0.64 for this experiment, but can be determined experimentally), and ∆F = F – Fo is the readings from the microplate reader where F is the reading with the constructs and cells, and Fo is the background fluorescence reading of just the construct (Robinson et al., 2019). It is recommended that these calculations are performed on Excel.
Using Prism 5 (GraphPad) statistical software, perform a one-way ANOVA and Tukey post-hoc analysis using a confidence level of 95% (p < 0.05).
Dopamine ELISA
Obtain a standard curve by plotting the mean absorbance readings on the y-axis (linear) against the corresponding standard concentrations (logarithmic) on the x-axis.
Obtain a spline curve fit using Prism 5 statistical software and interpolate the unknown concentration values for each unpaired absorbance reading.
This assay is a competitive assay, so as absorbance values decrease, the concentration increases.
Notes
Always label plates with either your full name or initials, the date (Month/Day/Year), what the plate contains, and cell passage number.
PLO is toxic to cells, so the rinsing steps are critical.
Always ensure laminin is cold. Do not let laminin come to RT. Avoid freeze/thaw cycles—laminin should not be frozen more than twice.
Laminin should be aliquoted prior to use. The volume and concentration of laminin varies from batch to batch. Always refer to the batch number before aliquoting.
Dilution and centrifugation of the cells after thawing is not recommended, since these actions are more harmful to the cells than the effect of residual DMSO in the culture. Therefore, add the cells directly to the media in the new cell culture plate and change the media the next day to remove any DMSO once the cells are attached.
The number of live cells is what you will be using to do your calculations for the number of cells that will be added to the wells. Do not use the total number of cells.
Seed enough wells so that you can have three replicates for each cell density and control groups.
Ensure that you have enough MSCs for your printing purposes. The seeding density can vary from 2 to 10 million cells per milliliter of bioink, depending on your cell line. This may require multiple weeks of passaging MSCs so that you can accumulate enough cells.
For best results, use within one hour. It is also recommended that small batches of bioink are made (approximately 4 mL at a time), so that if bioprinting experiences any setbacks, materials and cells are not wasted.
It is important that you prepare the PLO/Laminin plate a day before bioprinting, so that plates are ready during the bioprinting process.
Perform this step a day or two before printing, so that plates are ready for print day.
We used this method as 3D tissues cannot be easily patch-clamped. This analysis is performed on both control and experimental groups. It is also performed on cell-free constructs (3D) or blank wells (2D), which are a control for background fluorescence.
Our lab used the Abnova dopamine ELISA kit (refer to Materials and Reagents section for catalog number) because it could detect low concentrations of dopamine.
A titration of the primary and secondary antibodies is recommended, to determine the most optimal concentration of each antibody for your cell line.
It is not recommended to place the media in a water bath, as this can cause some components to degrade.
Recipes
Control Group Cell Culture Media
Neurobasal Media (NBM)
2% B-27 supplement
1% GlutaMAXTM
1% Penicillin-streptomycin
Experimental Cell Culture Media
Control Group Cell Culture Media
250 ng/mL purmorphamine
100 ng/mL of FGF8
100 nM of LDN-193189
10 µM of SB431542
Complete Mesenchymal Stem Cell (MSC) Medium
Mix all components of the MSC media kit, which include:
500 mL of MSC basal medium
25 mL of fetal bovine serum
5 mL of mesenchymal stem cell growth supplement
5 mL of penicillin/streptomycin solution
Diluting PLO in PBS
Depending on the lot number of your PLO, obtain the certificate of analysis (COA) from the Sigma-Aldrich website and determine the concentration of the purchased bottle.
Calculate the amount of PLO needed for a final volume of 6 mL (enough for a 6- or 12-well plate) using equation 4, where C1 is the stock concentration of PLO.
Add (6 - V1) mL of PBS to a sterile 15 mL conical.
Add V1 mL of PLO to the 15 mL conical.
Diluting Laminin in DMEM
Depending on the lot number of your laminin, obtain the COA from the Sigma-Aldrich website and determine the concentration of the purchased bottle.
Calculate the amount of laminin needed for a final volume of 6 mL (enough for a 6- or 12-well plate) using equation 5, where C1 is the stock concentration of PLO.
Add (6 - V1) mL of DMEM to a sterile 15 mL conical.
Add V1 mL of PLO to the 15 mL conical.
Making a Calcein AM and Ethidium Homodimer-1 solution
To make a 12 mL solution, add 11.97 mL of DPBS to a 15 mL conical.
To the same conical, add 6 µL of Calcein AM and 24 µL of ethidium homodimer-1.
Making a 0.1% Triton-X solution
To make a 25 mL solution of 0.1% Triton-X, add 24.975 mL of PBS to a 50 mL conical.
To the same conical, add 25 µL of 100% Triton X-100.
Making a 5% NGS solution
To make a 25 mL solution of 0.1% Triton-X, add 24.975 mL of PBS to a 50 mL conical.
To the same conical, add 25 µL of 100% Triton X-100.
Making the Primary Antibody solution
To make a 12 mL solution, add 11.856 mL of the above 5% NGS solution to a 15 mL conical.
To the same conical, add 120 µL of anti-TUJ1 and 24 µL of anti-TH.
Refer to Note 14.
Making the Secondary Antibody solution
To make a 12 mL solution, add 11.880 mL of the above 5% NGS solution to a 15 mL conical.
To the same conical, add 60 µL of Alexa Fluor® 488 and 60 µL of Alexa Fluor® 568.
Refer to Note 14.
Making a 300 nM DAPI solution
Add 2 mL of distilled water to the entire contents of the DAPI vial to make a 14.3 mM DAPI stock solution.
In a 1 mL Eppendorf tube, add 100 µL of PBS and 2.1 µL of the 14.3 mM DAPI stock solution to make a 300 µM DAPI solution.
Dilute the 300 µM DAPI intermediate dilution 1:1,000 in PBS as needed to make a 300 nM DAPI stain solution.
Making a 70% Ethanol solution
Add 4 L of 95% ethanol to a large container.
Add 1.43 L of distilled water to the same container.
Making a Potassium Chloride solution
Add 149.1 mg of KCl powder to 20 mL of distilled water to make a 100 mM solution.
Mix 560 µL of the 100 mM KCl solution to 440 µL of media to make a 56 mM solution of KCl.
Making a 10% DMSO Freezing Media solution
To make a 10 mL solution of 10% DMSO, add 9 mL of complete MSC media to a 15 mL conical.
To the same conical, add 1 mL of sterile 100% DMSO.
Acknowledgments
This work was funded by the Natural Sciences and Engineering Research Council (NSERC) Discovery Grant program, the Canadian Institutes for Health Research Project Grant program, Innovate BC’s Ignite program, Canada Research Chairs, the Michael Smith Foundation for Health Research, and Pacific Parkinson’s Research Institute’s Innovation to Commercialization grant. This protocol was derived from an original research paper from the Willerth laboratory (Restan Perez et al., 2021) and the differentiation protocol was modified from (Trzaska and Rameshwar, 2011). We would also like to thank Victor Allison da Silva for his help with the bioprinting videos.
Competing interests
Dr. Stephanie Willerth is the CEO and cofounder of Axolotl Biosciences, as well as a shareholder. She is also listed as an inventor on the pending patent entitled “MORPHOGENIC COMPOUND-RELEASING MICROSPHERES AND USE IN BIOINK.” Milena Restan Perez is currently employed by Axolotl Biosciences.
Ethics
This study was done with Human Ethics approval from the University of Victoria.
References
Abelseth, E., Abelseth, L., De la Vega, L., Beyer, S. T., Wadsworth, S. J. and Willerth, S. M. (2019). 3D Printing of Neural Tissues Derived from Human Induced Pluripotent Stem Cells Using a Fibrin-Based Bioink. ACS Biomater Sci Eng 5(1): 234-243.
Chrenek, J., Kirsch, R., Scheck, K. and Willerth, S. M. (2022). Protocol for printing 3D neural tissues using the BIO X equipped with a pneumatic printhead. STAR Protoc 3(2): 101348.
Dauer, W. and Przedborski, S. (2003). Parkinson's disease: mechanisms and models. Neuron 39(6): 889-909.
Gao, G., Hubbell, K., Schilling, A. F., Dai, G. and Cui, X. (2017). Bioprinting Cartilage Tissue from Mesenchymal Stem Cells and PEG Hydrogel. Methods Mol Biol 1612: 391-398.
Govindharaj, M., Hashimi, N. al, Soman, S. S., Kanwar, S., & Vijayavenkataraman, S. (2022). 3D Bioprinting of human Mesenchymal Stem Cells in a novel tunic decellularized ECM bioink for Cartilage Tissue Engineering. Materialia, 23, 101457.
Hamadjida, A., Frouni, I., Kwan, C. and Huot, P. (2019). Classic animal models of Parkinson's disease: a historical perspective. Behav Pharmacol 30(4): 291-310.
Maan, Z., Masri, N. Z. and Willerth, S. M. (2022). Smart Bioinks for the Printing of Human Tissue Models. Biomolecules 12(1).
Restan Perez, M., Sharma, R., Masri, N. Z. and Willerth, S. M. (2021). 3D Bioprinting Mesenchymal Stem Cell-Derived Neural Tissues Using a Fibrin-Based Bioink. Biomolecules 11(8).
Robinson, M., Valente, K. P. and Willerth, S. M. (2019). A Novel Toolkit for Characterizing the Mechanical and Electrical Properties of Engineered Neural Tissues. Biosensors (Basel) 9(2).
Tasnim, N., De la Vega, L., Anil Kumar, S., Abelseth, L., Alonzo, M., Amereh, M., Joddar, B. and Willerth, S. M. (2018). 3D Bioprinting Stem Cell Derived Tissues. Cell Mol Bioeng 11(4): 219-240.
Trzaska, K. A. and Rameshwar, P. (2011). Dopaminergic neuronal differentiation protocol for human mesenchymal stem cells. Methods Mol Biol 698: 295-303.
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Simultaneous Microendoscopic Calcium Imaging and EEG Recording of Mouse Brain during Sleep
ST Sasa Teng
Yueqing Peng
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4664 Views: 912
Reviewed by: Geoffrey C. Y. LauShengjin XuBo Liang
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Original Research Article:
The authors used this protocol in Nature Communications Aug 2022
Abstract
Sleep is a conserved biological process in the animal kingdom. Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology, important for the development of new treatments for insomnia and other sleep-related disorders. Yet, brain circuits controlling this process remain poorly understood. A key technique in sleep research is to monitor in vivo neuronal activity in sleep-related brain regions across different sleep states. These sleep-related regions are usually located deeply in the brain. Here, we describe technical details and protocols for in vivo calcium imaging in the brainstem of sleeping mice. In this system, sleep-related neuronal activity in the ventrolateral medulla (VLM) is measured using simultaneous microendoscopic calcium imaging and electroencephalogram (EEG) recording. By aligning calcium and EEG signals, we demonstrate that VLM glutamatergic neurons display increased activity during the transition from wakefulness to non-rapid eye movement (NREM) sleep. The protocol described here can be applied to study neuronal activity in other deep brain regions involved in REM or NREM sleep.
Keywords: Sleep Microendoscope GRIN lens EEG Calcium imaging Brainstem
Background
Sleep and wakefulness are actively controlled by the interplay of distinct neural circuits in the brain (Weber and Dan, 2016; Scammell et al., 2017). Centered on the mutual inhibition between sleep- and wakefulness-promoting circuits, a flip-flop model has been proposed to explain the mechanisms of sleep-wake state transitions (Saper et al., 2001; Saper et al., 2010). The discovery of the ascending reticular activating system (Moruzzi and Magoun, 1949), the orexin neurons (de Lecea et al., 1998; Sakurai et al., 1998), and further studies of neuromodulatory systems have greatly advanced our understanding of the neural circuits supporting wakefulness (Brown et al., 2012; Lee and Dan, 2012; Scammell et al., 2017). By contrast, the neural circuits controlling sleep have remained elusive. Several sleep-promoting regions have been identified, including the preoptic area (POA, particularly the ventrolateral preoptic area and median preoptic nucleus, or VLPO and MPO, respectively) (Sherin et al., 1996; John and Kumar, 1998; Lu et al., 2000; Alam et al., 2014; Kroeger et al., 2018), the parafacial zone (Anaclet et al., 2012 and 2014), and the basal forebrain (Xu et al., 2015). However, whether these brain structures are involved in sleep initiation or maintenance remains largely unknown. GABAergic neurons in these brain regions are sleep-active, and their activation promotes non-rapid eye movement (NREM) sleep (Scammell et al., 2017). In addition to the role of GABAergic neurons in sleep regulation, recent studies have identified glutamatergic neurons that promote NREM sleep in a few brain areas, including the perioculomotor region of the midbrain (Zhang et al., 2019), the ventrolateral periaqueductal gray of the midbrain (Zhong et al., 2019), and the posterior thalamus (Ma et al., 2019).
The POA is the most intensively studied sleep-active and sleep-promoting region. Immunohistochemistry studies in rats showed the existence of sleep-active GABAergic neurons in the VLPO and a positive correlation between the number of c-Fos-positive cells and the amount of NREM sleep (Sherin et al., 1996; Lu et al., 2002). Consistently, electrophysiological recordings demonstrated that the firing rate of VLPO sleep-active neurons correlates with the depth of NREM sleep [indicated by electroencephalogram (EEG) delta power] (Szymusiak et al., 1998; Alam et al., 2014). Notably, VLPO sleep-active neurons displayed lower activity in the wake-sleep transition period than in the subsequent sleep episode (discharge rates further increased significantly from light to deep NREM sleep) (Szymusiak et al., 1998). These findings suggest that VLPO neurons might be involved in sleep maintenance, whereas other neurons may be responsible for sleep initiation. Using c-Fos activity approach, retrograde tracing, calcium imaging, and optogenetic and chemogenetic manipulations, we recently identified a population of POA-projecting glutamatergic neurons in the ventrolateral medulla (VLM) that controls the transitions from wakefulness to NREM sleep (Teng et al., 2022).
Here, we describe a detailed protocol for deep brain calcium imaging in the brainstem with simultaneous EEG/electromyography (EMG) recording to monitor neuronal activity during wake and sleep cycles (Figure 1). Compared to fiber photometry recording, this microendoscopic calcium imaging technique provides single-cell resolution to distinguish the function of subpopulations of VLM neurons in behaving mouse. Compared to in vivo electrophysiological recording, this imaging method allows the examination of neuronal activity in genetically defined cells. Similar in vivo calcium imaging techniques have been used to study sleep-related neuronal activity in other brain regions, including galanin-expressing GABAergic neurons in the dorsomedial hypothalamus (Chen et al., 2018) and neurotensinergic neurons in the midbrain (Zhong et al., 2019). A detailed imaging protocol in the cortical and subcortical areas has been published (Resendez et al., 2016). However, imaging the brainstem in freely moving animals typically involves significant movement artifacts. To overcome this issue, a previous study used anchoring tungsten wires glued to the gradient refractive index (GRIN) lens to stabilize the field of view (Gong et al., 2020). Here, we adopted this method to image VLM neurons in wake and sleep cycles. The whole experimental procedure takes approximately two months (Figure 1A). Our imaging protocol should be applicable to other deep brain regions that are involved in sleep regulation.
Figure 1. Imaging medulla glutamatergic neurons during sleep. A. Timeline of experimental design. B. Top: schematic of microendoscopic calcium imaging in the brainstem. Tungsten wires attached to the gradient refractive index (GRIN) lens were used to stabilize the field of view. Bottom: averaged GCaMP fluorescence image in the field of view and activity map of a representative imaging session in a Vglut2-Cre mouse. Scale bar, 50 μm. C. Example data showing electroencephalogram (EEG), electromyography (EMG), and representative calcium traces (DF/F) in an imaging session. Bottom: enlarged window showing calcium activity in three cells.
Materials and Reagents
Materials
GRIN lens and adapter, 0.5 mm diameter, 8.4 mm length (Inscopix, catalog number: 100-004155, adapter included)
Base plate (Inscopix, catalog number: 100-004096)
Base plate cover (Inscopix, catalog number: 100-002388)
5-position connectors (Mouser Electronics, catalog number: 437-8618300510001101)
Bone screws (J.I. Morris, catalog number: F000CE094)
Stainless wires, insulated, 0.003" diameter (A-M Systems, catalog number: 791100)
Silver wires, insulated, 0.005" diameter (A-M Systems, catalog number: 786000)
Flexible piano wires, 0.004" diameter (Precision Fiber Products, catalog number: SMWL-004-01)
Stainless steel custom-built headpost
Reagents
AAV1-syn-FLEX-GCaMP6f (Addgene, catalog number: 100833-AAV1)
Mineral oil (Fisher Scientific, catalog number: O121-1)
Contemporary Ortho-JetTM BCA powder (Lang Dental Manufacturing, catalog number: 1530BLK)
Ortho-JetTM liquid (Lang Dental Manufacturing, catalog number: 1304CLR)
Kwik-Cast silicone sealant (WPI, catalog number: KWIK-CAST)
3MTM Scotch-Weld epoxy adhesive DP100 clear (3M, model: DP-100)
Krazy glue (Krazy Glue, catalog number: KG94548R)
Ophthalmic ointment (Akorn, catalog number: 59399-0162-35)
Ketamine (Butler animal health holding, catalog number: 071069)
Xylazine (Butler animal health holding, catalog number: 061035)
Povidone iodine (MedSupply Partners, catalog number: D02-1202)
Equipment
Micropipette puller (Sutter Instrument, model: P-1000)
Surgical microscope (Leica, model: M60)
Stereotaxic instruments (David Kopf Instruments, model 900-U)
Nanoliter 2020 injector (WPI, NANOLITER2020)
DC temperature controller (FHC, 40-90-8D)
Micro drill (WPI, model: OmniDrill35)
Micromanipulator (Sutter Instrument, model: MP-285A)
Microendoscopic calcium imaging system (Inscopix, model: nVista3)
Electrophysiological recording system (Neuralynx, model: Digital Lynx 4S)
MDF sound attenuating cubicle (Med Associates, model: ENV-018MD)
Custom-built head-fixation platform (Figure 2, see components below):
1" XYZ translation stage (Thorlabs, catalog number: PT3)
Small dual-axis goniometer, 1/2" distance to point of rotation (Thorlabs, catalog number: GN2)
C-clamp (Siskiyou, catalog number: CC-2)
Post, 1/2" diameter, 8" length (Thorlabs, catalog number: TR8)
Parallel clamp for Ø1/2" posts, #8 counterbore and 3/16" hex (Thorlabs, catalog number: RA360)
Right-angle clamp for Ø1/2" posts, 3/16" hex (Thorlabs, catalog number: RA90-P5)
Aluminum breadboard, 6" × 12" × 1/2", 1/4"-20 taps (Thorlabs, catalog number: MB612F)
Mini-series aluminum breadboard, 3" × 4" × 3/8*, 8-32 and 1/4"-20 high-density taps (Thorlabs, catalog number: MSB34)
Custom-built metal adapter for headpost
M2.5 socket head cap screws for headpost
Figure 2. Head-fixation platform. Different views of the platform.
Software
Cheetah (Neuralynx, v5.7.4)
MATLAB (MathWorks, version 2021)
IDSP software (Inscopix, IDSP 1.80)
Procedure
Preparation of GRIN lens and EEG electrodes
Cut piano wires to ~5 mm length (Figure 3A and 3B).
Position two wires on the side of the GRIN lens, with the help of a small piece of Scotch tape (3 × 5 mm) under the microscope. The wires should be parallel to the lens, with the tips extending 200–300 μm from the GRIN lens (Figure 3C).
Add 1–3 μL of Krazy glue to the wires.
Wait until the Krazy glue dries.
Carefully remove the Scotch tape.
Keep finished GRIN lens (Figure 3D) in a container for surgical use.
Cut stainless steel wires and silver wires to ~2 cm length.
Solder three stainless steel wires and two silver wires to a 5-position connector. The stainless steel wires will be used as EEG electrodes, while the silver wires will be used as EMG electrodes (Figure 3E).
Apply epoxy around the soldering area to secure all wires to the connector.
Keep finished EEG electrodes (Figure 3E) in a container for surgical use.
Figure 3. Assembled gradient refractive index (GRIN) lens and EEG electrodes. A. Tools and materials for GRIN lens preparation. B. GRIN lens and anchoring wires. C. Attachment of anchoring wires to the GRIN lens. D. Assembled GRIN lens. E. Assembled EEG/EMG electrode, inset: diagram of channel configuration. The middle wire is used as the reference (ref) for EEG.
Viral injection
Pull the glass capillary into a needle shape with Sutter P-1000 and load ~500 nL of AAV1-FLEX-GCaMP6f virus into the glass pipette of the nano injector.
First weigh and then anesthetize mouse by intraperitoneal injection of a mixture of ketamine and xylazine (100 and 10 mg/kg).
Once the mouse is anesthetized, lubricate their eyes with an application of ophthalmic ointment.
Mount the animal onto a stereotaxic frame (Figure 4A) and clip the hair in a 1 × 2 cm area encircling the surgical site. Maintain normothermia by a feedback-controlled thermostatic heating pad.
Disinfect the skin by swabbing three times with povidone iodine alternated with 70% alcohol, followed by a final application of povidone iodine. Then, remove the skin on the dorsal aspect of the head.
Level the antero-posterior (A-P) and left-right (L-R) axis of the skull.
Drill a hole of 1 mm diameter into the skull above the target coordinate (VLM, bregma -6.9 mm, lateral 1.1 mm, ventral 5.6 mm, the ventral coordinate is relative to the pial surface).
Position the nano injector to the target coordinate (Figure 4B) and lower the injector slowly until its tip reaches the depth of 5.6 mm below the dorsal surface of the brain (Figure 4C). Use a low speed to minimize the damage to the surrounding brain tissues.
Figure 4. Stereotaxic viral injection. A. Equipment for viral injection. B. Positioning the nano injector to the target brain region. C. Inserting the glass capillary into the drilled brain region.
Inject 200 nL of virus at a rate of 50 nL/min.
Wait 5 min to allow the spread of the virus.
Lift the injector slowly until the tip is out of the brain.
GRIN lens implantation
We used a custom-made stainless steel metal pin (400 μm in diameter, 20 mm in length, tip polished into a sharp edge) to create a path for facilitating the implantation of the GRIN lens, by inserting the metal pin 400 μm above the injection site and retracting it 2 min after the insertion (Figure 5A).
Attach the GRIN lens to an Inscopix holder through the provided adapter (Figure 5B). Attach the holder on the micromanipulator.
Slowly insert the GRIN lens at the speed of 10 μm/s into the brain path created by the metal pin, until it reaches 200 μm above the target coordinate (Figure 5C).
Fix the GRIN lens onto the skull with the dental cement surrounding the lens (Figure 5D). Note that only the necessary amount of dental cement should be applied, to ensure most of the skull is still exposed for the following procedure. A minimum of 20 min is recommended to make sure the dental cement dried completely for the strong stabilization of the mount.
Figure 5. Implantation of gradient refractive index (GRIN) lens. A. Insert a metal pin to create a path for GRIN lens. B. Insert the GRIN lens. C. Secure the GRIN lens with black dental cement.
EEG/EMG electrodes implantation
Trim stainless steel and silver wires if needed. Remove the plastic insulation at the end of the wires with ~0.3 mm length.
Drill (drill size: 0.5 mm diameter) three holes into the skull: two holes for EEG recording on top of the cortex (EEG1: 1 mm from midline, 1.5 mm anterior to the bregma; EEG2: 1 mm from midline, 1.5 mm posterior to the bregma). The third hole, for reference, should be on top of the cerebellum.
Position the electrodes on top of the brain using a holder.
Insert the EEG stainless wires into the holes and then secure them with bone screws. Use a screwdriver to make 2.5–3 turns for epidural position over the cortex (Figure 6A).
Insert two EMG electrodes bilaterally into the neck musculature (Figure 6A).
Cover the skull and implanted GRIN lens and EEG/EMG electrodes with black dental cement.
Attach a metal headpost with dental cement.
Cover the top of the Proview GRIN lens with silicon adhesive (Figure 6B and 6C).
Keep the mouse on the heat pad until recovery.
Figure 6. Implantation of EEG electrodes and gradient refractive index (GRIN) lens. A. Implanted EEG wires on the skull and EMG wires into the musculature. B. Cover the GRIN lens with silicon adhesive (green). C. Implants and headpost secured with black cement.
Mounting base plate for microendoscopic calcium imaging
After 3–4 weeks, fix the awake mouse on a custom-built head-fixation platform.
Attach the baseplate to the bottom of the Inscopix miniature microendoscope.
Remove the silicon cover on the GRIN lens.
Position the microendoscope above the GRIN lens (Figure 7A).
Adjust the angle of the head until the focal plane is parallel to the surface of the GRIN lens (Figure 7B).
Find the best focal plane of the active cells (Figure 7B) by a) maximizing the number of cells in the field of view, and b) using the DF/F mode to clearly visualize real-time activity.
Attach the baseplate to mouse head with dental cement (Figure 7C). Do not move the microendoscope until the dental cement is totally dry.
Remove the microendoscope and cover the GRIN lens with baseplate cover (Figure 7D).
Return the mouse to its home cage to rest.
Figure 7. Mounting base plate for microendoscopic imaging. A. Align the baseplate with the gradient refractive index (GRIN) lens. B. Mount baseplate and microendoscope on a head-fixation platform. Inset: screenshot of a live focal plane. C. Secure the baseplate to the GRIN lens with black cement. D. EEG implants, baseplate, and baseplate cover secured with black cement.
Simultaneous microendoscopic calcium imaging and EEG recording
On the day of recording, connect the microendoscope and the EEG cables to the mouse on a head-fixation platform (Figure 8A).
Habituate the mouse in the behavioral arena for at least 90 min.
Start the imaging acquisition and sleep recording for 1–2 h as follows.
Calcium activity is acquired using the nVista 3.0 hardware and IDPS software (Inscopix) with 475 nm LED illumination (10 Hz, 0.4–1.2 mW/mm2). EEG and EMG are recorded, bandpass filtered at 0.5–500 Hz, and digitized at 1,017 Hz using Neuralynx Digital Lynx 4S controlled by a custom-built MATLAB program via Neuralynx API.
A TTL signal delivered from the Inscopix system to the Neuralynx system is used to synchronize the timing between the imaging and EEG/EMG recordings (Figure 8B).
Repeat 2–3 imaging sessions to capture both wake and sleep states.
Figure 8. Concurrent microendoscopic calcium imaging and EEG recording. A. Left: Connect cables on a head-fixation platform. Right: Freely moving mouse with attached cables in a recording chamber. B. Schematic of recording setup.
Mice are perfused for histology to examine viral expression and GRIN lens placement after recording (Figure 9).
Figure 9. Placement of gradient refractive index (GRIN) lens and expression of GCaMP6f in the brainstem. A. Fluorescence image of a brain coronal section showing the lesion track (GRIN lens) and GCaMP6f expression. Scale bar, 0.5 mm. B. Enlarged view of the brain area under the GRIN lens (red box in A). Scale bar, 50 μm.
Data analysis
Calcium imaging analysis
The isxd-format video file recorded during microendoscopic imaging is first processed in the IDSP software (Inscopix, IDSP 1.80). We use the default parameters in the IDSP software except those that are specifically mentioned in this protocol. Then, imaging data is further processed in MATLAB along with EEG data.
Preprocessing: The maximum resolution in Inscopix nVista3 is 1,280 × 800 pixels. To speed up the whole data acquisition and analysis process, our calcium imaging was mostly recorded at the resolution of 320 × 240 pixels.
Note: The imaging data should be spatially down-sampled to 1/4 of the original resolution if the maximum resolution is used. Use a down-sample factor of 2 if the imaging data has already been binned by 2 during image acquisition. The frame rate should be down-sampled to 10 Hz.
Spatial filter: To remove the low-frequency component (background) and the high-frequency component (noise), we process the image with the Spatial filter function in the IDSP with default bandpass filter cut-off (pixel-1): low 0.005, high 0.5.
Motion correction: Run Motion correction function in the IDSP to minimize motion artifact between frames. Set the 100th frame as the reference frame.
ROI segmentation: To detect cells in the field of view, we first convert images to DF/F in the IDSP, then calculate the activity map using the max-intensity of each pixel of DF/F images in the whole imaging session. The cells in the field of view are relatively sparse, so we manually define ROIs to contain all the cells (Figure 10A).
Convert raw calcium signals to DF/F: Convert the calcium signals in each ROI to DF/F in the IDSP software. The DF/F for each ROI is calculated as the difference between the calcium activity at each bin and the averaged calcium activity of the whole recording time, divided by the average (Figure 10B).
The calcium traces are then deconvolved by the OASIS fast deconvolution algorithm (Friedrich et al., 2017) in MATLAB to extract calcium events (Figure 10C). Locomotion-induced artifact events are excluded by using a custom-built MATLAB program (EventReviewer, available from DOI: 10.5281/zenodo.6870710).
Figure 10. Calcium data processing. A. ROI segmentation in a recording session. Images from left to right: maximum intensity of pixels across the image session, maximum projection of fluorescent changes indicated by DF/F, DF/F image superimposed with ROI labels. Segmented ROIs for each cell. B. Calcium traces presented with Z-scored DF/F after motion-correlation. C. Deconvoluted calcium traces (red) with the OASIS algorithm.
The integrated area under curve (AUC) of detected events is used for quantification.
Processed data are saved as MATLAB data files.
EEG data analysis
Spectral analysis is carried out using fast Fourier transform (FFT) over a 5 s sliding window, sequentially shifted by 2 s increments (bins) for the whole recording session.
Brain states are semi-automatically classified into wake, NREM sleep, and REM sleep states using a custom-written MATLAB program (DOI: 10.5281/zenodo.6870666) (Peng, 2022). Semi-autoclassification is validated manually by trained experimenters (Figure 11).
Figure 11. EEG data processing. A screenshot of NLXEEGgui software.
The criterion for sleep/wake states are:
Wake: desynchronized EEG and high EMG activity;
NREM sleep: synchronized EEG with high-amplitude, delta frequency (0.5–4 Hz) activity, and low EMG activity;
REM sleep: high power at theta frequencies (6–9 Hz) and low EMG activity.
Processed EEG and EMG data are saved as MATLAB data files.
Alignment of microendoscopic imaging and EEG data
Load saved imaging data and EEG data into MATLAB.
Align time points of imaging data and EEG data using the TTL signal in EEG data (Figure 12A and 12B).
Figure 12. Identify sleep-related neuronal activity in the ventrolateral medulla (VLM). A. Align calcium traces with EEG data. From top to bottom: brain states (gray: wake; orange: NREM sleep; purple: REM sleep), EEG spectrogram (0–25 Hz), EMG amplitude, representative calcium traces. B. Enlarged time windows showing EEG, EMG, and calcium signals in a NREM sleep period (left) and a wake period (right). Calcium events detected by the deconvoluted algorithm are highlighted in red.
To compare the activity in different brains states, we used the AUC and normalized it to the duration (in minutes) of each state, which yields relative activity per minute.
We then calculated the selectivity index between brain states as:
Index = (AUCa - AUCb)/(AUCa + AUCb)
Where AUCa and AUCb refer to AUC activity in brain state a (e.g., NREM sleep) and b (e.g., wake) respectively. Index ranges from -1 to 1, with 0 indicating no selectivity between two states.
To analyze calcium activity during the transitions, we calculated AUC activity in the NREM active cells before and after wake-to-NREM or NREM-to-wake transitions. A 30 s window in each brain state (e.g., 30 s wake, 30 s NREM for wake-to-NREM transitions) is used for quantification. Transitions with less than 30 s episodes in either state are excluded for analysis. Overall, we found that 57% of VLM glutamatergic cells were selectively active during wakefulness, 27% during REM sleep, and 16% during NREM sleep. Further analysis demonstrated that NREM-active neurons displayed increased activity during the transitions from wakefulness to NREM sleep, whereas their activity remains almost unchanged during the NREM-to-wake transitions. For detailed results, please refer to the published paper (Teng et al., 2022).
Acknowledgments
We thank Charles Zuker at Columbia University for sharing the Inscopix equipment. We thank Li Wang for his assistance with the GRIN lens implantation surgery and microendoscopic imaging data analysis. This work was supported by startup funds from Columbia University and by National Institute of Neurological Disorders and Stroke grant R01NS129997. This protocol was adapted from previous work (Gong et al., 2020, Teng et al., 2022).
Competing interests
The authors declare no competing interest.
Ethics
All procedures were carried out in accordance with the US National Institute of Health (NIH) guidelines for the care and use of laboratory animals and approved by the Animal Care and Use Committees of Columbia University.
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Long-term in toto Imaging of Cellular Behavior during Nerve Injury and Regeneration
WT Weili Tian
AG Andrés González-Suarez
HL Hernán López-Schier
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4665 Views: 629
Reviewed by: Nafisa M. JadavjiXiaochen SunWenyang Li
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Original Research Article:
The authors used this protocol in microPublication Biology Mar 2021
Abstract
Accidental wounding of the peripheral nervous system leads to acute neural dysfunction. Normally, chronic deficits are overcome because peripheral nerves naturally regenerate. However, various genetic and metabolic defects can impair their natural regenerative capacity, which may be due to neuron-extrinsic mechanisms. Therefore, characterizing the behavior of multiple cells during nerve injury and repair in vivo is a pressing need in regenerative medicine. Here, we detail a method for precise wounding of sensory axons in zebrafish, followed by high-resolution in toto long-term quantitative videomicroscopy of neurons, Schwann cells, and macrophages. This protocol can be easily adapted to study the effects of targeted genetic or metabolic disruptions in zebrafish and other suitable organisms, as well as for screening pharmacological agents with therapeutic potential.
Graphical overview
Keywords: Zebrafish Macrophages Glia Nerve Injury Repair Microscopy
Background
The peripheral nervous system communicates sensory cues to the brain. It therefore mediates organismal responses to changes in the body’s internal and external environments (Handler and Ginty, 2021; Elias and Abdus-Saboor, 2022; Prescott and Liberles, 2022). Sensory neurons must maintain functionality throughout the life of the organism despite stress and trauma. Although acute loss of integrity of these neurons occurs persistently, animals normally overcome chronic neurological dysfunctions via effective repair (Johnson et al., 2005). By contrast, repair is normally extremely limited in the central nervous system (Varadarajan et al., 2022). Peripheral nerve wounding triggers dynamic changes in the behavior of Schwann cells as well as the recruitment of other cells including macrophages, which together resolve injury, initiate repair, and enhance regeneration (Abdo et al., 2019). In recent studies, we have combined genetic and chemical perturbations, transgenic markers, and high-resolution live imaging to characterize nerve injury and regeneration in larval zebrafish (Lozano-Ortega et al., 2018; Tian and López-Schier, 2020; Xiao et al., 2015a). Furthermore, we discovered that Schwann cells are important but not essential for sensory nerve regeneration (Tian et al., 2020b; Xiao et al., 2015b). Moreover, we revealed fast recruitment of macrophages to the wound (Tian et al., 2020). We have also discovered that blocking the degradation of axons severed from the neuronal cell body has no significant impact on nerve repair (Tian et al., 2020). Our finding that loss of the pro-degenerative protein Sarm1 accelerates axonal regeneration may have important implications for the development of therapies aimed at improving neural repair in humans.
The zebrafish larva is a powerful vertebrate experimental system for studies of neuronal wounding and regeneration (Rieger and Sagasti, 2011; Cardozo et al., 2017; Cigliola et al., 2020; González and Allende, 2021). Spatial and temporal control of injury can be done by laser-mediated axon transection or neuronal ablation. Axonal injury triggers fast degeneration of the part of the axon detached from the cell body, followed by regenerative growth of the proximal stump. In several previous studies, we used the mechanosensory lateral line in larval zebrafish to perform nerve wounding using an ultraviolet laser, and live microscopy in transgenic animals expressing fluorescent markers to track relevant cellular populations during injury resolution and repair (Figures 1 and 2). Of note, laser-mediated axon severing is a powerful technique but it suffers from the fact that both proximal and distal axon fragments are immediately cauterized. This is unlikely to occur during physical injury, when axoplasmic spillage may trigger different responses from the Schwann cells situated in proximity to the damaged nerve. Nevertheless, the precise spatiotemporal control of injury and simultaneous live imaging that this protocol allows vastly overcome this potential shortcoming. Therefore, we present a detailed protocol for facile implementation of laser-mediated microsurgery of sensory axons and in toto imaging of subsequent responses by axons, glia, and macrophages (Figure 3 and 4, and Videos 1 and 2). This protocol uses 5-day-post-fertilization (5 dpf) zebrafish Danio, but it can be equally used in older animals and other specimens of similar characteristics, including Oryzias, Astyanax, or Danionella.
An ultraviolet laser is directed to the target through a high numerical aperture objective lens mounted on a spinning-disk confocal microscope. However, a point-scanning single- or two-photon confocal microscope will be useful. Targeting is guided by the fluorescence from stable expression of EGFP or RFP using a lateralis afferent neuron-specific enhancer. However, mosaic expression of a fluorescent protein using pan-neuronal genetic control elements, for instance the widely employed NeuroD, Ngn1, or HuC, is also suitable.
Materials and Reagents
Cover glass–bottomed mini dish (MatTek, catalog number: p35G-1.0-14-c)
Petri dishes (Greiner Bio-One, catalog number: G180535A/01889)
Plastic strainer (ZM Systems, Product code: teastrain15)
Plastic Pasteur pipettes
Tricaine (MS-222) (25× stock solution) (PharmQ, UK)
Low-melting agarose (1% M/V) (Sigma-Aldrich, catalog number: A9414-100G)
Sodium chloride (NaCl) (Merck KGaA, catalog number: 1.06404.5000)
Potassium chloride (KCl) (Sigma-Aldrich, P9541-500G)
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306-250G)
Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M5921 500G)
Trizma (Tris base) (Sigma-Aldrich, catalog number: T1503-1KG)
HCl (Sigma-Aldrich, catalog number: H1758-500ML)
Plasmids UAS:EGFP, SILL:mCherry; mfap4:EGFP
Fish lines: Casper, Tg[UAS:EGFP], Tg[SILL:mCherry], Tg[gSAGFF202A], Tg[ mfap4:EGFP]
E3 embryo medium (10×) (see Recipes)
Tris-HCl (1 M pH 9.0) (see Recipes)
Tricaine (MS-222) (25×) (see Recipes)
Low-melting agarose (1% w/v) (see Recipes)
Equipment
Incubator set at 28.5 °C
Fine forceps
Optically-clear polystyrene 90 mm Petri dishes
Plastic transfer pipettes
Microwave oven
100 mL glass bottle
Handmade 1-eyelash brush
Microinjection system
Microscopes:
Upright stereomicroscope (e.g., ZEISS Stemi SV 11) equipped with fluorescent-light source and a filter set for GFP and RFP/Cherry.
Spinning-disk microscope confocal microscope (ZEISS) with an iLasPulse laser system (Roper Scientific SAS) and a temperature-control system.
Procedure
Prepare zebrafish larva (Tg[gSAGFF202A:UAS-EGFP; Tg[SILL-mCherry] line for imaging Schwann cells and afferent neurons, or Tg[mfap4:EGFP; Tg[SILL:mCherry] line for imaging macrophages and afferent neurons)
Collect embryos and keep them in the incubator at 28.5 °C for standard development to the 5–6 days-post-fertilization (dpf) larval stage. Embryos should be kept in 90 mm Petri dishes. For optimal survival, keep around 50 embryos per dish.
At 4 dpf, screen specimens using a fluorescence stereomicroscope (Figures 1 and 2).
Using a transfer pipette, isolate the positive larva expressing the mCherry (red) in the lateralis neurons and EGFP (green) in Schwann cells or macrophages.
Note: Plastic materials intended for single use can be washed and re-used. This includes Petri dishes and transfer pipettes.
Figure 1. Main tools for the protocol. A. Photograph exemplifying the placement of a plastic Petri dish containing eggs into an incubator. B. Photograph of a standard Petri dish, glass pipette with a hair loop attached to its tip, a forceps and a plastic transfer pipette. C. Example of a stereomicroscope with a typical microinjection apparatus. D. Photograph of an inverted spinning-disc microscope for long-term imaging.
Figure 2. Procedures for collecting eggs and raising specimens. A. Photograph of a breeding tank, with a bottom grid to enable eggs to fall outside the reach of adult fish (which may otherwise eat them), a Petri dish and an egg-harvesting strainer. B. Once the inner container is removed with the breeding adults, the water with the eggs contained in the breeding tank is gently poured through the strainer to collect eggs. C. The eggs are gently and briefly washed with clean water from the facility system. D. The strainer is then placed inside down onto the inner (bottom) part of the Petri dish and eggs are flushed into this dish applying a gently flow of water using a plastic bottle. E. The eggs will now be visible in the dish. F. The dish with the eggs can then be moved to a stereomicroscope of fluorescence for screening and cleaning.
Mosaic-labelling, an alternative to stable transgenics lines for marking a selected population of neurons and Schwann cells or macrophages
Prepare Casper zebrafish eggs from the Tg[gSAGFF202A] driver line for Schwann cells.
Prepare the plasmids for injection. Mix well 20 ng/μL each of the UAS:EGFP and SILL:mCherry plasmids before loading into the capillary for injection.
For macrophage and neurons, mix mfap4:EGFP and SILL:mCherry constructs for injection (10 ng/μL of each plasmids).
Inject around 1–2 nL plasmids into the one-cell stage embryos.
Screen the injected eggs that have developed as morphologically normal larva at 4 dpf and pick the EGFP and mCherry double-positives under a stereomicroscope of fluorescence. Then, transfer the positive larva to a new plate for growth to desired stage (Figure 2).
Laser-mediated nerve transection and imaging
Anesthetizing and mounting the larva
Anesthetize the larva with 1× MS-222 solution (0.016% w/v) diluting with E3 medium. The E3 embryo medium is prepared as a stock solution (10×) from which a working solution (1×) is freshly made (see Recipe 1). Put one large drop (around 100 μL) of 1% low melting agarose (melted at no more than 37 °C) onto the cover glass–bottomed dish. Move the anesthetized larva into the agarose drop using a transfer pipette with the minimal amount of water possible, to prevent too much dilution of the agarose. Using a hair loop glued to the tip of a glass pipette, gently push the larva to the bottom of the dish so that it touches the glass. Then, orient and position the specimen as desired. Generally, the resulting ~1% low melting agarose will allow enough time (30–45 s) for the procedure. With practice, up to three fish can be mounted almost simultaneously.
After 5 min at room temperature, cover the now solidified agarose with E3 embryo medium containing MS-222.
Move the dish with extreme care because, if shaken, the agarose block could detach from the glass, rendering the preparation unusable.
Note: We noticed a significant drop of survival during imaging when more than three fish are mounted on the same dish.
Put the dish containing fish on a holder adapted to the microscope stage. Put the heating system at 28.5 °C (or the ideal temperature for the specific experiment). Use the 20× objective to adjust the position of the dish by looking at the red or green fluorescence.
Transect the axons with a laser beam by using a water-immersion 63× objective (see Notes). It is important to avoid oil-immersion objectives because they will prevent repeating the procedure on multiple fish mounted on the same dish, and also significantly complicate later imaging using lower magnification objectives.
For transection, we use the iLas-Pulse ultraviolet laser system, controlled by VISION software. A 350 nm laser with 400 ps/2.5 μJ per pulse and adjusted to power of 27 mW is used for nerve transections.
Using the VISION software, draw a line or a rectangle to mark the region to be targeted by the laser beam. This can be saved in the system for subsequent equivalent samples. To fully sever the axons, the laser pulses should be applied repeatedly until there is a clear gap in the rectangle region (see Notes). We normally set the pulse at 0.05 ms and hit three times per mouse click. If needed to generate a larger damage, move the line/rectangle to the next position for a new transection. We normally use a distance of 40 µm between multiple transections of the same nerve (Figure 5).
Set up the parameters for intravital imaging
After ensuring the axons are completely severed, select a 40× (air) or 63× (water) objective for live imaging. Put the region of interest in the center of the field of view and focus to the plane of interest. When Z-stacks’ parameters are fixed, the intervals of one stack can be set to 0.8–1.3 μm. The intervals of imaging are set to 5–10 min.
If long-term imaging (more than 1 h) will be performed, replace the 63× water-immersion objective by an equivalent objective that can tolerate an immersion medium that will not evaporate. Given that high-grade low-melting-point agarose has a refractive index (RI) of 1.33 (similar to water) and that borosilicate glass has a RI of 1.47, the use of 100% glycerol with a RI of 1.47 is optimal.
Image quantification
The live imaging raw data is sent to Fiji (https://imagej.net/software/fiji/) for analysis.
Use the plugin “Tracking” to analyze the dynamics of nerve repair and the behavior of Schwann cells or macrophages. Open Plugins – Tracking – Manual Tracking.
Select the cell(s) of interest at the starting point for tracing over the course of the time series. This can be done from a single focal plane or a maximal projection of the entire or partial Z-stack.
With information we got from tracking, we can make conclusions on how many Schwann cells were attracted to the damaged site and how long these cells stayed (Tian et al., 2020b; Figures 3–5, Videos 1 and 2).
Figure 3. Example of in toto videomicroscopy of axons and macrophages. A–F. Still images from a high-resolution time-lapse recording of a macrophage (green) and a lateralis sensory nerve (red) in the trunk of a larval zebrafish. A single macrophage is seen migrating towards the nerve immediately after it has been severed with a laser. A second macrophage is seen below, which never makes contact with the nerve. Over time, the macrophage moves along the nerve, surveilling damage (which can be seen as a discontinuity of the nerve on the left-hand side of the panels (C and D). The drawings below each panel (dotted lines) describe the two-dimensional shape changes ofthis single macrophage during the course of videomicroscopic imaging. Scale bars is 50 μm.
Figure 4. Example of in toto videomicroscopy of axons and Schwann cells. A–C. Fluorescence images from a high-resolution time-lapse recording of the movement of Schwann cells (green) and the regeneration of a lateralis afferent nerve (red) after transection. A shows the merge of the two fluorescence channels, labelling the sensory nerve (Red) and the Schwann cells (green).The respective individual channels are show in B and C. D shows the tracks of the moving Schwann cells. t1, t2, t3, t4, t5, t6 indicate different time points extracted from the time-lapse. Dt6. It a zoom-in of the entire track of Schwann cells of at t6.
Figure 5. Example laser-mediated nerve transection. A-1 and A-2. Screenshots from the transection, before and after laser-mediated wounding, respectively. Images show the VISION software settings used during this procedure, as well as the targeted region within the nerve axon (yellow rectangle). A-1.1 and A2.1. Spinning-disk images render the nerve axon targeted by the bean laser (yellow rectangle), from before and after transection.
Video 1. Intravital videomicroscopy of neurons and macrophages
Video 2. Intravital videomicroscopy of neurons and Schwann cells
Notes
Prepare fresh plasmids and check the quality of the DNA one day before the injections. Plasmid DNA quality needs to be check first by analysis of its correct nucleotide sequence data using a bio-tool software (e.g., snapgene, https://www.snapgene.com/). The plasmids were extracted from fresh overnight-cultured transformed DH5alpha. The quality of plasmids should be confirmed with Nanodrop. The 260/280 ranges from 1.8–2.0. The same plasmid preparation, kept in cold storage, can be used for 1–2 consecutive days if no sign of degradation is seen.
Keep the low-melting-point agarose in a small glass Erlenmeyer partially submerged in a water bath set at 40–42 °C.
Aim the laser beam to the axons. Ensure full transection by looking at a gap along the axons and evident signs of peripheral axon degradation (pearling) between 10 and 30 min after exposure to the laser.
For long-term live imaging, the laser power must be adjusted to around 10 mW. Higher power or more frequent imaging intervals cause tissue damage and decrease fish survival.
After imaging, check the condition of fish. Only data from healthy fish with no signs of physical injury and that can swim after being released from the agarose should be used. This, of course, is not applicable if the experiment itself must result in a behavioral dysfunction, for instance if transecting the spinal cord.
In case images are shifting within the imaging plane, use the Template matching plugin in Fiji to re-align the images.
Recipes
E3 embryo medium (10×)
50 mM NaCl
1.7 mM KCl
3.3 mM CaCl2·2H2O
3.3 mM MgSO4, dissolved in 1 L deionized water
Tris-HCl (1 M pH 9.0)
12.1 g of Tris base dissolved in 80 mL of deionized water. Adjust the pH to 7.0 with HCl and then make the final volume to 100 mL.
Tricaine (MS-222) (25×)
2 g of MS-222 in 480 mL of deionized water; adjust the pH to 7.0 with 1 M Tris-HCl (pH 9.0) and then make the final volume to 500 mL. Store at 4 °C.
Low-melting agarose (1% w/v)
30 mg of low-melting agarose dissolved in 30 mL of 1× E3 medium by heating in microwave oven. Keep in a 40–42 °C water bath.
Acknowledgments
This work was supported by the Helmholtz Association and BMBF grant 01GQ1904.
This protocol has been adapted from previous work by our laboratory (Xiao et al., 2015a and 2015b; Lozano-Ortega et al., 2018; Tian and Lopez-Schier, 2020; Tian et al., 2020; Asgharsharghi et al., 2021).
Competing interests
HL-S is scientific advisor and paid consultant for Sensorion (France). The company had no role in this study. No competing interests exist with any vendor cited in this work.
Ethics
Procedures were conducted in accordance with the Ethical Committee of Animal Experimentation of the Helmholtz Zentrum München, the German Animal Welfare act Tierschutzgesetz §11, Abs. 1, Nr. 1, Haltungserlaubnis, to European Union animal welfare, and to protocols number Gz.:55.2-1-54-2532-202-2014 and Gz.:55.2-2532.Vet_02-17-187 from the “Regierung von Oberbayern”, Germany.
References
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Asgharsharghi, A., Tian, W., Haehnel-Taguchi, M. and López-Schier, H. (2021). Sarm1 is dispensable for mechanosensory-motor transformations in zebrafish. MicroPubl Biol. DOI: 10.17912/micropub.biology.000369
Cardozo, M. J., Mysiak, K. S., Becker, T. and Becker, C. G. (2017). Reduce, reuse, recycle - Developmental signals in spinal cord regeneration. Dev Biol 432(1): 53-62.
Cigliola, V., Becker, C. J. and Poss, K. D. (2020). Building bridges, not walls: spinal cord regeneration in zebrafish. Dis Model Mech 13(5). doi: 10.1242/dmm.044131.
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Article Information
Copyright
© 2023 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Category
Neuroscience > Cellular mechanisms
Developmental Biology > Cell growth and fate > Regeneration
Cell Biology > Cell imaging > Fluorescence
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LD Lance S. Davidow
AA Anthony C. Arvanites
LR Lee L. Rubin
JP Josef M. Penninger
CW Clifford J. Woolf
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4666 Views: 698
Reviewed by: Joana Alexandra Costa ReisNader GhasemlouKaustav Mukherjee
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Original Research Article:
The authors used this protocol in Science Translational Medicine Aug 2022
Abstract
Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene, Gch1, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers. Gch1 is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced Gch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell–derived sensory neurons.
Graphical overview
Keywords: BH4 DRG neurons GCH1 Drug screen Neuropathic pain
Background
Tetrahydrobiopterin (BH4) is a co-factor metabolite used by the aromatic amino acid hydroxylases and nitric oxide synthases in the production of dopamine, serotonin, noradrenaline, and nitric oxide (Werner et al., 2011). BH4 synthesis is increased in dorsal root ganglion (DRG) sensory neurons after nerve injury, where its levels correlate with pain hypersensitivity in rodent neuropathic pain models, as well as in patients with chronic pain (Tegeder et al., 2006; Campbell et al., 2009; Kim et al., 2010; Lötsch et al., 2010; Heddini et al., 2012; Nasser and Møller, 2014; Belfer et al., 2015; Sadhu et al., 2018; Zheng et al., 2019). Targeting BH4 synthesis peripherally in injured sensory neurons represents a novel and potentially safe approach to combat certain neuropathic pain conditions (Latremoliere et al., 2015; Fujita et al., 2019). Since the early 1990s, the focus of drug discovery has been driven by target-based screening, using structural modeling and in silico computational analyses to design and screen small molecules using simple target-dependent assays, to find those that enter and block a certain enzymatic domain or binding motif (Sams-Dodd, 2005; Swinney, 2013). However, before this target-based approach, phenotypic screening was prevalent, often with limited information on the underlying mechanisms involved in the disease in question, and in which no targets were known or identified. Indeed, the success of these phenotypic screens over the more precision-targeted screens is revealed by the fact that the majority of first-in-class drugs actually came from phenotypic screens (Swinney and Anthony, 2011). Here, we describe a phenotypic screening platform using primary DRG sensory neurons from transgenic Gch1-GFP reporter mice that allowed us to monitor the effects of chemical libraries on the regulation of Gch1 expression in individual DRG neurons after axonal injury (axotomy). Using this platform, we provided not only novel insights into the biology of GCH1 expression and BH4 synthesis in injured sensory neurons, but also identified FDA-approved compounds with existing safety and pharmacokinetic profiles, which could potentially be repurposed to block the GCH1/BH4 pathway in neuropathic pain (Cronin et al., 2022). This protocol can be adapted to monitor the effects of compounds on the expression of multiple algesic genes in primary DRG cultures from transgenic mice or on human stem cell–derived sensory neurons.
Materials and Reagents
1.5 mL Eppendorf tubes (Eppendorf, catalog number: 0030121872)
Pipette tips (Biozym)
Glass Pasteur pipettes (Thermo Fisher, catalog number: 1367820C)
50 mL conical centrifuge tubes (Falcon, catalog number: 352070)
100 μm cell strainer (Corning, catalog number: 352360)
Gch1-GFP reporter mice [GENsat.org; stock name: Tg(Gch1-EGFP)GU68Gsat/Mmucd; stock number: 012014-UCD]
Neurobasal media (Thermo Fisher, catalog number: 21103049)
Black flat-bottomed TC-treated 384-well plate pre-coated with poly-D-lysine (BD Biocoat, Corning, catalog number: 354663)
Laminin (Sigma, catalog number: L2020)
Phosphate-buffered saline (PBS) (Thermo Fisher, 10010023)
Liberase-TH (Roche, catalog number: 5401135001)
Trypsin 0.5% (Gibco, catalog number: 15400-054)
Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher, catalog number: 12491015)
Fetal bovine serum (FBS) (Gibco, catalog number: 26140079)
L-glutamine (Gibco, catalog number: 25030-024)
Penicillin and streptomycin (Sigma, catalog number: P0781)
DNase-I (Sigma, catalog number: D5025)
30% BSA solution (Sigma, catalog number: A9205)
50× B-27 supplement (Thermo Fisher, catalog number: 17504044)
Trypan blue (Invitrogen, catalog number: T10282)
Propidium iodide (Thermo Fisher, catalog number: P1304MP)
Phorbol 12-myristate 13-acetate (PMA) (Sigma, catalog number: P1585)
Go6976 (Tocris, catalog number: 2253)
Complete media (see Recipes)
DRG media (see Recipes)
Equipment
Pipettes (P20, P200, and P1,000) (Gilson, catalog number: F167360)
Centrifuge (Eppendorf, model: 5810R)
Heidolph Unimax 1010 plate shaker
Bunsen burner (USBECK, W. Germany)
Hemocytometer (Marienfeld, catalog number: 0630010)
PerkinElmer/Evotec Opera QEHS model 2.0 laser spinning-disk confocal fluorescence microscope
Sterile tissue culture cabinet (Szabo Scandic, SafeFAST Premium 212)
Software
PerkinElmer Acapella language (PerkinElmer)
Procedure
DRG culture in 384-wells
Below is an optimized protocol for DRG culture used to coat one 384-well plate; work should be performed under sterile conditions in a tissue culture cabinet.
Coat a poly-D-lysine-coated 384-well plate additionally with 40 μL of laminin (10 μg/mL in PBS) for 3 h at 37 °C.
Isolate DRGs from eight Gch1-GFP reporter mice and collect evenly into two (for balancing purposes during centrifugation) 1.5 mL Eppendorf tubes filled with 1.4 mL of neurobasal media.
When all DRGs are collected, allow them to settle by gravity and remove as much neurobasal media as possible by pipetting with a 1,000 μL tip.
Add 350 μL of liberase-TH solution to the DRGs and incubate at 37 °C for 80 min.
Remove liberase-TH solution by pipetting and wash DRGs slowly in 1 mL of PBS, again allowing the DRGs to settle by gravity.
Remove PBS by pipetting.
Dilute 250 μL of 0.5% trypsin 1:1 in PBS. Add 350 μL of the resulting 0.25% trypsin solution to the tubes and incubate them at 37 °C for 7 min.
Add 1 mL of complete media (see Recipes) to stop trypsin reaction.
Allow DRGs to settle by gravity and remove supernatant by pipetting carefully.
Wash with 1 mL of complete media.
Allow DRGs to settle by gravity and remove supernatant by pipetting carefully.
Add 1 mL of complete media with DNase-I (10 μg/mL).
Flame-polish the tip of three Pasteur pipettes over a Bunsen burner flame, to achieve varying diameters of pipette opening: large, medium, and small (Figure 1).
Figure 1. Flame-polished (using a Bunsen burner) pipettes with three comparative sizes of pipette opening
Mechanically triturate the DRGs, progressing from the normal diameter pipette to the medium and finally to the small, to dissociate the DRGs into single-cell suspension (with each pipette size, triturate 10–15 times).
Prepare 10% BSA solution in a 50 mL tube by mixing 2 mL of 30% BSA solution with 4 mL of PBS.
Layer 1 mL of dissociated DRG suspension over the 10% BSA solution.
Wash tube with 500 μL of complete media and transfer and overlay to the 10% BSA tube.
Centrifuge at 800× g for 15 min at 4 °C.
Carefully remove BSA interface first and then the rest of BSA supernatant (top layer). Then, carefully remove all the BSA lower layer ensuring not to disrupt the cell pellet.
Carefully resuspend the DRG pellet first in 2 mL of complete media and bring up to 20 mL. At this point, combine DRG pellets in a total of 20 mL.
Pass the cell suspension through a 100 μm cell strainer into a fresh 50 mL conical tube.
Centrifuge at 800× g for 15 min at 4 °C.
Carefully remove supernatant.
Resuspend cell pellet in 2 mL of DRG media (see Recipes).
Count DRG neurons using trypan blue staining (in a 1:1 ratio, i.e., 10 μL each) and add 10 μL of resulting mixture to a hemocytometer.
Adjust volume to ensure density of 25,000 neurons/mL.
Remove laminin solution from the wells completely.
Add 40 μL (~1,000 neurons) of the DRGs in DRG media to each well.
Incubate plate at 37 °C with 5% CO2 (timepoint set as Day 0).
Compound and propidium iodide (PI) addition
Twenty-four hours after plating the neurons (i.e., on Day 1), add compounds from chemical library. Compounds should be prepared in such a way that 10 μL of each compound is added to the 40 μL of DRGs to give a desired final working concentration. Add compounds at a rate of approximately 1 mL/min to minimize cell disturbances.
Shake plate for 5 min on a shaker at 120 rpm.
Incubate plate at 37 °C with 5% CO2.
On Day 3, prepare a working solution of PI viability stain (0.5 μg/mL) to identify dead cells.
Add 50 μL of PI solution to the DRGs (i.e., 1:1 mixture).
Shake plate for 1 min on a shaker at 120 rpm and incubate plate at 37 °C with 5% CO2 for 20 min.
Neuronal imaging
After PI staining, transfer the plate immediately to PerkinElmer/Evotec Opera QEHS model 2.0 laser spinning-disk confocal fluorescence microscope.
Visualize and image Gch1-GFP cells with a 488 nM laser and PI with a 561 nM laser. Divide each well into 15 sections and acquire each section (Figure 2).
Figure 2. Confocal imaging of individual Gch1-GFP-expressing dorsal root ganglion (DRG) neurons. A. Each 384-well was divided into 15 regions; confocal fluorescent images of GFP and propidium iodide (PI) were taken of each section. All 15 sections were combined, and the various parameters extracted for each well. B. Representative GFP fluorescent images of a single-well subsection depicting high-expressing GFP DRG neurons (red arrows) induced by vehicle (DMSO), PMA, and Go6976. Scale bar, 30 µm.
Proceed to image analysis.
Data analysis
Image analysis
Analyze the resulting images with scripts written in the PerkinElmer Acapella language that is packaged with the automated microscope. Use the GFP channel images for subroutines for nuclei detection. The first-round candidate nuclei that were too small, large, wide, or narrow were filtered out using the follow scripts [please refer to Supplemental Figure 3 in original paper (Cronin et al., 2022)]:
// User script STARTS
input(IN_Green_lo_pos_thresh, 300,"Green Low Positive Threshold","i","Minimum Green intensity to qualify as Low Positive")
input(IN_Green_hi_pos_thresh, 1000,"Green High Positive Threshold","i","Minimum Green intensity to qualify as High Positive")
input(IN_max_nuc_area, 2000)
input(IN_min_nuc_area, 300)
input( IN_LowerWidthToLengthThresh, 0.3, "Lower WidthToLength Threshold")
input(IN_Green_channel, 1,"Green Channel","i","Channel with Green Nuclear Marker")
input(IN_viability_channel, 2,"viability_channel","i","Channel with viability Nuclear Marker")
input(IN_Green_neg_thresh, 60,"Green False Nuclei Threshold","i","Minimum Green intensity to qualify as a Real Nucleus")
input(IN_Viability_thresh, 75,"Viability Threshold","i","Minimum Viability intensity to qualify as Dead")
set(Green_channel= _["IM_CH" & IN_Green_channel])
//set(viability_channel= _["IM_CH" & IN_viability_channel])
set(viability_channel= IM_CH2)
Nuclei_Detection_Select(green_channel)
//Make all the measurements before starting the object filters
set(objects=nuclei)
CalcIntensity(image=Green_channel, objects=objects)
RenameAttr(Green_intensity=intensity, objects=objects)
CalcIntensity(image=viability_channel, objects=objects)
RenameAttr(viability_intensity=intensity, objects=objects)
set(nuclei=objects)
CalcAttr( "Roundness" )
CalcWidthLength()
DefineAttr( WidthToLength, "half_width / full_length" )set(nuclei=objects)
// Collects results for Nuclei
if (defined("all_Nuclei")) // Not the first evaluated field
AddObjects(Nuclei, objects=all_Nuclei, CheckOverlap=no)
set(all_Nuclei=objects) // Renames output from AddObjects()
else()
set(all_Nuclei=Nuclei) // The first evaluated field
end()
end() // end of the analysis of valid image fields
end() // end of the foreach loop over image fields
//Start Filtering now that we have all the nuclei collected. do imageviews of each individual classification
objectfilter(area>=IN_max_nuc_area, objects=all_Nuclei)
set(too_big=objects)
objectfilter(area<IN_max_nuc_area, objects=all_Nuclei)
set(all_Nuclei=objects)
objectfilter(area<=IN_min_nuc_area, objects=all_Nuclei)
set(too_small=objects)
objectfilter(area>IN_min_nuc_area, objects=all_Nuclei)
set(all_Nuclei=objects)
objectfilter(WidthToLength<IN_LowerWidthToLengthThresh, objects=all_Nuclei)
set(too_skinny=objects)
objectfilter(WidthToLength>=IN_LowerWidthToLengthThresh, objects=all_Nuclei)
set(all_Nuclei=objects)
objectfilter( (Green_intensity <=IN_Green_neg_thresh) , objects=all_Nuclei)
set(GreenFalseNuc=objects)
objectfilter( (Green_intensity >IN_Green_neg_thresh) , objects=all_Nuclei)
set(all_Nuclei=objects)
//find all 12 phenotypic classes for each of the 3 markers (lo_pos and hi_pos for ubiq)
//GreenNeg
objectfilter( (Green_intensity <=IN_Green_lo_pos_thresh) , objects=all_Nuclei)
set(GreenNeg=objects)
objectfilter( (Green_intensity <=IN_Green_lo_pos_thresh) AND (Viability_intensity > IN_viability_thresh) , objects=all_Nuclei)
set(GreenNeg_Dead=objects)
objectfilter ((Green_intensity <=IN_Green_lo_pos_thresh) AND (Viability_intensity <= IN_viability_thresh) , objects=all_Nuclei)
set(GreenNeg_Alive=objects)
//GreenLoPos
objectfilter((Green_intensity >IN_Green_lo_pos_thresh) AND (Green_intensity <=IN_Green_hi_pos_thresh) , objects=all_Nuclei)
set(GreenLoPos=objects)
objectfilter((Green_intensity >IN_Green_lo_pos_thresh) AND (Green_intensity <=IN_Green_hi_pos_thresh) AND (Viability_intensity > IN_viability_thresh), objects=all_Nuclei)
set(GreenLoPos_Dead=objects)
objectfilter((Green_intensity >IN_Green_lo_pos_thresh) AND (Green_intensity <=IN_Green_hi_pos_thresh) AND (Viability_intensity <= IN_viability_thresh), objects=all_Nuclei)
set(GreenLoPos_Alive=objects)
//GreenHiPosobjectfilter((Green_intensity > IN_Green_hi_pos_thresh), objects=all_Nuclei)
set(GreenHiPos=objects)
Divide the remaining accepted nuclei into high-intensity, low-intensity, or GFP-negative based on the average intensities in the GFP channel (and also use non-transgenic mice to set baseline GFP levels).
Subgroup the cells further into alive or dead based on their PI fluorescence intensity. Only living, PI-negative cells are included in further analysis.
The following parameters will depend on the specific target gene(s) being analyzed; for Gch1-GFP, these were individually optimized. See Notes.
Calculate three different parameters for each compound well: the number of high-expressing GFP neurons (#GFPHi), the mean GFP intensity per well (mean GFP intensity), and the percentage of high-expressing GFP neurons of total neurons identified per well (%GFPHi/total nuclei).
For each parameter, three standard deviations above and below (± 3 SD) that of the vehicle (DMSO) control samples were scored.
For each threshold passed, assign the following scoring: #GFPHi = 1; mean GFP intensity = 3; %GFPHi/total nuclei = 5. The total rank sum was assigned to each compound. For decreasers, compounds that acquired a total score > 1 were considered a hit; for increasers, a stricter total score >5 was used.
Notes
Positive [PMA; phorbol 12-myristate 13-acetate; protein kinase C (PKC) activator], negative (Go6976; PKC inhibitor), and appropriately diluted vehicle (DMSO) control chemicals were included as controls on each plate to validate the algorithm thresholds and data consistency on each plate.
In our screen, Gch1-GFP expression could be classified into high and low GFP expression, as described in the paper. We focused on the effect of compounds on the GFP-high-expressing neurons.
The section of the acapella script we added to the PerkinElmer supplied Multifield Analysis script is shown above. Presumably, any image analysis scripting language that finds nuclei, measures sizes, and measures intensity at different wavelengths could be used. Moreover, the calculations and thresholds we used to select cells for analysis inclusion could vary with different biological experimental systems.
Recipes
Complete media (suggested volume to prepare: 100 mL)
DMEM + 10% FBS + L-glutamine (5 mM) + Penicillin and streptomycin (5 mM)
DRG media (suggested volume to prepare: 50 mL)
Neurobasal media + 50× B-27 supplement + L-glutamine (5 mM) + Penicillin and streptomycin (5 mM)
Acknowledgments
We acknowledge the Harvard Stem Cell Institute (HSCI) for their expertise and infrastructure and chemical libraries in running the screen. We also would like to acknowledge all the members of the Woolf, Penninger, and Rubin labs for all their advice and help in generating the original publication from which this protocol was derived (Cronin et al. 2022). CJW is supported by NIH R35NS105076 and an HMS Blavatnik Award. JMP is supported by the Austrian Federal Ministry of Education, Science and Research, the Austrian Academy of Sciences, and the City of Vienna and grants from the Austrian Science Fund (FWF), Wittgenstein award (Z 271-B19), and the T. von Zastrow foundation. LR and HSCI is supported by the HSCI Therapeutic Screening Center (TSC) grant (# CF-0009-17-03).
Competing interests
The authors have no competing financial interests.
Ethics
All experiments were performed with approval from the Boston Children’s Hospital Institutional Animal Care and Use Committee.
References
Belfer, I., Dai, F., Kehlet, H., Finelli, P., Qin, L., Bittner, R. and Aasvang, E. K. (2015). Association of functional variations in COMT and GCH1 genes with postherniotomy pain and related impairment. Pain 156(2): 273-279.
Campbell, C. M., Edwards, R. R., Carmona, C., Uhart, M., Wand, G., Carteret, A., Kim, Y. K., Frost, J., and Campbell, J. N. (2009). Polymorphisms in the GTP cyclohydrolase gene (GCH1) are associated with ratings of capsaicin pain.Pain 141(1-2):114-8.
Cronin, S. J. F., Rao, S., Tejada, M. A., Turnes, B. L., Licht-Mayer, S., Omura, T., Brenneis, C., Jacobs, E., Barrett, L., Latremoliere, A., et al. (2022). Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.Sci Transl Med 14(660): eabj1531.
Fujita, M., da Luz Scheffer, D., Lenfers Turnes, B., Cronin, S. J. F., Latrémolière, A., Costigan, M., Woolf, C. J., Latini, A. and Andrews, N. A. (2019). Sepiapterin reductase inhibition selectively reduces inflammatory joint pain and increases urinary sepiapterin. Arthritis Rheumatol 72(1): 57-66.
Heddini, U., Bohm-Starke, N., Grönbladh, A., Nyberg, F., Nilsson, K.W., and Johannesson, U. (2012). GCH1-polymorphism and pain sensitivity among women with provoked vestibulodynia.Mol Pain 8: 68.
Kim, D. H., Dai, F., Belfer, I., Banco, R. J., Martha, J. F., Tighiouart, H., Tromanhauser, S. G., Jenis, L. G., Hunter, D. J., and Schwartz, C. E. (2010). Polymorphic variation of the guanosine triphosphate cyclohydrolase 1 gene predicts outcome in patients undergoing surgical treatment for lumbar degenerative disc disease.Spine (Phila Pa 1976) 35(21):1909-14.
Latremoliere, A., Latini, A., Andrews, N., Cronin, S. J., Fujita, M., Gorska, K., Hovius, R., Romero, C., Chuaiphichai, S., Painter, M., et al. (2015). Reduction of Neuropathic and Inflammatory Pain through Inhibition of the Tetrahydrobiopterin Pathway. Neuron 86: 1393-1406.
Lötsch, J., Klepstad, P., Doehring, A., and Dale, O. (2010). A GTP cyclohydrolase 1 genetic variant delays cancer pain. Pain 148(1):103-106.
Nasser, A., and Møller, L.B. (2014). GCH1 variants, tetrahydrobiopterin and their effects on pain sensitivity.Scand J Pain 5(2): 121-128.
Sadhu, N., Jhun, E. H., Yao, Y., He, Y., Molokie, R. E., Wilkie, D. J. and Wang, Z. J. (2018). Genetic variants of GCH1 associate with chronic and acute crisis pain in African Americans with sickle cell disease. Exp Hematol 66:42-49.
Sams-Dodd, F. (2005). Target-based drug discovery: Is something wrong?Drug Discov Today 10(2): 139-47.
Swinney, D.C. (2013). Phenotypic vs. Target-based drug discovery for first-in-class medicines.Clin Pharmacol Ther 93(4): 299-301.
Swinney, D.C., and Anthony, J. (2011). How were new medicines discovered?Nat Rev Drug Discov 10(7):507-19.
Tegeder, I., Costigan, M., Griffin, R.S., Abele, A., Belfer, I., Schmidt, H., Ehnert, C., Nejim, J., Marian, C., Scholz, J., et al. (2006). GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat Med 12(11):1269-77.
Werner, E.R., Blau, N., and Thöny, B. (2011). Tetrahydrobiopterin: biochemistry and pathophysiology. Biochem J 438(3): 397-414.
Zheng, N.N., Zhang, R.C., Yang, X.X., and Zhong, L.S. (2019). Association of rs3783641 single-nucleotide polymorphism in GTP cyclohydrolase 1 gene with post-herpetic neuralgia. J Dermatol 46(11): 993-997.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Drug Discovery > Drug Screening
Neuroscience > Nervous system disorders
Cell Biology > Cell isolation and culture
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Peer-reviewed
A Simple, Reproducible Procedure for Chemiluminescent Western Blot Quantification
DC Daniel Cañada-García
JA Juan C. Arévalo
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4667 Views: 2532
Reviewed by: David Paul Anonymous reviewer(s)
Download PDF
Ask a question
Favorite
Cited by
Original Research Article:
The authors used this protocol in Pain Mar 2023
Abstract
Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture. However, there is no clear and common procedure to quantify the results obtained, resulting in variations due to the different software and protocols used in each laboratory. Here, we have developed a procedure based on the increase in chemiluminescent signal to obtain a representative value for each band to be quantified. Images were processed with ImageJ and subsequently compared using R software. The result is a linear regression model in which we use the slope of the signal increase within the combined linear range of detection to compare between samples. This approach allows to quantify and compare protein levels from different conditions in a simple and reproducible way.
Graphical overview
Keywords: Western blot Protein Quantification Analysis Data normalization Linear regression model
Background
Proteins are key biomolecules for the correct functioning of living animals; therefore, it is required not only to detect but also to accurately quantify them. Western blot is the most widely used and accepted method to detect proteins of interest using specific antibodies. Since the development of western blotting in 1979 (Towbin, H, PNAS 1979), more than 382,000 articles have been published using this technique (Western blot, PubMed, October 11, 2022). Since 2000, when 7,300 papers were published, there has been a constant increase in the number of articles reporting the use of this method (Moritz, 2020), reaching almost 22,600 in 2021. However, a proper quantification to determine the amount or the corresponding protein modification detected is still missing in most of the articles. In addition, reviewers and editors are eager to have trusty quantitative data regarding western blot analyses (Murphy and Lamb, 2013; Taylor et al., 2013;Janes, 2015; McDonough et al., 2015; Bass et al., 2017; Pillai-Kastoori et al., 2020). To assess accurate quantification, it is required to perform data normalization using proteins that do not vary due to experimental conditions. Usually, these are housekeeping proteins including actin, tubulin, and glyceraldehyde 3-phosphate dehydrogenase. Previous articles quantifying chemiluminescence western blots have relied on the use of single images of signals from different samples that are sometimes saturated, leading to incorrect outcomes. Fluorescence-based techniques using fluorophore-conjugated secondary antibodies are not commonly employed in most laboratories but provide a wider range of linear correlation between signal and protein abundance (Huang et al., 2019). Therefore, it is urgent to establish a simple, reliable method to quantify western blot signals to draw accurate conclusions in the studies.
Here, we describe an improved, simple protocol for western blot quantification taking advantage of chemiluminescent detection by ccd cameras and ImageJ and R software. Different images with different exposure times were utilized to generate the linear range for each protein to be quantified. The advantage of our procedure is that the signals are quantified across an interval of different exposures within the linear range for all the samples, where the differences between them are constant. Comparison of the slopes obtained provides a more accurate representation of the relative amounts of proteins in different samples than would be achieved by comparing individual images.
Materials and Reagents
The following list provides examples of the materials and equipment that we routinely use in our laboratory. Reagents and equipment with similar specifications will work as well.
All the materials and reagents listed were handled and stored following manufacturer’s instructions.
Polyvinylidene difluoride transfer membranes, 0.2 µm (PVDF membranes) (Thermo Fisher Scientific, catalog number: 88520)
Whatman 3MM CHR paper (Cytiva, catalog number: 3030-335)
β-glycerophosphate (AppliChem, catalog number: A2253, CAS number: 819-83-0)
β-mercaptoethanol (VWR BHD Chemicals, catalog number: 25351.182, CAS number: 60-24-2)
Acrylamide-bis solution (BioAcryl-P 30%, 37.5:1) (Alfa Aesar, catalog number: J61505)
Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678, CAS number: 7727-54-0)
Aprotinin (Sigma-Aldrich, catalog number: A1153, CAS Number: 9087-70-1)
BCA protein assay: reagent A (Thermo, catalog number: 23228) and reagent B (Thermo, catalog number: 23224)
Bis-Tris ultrapure (Thermo Fisher Scientific, catalog number: J12112.22, CAS number: 6976-37-0)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906, CAS number: 9048-46-8)
Bromophenol blue (Amresco, catalog number: 0449-25G, CAS number: 115-39-9)
Dye reagent concentrate (Bio-Rad, catalog number: 500-0006)
Ethylenediaminetetraacetic acid (EDTA) (VWR Life Science, BHD Prolabo Chemicals, catalog number: 443885J, CAS number: 6381-92-6)
Glycerol (Thermo Fisher Scientific, catalog number: BP229-1, CAS number: 56-81-5)
Glycine (Sigma-Aldrich, catalog number: G8898-1KG, CAS number: 56-40-6)
HRP-conjugated anti-mouse antibody (Jackson ImmunoResearch, catalog number: 115-035-003, concentration used: 1:5,000)
HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch, catalog number: 111-035-003, concentration used: 1:5,000)
Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: H/1200/PB15, CAS number: 7647-01-0)
Isopropanol (Sigma-Aldrich, catalog number: 1.09634.1011, CAS number: 67-63-0)
Leupeptin (Sigma-Aldrich, catalog number: L9783, CAS number: 24125-16-4)
MES-SDS running buffer (Thermo Fisher Scientific, catalog number: NP0002)
Methanol (Fisher Chemical, catalog number: M/4000/PB17, CAS number: 67-56-1)
N,N,N',N'-tetramethyl-ethane-1,2-diamine (TEMED) (Thermo Fisher Scientific, catalog number: BP150-20, CAS number: 110-18-9)
Non-fat dry milk (Nestlé Sveltesse)
NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I3021, CAS number: 9002-93-1)
Phenylmethanesulfonyl fluoride (PMSF) (CalbioChem, catalog number: 52332, CAS number: 329-98-6)
Sodium azide (MERK, catalog number: 1066880100, CAS number: 26628-22-8)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71383-5KG, CAS number: 7647-14-5)
Sodium dodecyl sulfate (SDS) (Bio-Rad, catalog number: 1610302, CAS number:151-21-3)
Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920-100G, CAS number: 7681-49-4)
Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508, CAS number: 13721-39-6)
Trizma base (Tris) (Sigma-Aldrich, catalog number: T1503-1KG, CAS number: 77-86-1)
Tween-20 (Sigma-Aldrich, catalog number: P5927, CAS number: 9005-64-5)
WesternBrightTM ECL HRP Substrate (Advansta, catalog number: K-12045-D50)
Lysis buffer (see Recipes)
5× Laemmli buffer (see Recipes)
Polyacrylamide gels (see Recipes)
Transfer buffer (see Recipes)
TBST washing buffer (see Recipes)
Primary antibody blocking solution (see Recipes)
Secondary antibody blocking solution (see Recipes)
Equipment
Mini-PROTEAN® Tetra Vertical Electrophoresis Cell, 4-gel, for 1.0 mm thick handcast gels (Bio-Rad Laboratories Inc., catalog number: 1658001FC)
Mini-PROTEAN Tetra Companion Running Module (Bio-Rad Laboratories Inc., catalog number: 1658038)
Casting frame (Bio-Rad Laboratories Inc., catalog number: 1653304)
Casting stand gaskets (Bio-Rad Laboratories Inc., catalog number: 1653305)
Glass plates (Bio-Rad Laboratories Inc., catalog number: 1653311)
Short plates (Bio-Rad Laboratories Inc., catalog number: 1653308)
Combs, 15-well, 1.0 mm (Bio-Rad Laboratories Inc., catalog number: 1653360)
Buffer tank and lid with power cables (Bio-Rad Laboratories Inc., catalog number: 1658040)
Gel releasers (Bio-Rad Laboratories Inc., catalog number: 1653320)
Blot roller (Bio-Rad Laboratories Inc.)
Power supply (VWR, catalog number: HITAK622113)
MicroChemi System (Bio-Imaging Systems)
Software
ImageJ and R software
Procedure
The composition of solutions and buffers used are shown below under the section “Recipes”.
Western blot
Since this protocol aims to quantify the signal from membranes, we provide here a general western blotting guideline, based on which each user should apply the optimal conditions for his or her interest. In brief, proteins are electrophoretically separated in an acrylamide gel according to their size and then transferred to a membrane, to be probed with antibodies that recognize specifically antigenic epitopes displayed by the target protein.
The steps are the following:
Extract proteins from HEK293FT cells (at 80% confluency) seeded in a 6 cm plate adding 500 µL of cold lysis buffer with protease inhibitors and incubate for 30 min at 4 °C with gentle shaking. Collect the solution with the cells in a cold Eppendorf tube and centrifuge at 16,000× g for 10 min at 4 °C.
Note: To avoid protein degradation, include protease inhibitors and work in cold conditions, at 4 °C. Lysis can be performed using different lysis buffers including RIPA if e.g., immunoprecipitation will be performed.
Quantify protein concentration.
Note: Quantification of proteins needs to be done with an appropriate method depending on the lysis buffer used, i.e., BCA protein assay (for SDS-containing lysis buffer) and protein assay dye reagent concentrate (lysis buffer without SDS).
Collect the desired quantity of lysates (15–50 µg) in a new Eppendorf, add Laemmli buffer to a final 2× concentration up to 25 µL of volume, and mix well. Then, boil samples at 100 °C for 6 min, let them cool at room temperature and centrifuge at 16,000× g for 30 s.
Note: Samples can be stored at -20 °C if not used immediately.
Load the desired quantity of protein lysates in the corresponding acrylamide gel to perform SDS-PAGE. Place the gel in the tank filled with MES-SDS running buffer and apply current to allow protein separation until the front dye reaches the bottom of the gel (Figure 1).
Note: Depending on the size and proteins to be detected, the percentage and kind of acrylamide gels to be used will be different. For example, to detect proteins with high molecular weight (above 80 kDa), 6%–8% acrylamide gels are preferred. In case it is necessary to detect low (below 30 kDa) and high molecular weight proteins in the same gel, it is recommended to use gradient gels with a percentage of acrylamide from 4% to 12%. Depending on whether the acrylamide gel is a Bis-Tris or Tris-Glycine, the resolving range and the time required to run the samples will vary (30–40 min in Bis-Tris gels at 200 V to 75–120 min in Tris-Glycine gels at 90 V).
Figure 1. SDS-PAGE gel running; at loading (left panel) and after running for 30 min (middle panel) and 90 min (right panel). Note the molecular weight standards (blue) appearing on the left and right edges once the samples have run. Arrows point to the front dye and arrowheads to 75 kDa molecular weight marker.
Transfer proteins from gel to PVDF membranes. Before obtaining the gel, label and activate the PVDF membrane by soaking it in methanol for a few seconds, rinse twice with ddH 2 O for 3–4 min, and incubate with transfer buffer until use. Stop current once the front dye has reached the bottom part of the gel. Assemble the transfer cassette in a container with transfer buffer as shown in Video 1.
Note: It is crucial to avoid air bubbles when setting the cassette by covering the sponge, Whatman paper, and the gel with transfer buffer. Failure to remove air bubbles in the assembled cassette will jeopardize the detection of proteins. After placing each component, remember to run the blot roller gently to avoid the presence of air bubbles during the transfer. Be careful with the orientation of the gel so as not to lose the order in which the samples were loaded; a different volume of molecular weight marker can be added in the first and last lane to aid this purpose.
Video 1. Transfer preparation
Place the cassette in the frame in the right orientation located in the chamber filled with transfer buffer and an ice package. Include a magnetic stirrer and stir while applying a 200–400 mA current for at least 1.5 h in a cold room to avoid overheating. After that, remove PVDF membrane with the proteins attached and start the procedure to detect them.
Note: Protein transfer from gel to membrane can be done with different equipment, including semi-dry transfer. Insertion of the cassette in the wrong orientation will make proteins from the gel to migrate in the opposite direction of the PVDF membrane; therefore, proteins will be lost.
Block membrane with blocking solution with agitation for at least 30 min at room temperature.
Note: Blocking solution will depend on the primary antibody to be used. Check manufacturer datasheet for that info.
Incubate the membrane with the primary antibody in blocking solution with agitation for 2–3 h at room temperature or overnight at 4 °C.
Note: The quality of the signal and the amount of background will depend in a great manner on the quality/affinity of the antibodies. If possible, use antibodies that have been previously tested by you or other laboratories.
Wash with gentle agitation at least three times with TBST solution for at least 15 min, block membrane using TBST + 5% non-fat milk for 10 min with agitation, add the corresponding secondary antibody conjugated with HRP, and incubate for at least 45 min.
Note: The secondary antibody to be used can be coupled with different fluorophores instead of HRP, but extra care must be taken with light to avoid bleaching of signal.
Repeat the washes with TBST and leave the membrane in this solution until development.
Set up the settings for image acquisition in the MicroChemi apparatus using different exposure times.
Note: Acquisition of images can be performed with other equipment. The exposure times will depend on the quantity of protein and/or selectivity of the primary antibody. Routinely, we set time differences between exposures in the Chemidoc program of 1, 3, 5, 15, 30, 60, 180, 300, 600, and 900 s. The signal of each image corresponds to the accumulated time from the beginning, i.e., images 3 and 4 will have an exposure of 9 and 24 s, respectively.
Incubate membrane with the WesternBrightTM ECL HRP Substrate for 1 min.
Place the membrane in the MicroChemi chamber and acquire images (Figure 2).
Note: At the end of the experiment, take a picture with bright light to save an image with the molecular weight markers position in the membrane. This will help identifying the bands of interest.
Copy images and transfer to a computer with ImageJ program to process them.
Figure 2. Images obtained with MicroChemi equipment and GelCapture software. Western blots for actin in HEK293FT cell lysates with different exposure times (from 1 to 4,128 s) showing original images (two left panels, the first being inverted), saturation image profile (third panel from the left), and pseudo color image (fourth panel from the left). Note that the signal becomes saturated (red signal) in 37.5 µg sample with 828 s of exposure.
Image processing and band quantification
The aim is to obtain a numerical value for the intensity of each band in each image captured during membrane exposure, using the analysis tools of ImageJ program (Video 2).
Video 2. Band quantification
Open all images using ImageJ with the commands: File > Import > Image Sequence… choosing the folder directory where the images are located, the first one to start with, and the number of photos to import.
Note: Do not include the “Molecular Weight Markers Picture” taken with clear light, only the ones acquired with chemiluminescence.
Choose the parameters of interest to be recorded from the images. To do this, select Analyze > Set Measurements… clicking the ones that ImageJ should record for the analysis.
Note: In our case, we have selected Area, Mean gray value, Integrated density, and Display label. The analysis is going to be performed with Integrated density values, but as many parameters as desired can be recorded.
Select an image with visible bands, but not saturated (Figure 2, overexposure images), and delimit a rectangle that fits one of them with the "Rectangle" tool. Then, move the rectangle over an area of the membrane close to the bands but without signal, to capture the background. Record the measurements with the commands: Image > Stacks > Measure Stack…
Note: The less the area of the rectangle fits the band, the greater the possibility of introducing deviations due to the background (Gassmann et al., 2009). The size of the rectangle should not be modified, so that the same area is always quantified for all bands. If there are free wells in the gel, a lane with the same lysis buffer and Laemmli buffer can be included, which will serve to quantify the background.
Move the rectangle over each band and repeat the same commands to obtain measurements. As a result, a table with all the values collected for each of the bands in each sequence image will appear.
Note: Consider the order in which the bands have been quantified to know to which of the samples the value corresponds.
Save the data in .csv format with the commands File > Save As…
Linear model optimization
The aim of the analysis is to obtain a representative value for each band in the western blot. This value will be the slope of the signal increment within the linear range of the selected band.
Load the required packages for R software to work.
Note: If the packages are not installed, the command install.packages() needs to be used before loading them.
my_packages <- c("dplyr", "ggplot2", "openxlsx")
lapply(my_packages, require, character.only = TRUE)
Set the working directory, the folders where the data file is located, and where the result of the analysis is going to be exported.
Note: Remember that quotation marks should be used.
WorkingDirectory <- "/Users/xxxxx/Desktop/Folder1/Folder2/Actin"
setwd(WorkingDirectory)
Load the data from the image analysis in .csv format, copying the file name in the corresponding lane.
Note: Quotation marks should be used.
File_Name <- "Results.csv"
Results <- read.table(file = File_Name, sep = ",", header = T)
Define the conditions of the experiment: the background and the samples that have been loaded on the gel. The script is going to assign each name to each set of measurements in a new column called “conditions”.
Note: Be careful to write them in the same order in which the quantification has been done from the images and use quotation marks so that R interprets them as text.
conditions <- c("Background", "6μg", "9μg", "15μg", "18μg", "22.5μg", "30μg", "37.5μg")
Results$Conditions <- factor(x = rep(conditions, each = max(Results$Slice)), levels = conditions)
Introduce the exposure time for each image that has been quantified. The script is going to assign each value to its corresponding image in a new column called “Exposure.”
Note: Remember that it is not the value that has been introduced in the Chemidoc software, but the accumulated time from the beginning. The units to be used can be seconds or minutes; if using minutes, convert the values to decimal system and use “.” for decimals.
Exposure <- c(1, 3, 8, 18, 33, 63, 108, 168, 228, 288, 408, 588, 828, 1128, 1428, 1728, 2028, 2628, 3228, 4128)
Results$Exposure <- rep(exposure, times = length(conditions))
Perform background correction. The code will subtract the signal from the background of each image in all analyzed bands. The result will be written in a new column called “BckgCorrect.”
Results$BckgCorrect <- rep(0, dim(Results)[1])
for (i in 1 :dim(Results)[1]) {
Bkgnd <- Results$Conditions == "Background" & Results$Exposure == Results[i,"Exposure"]
Results$BckgCorrect[i] <- Results[i,"IntDen"] - Results[Bkgnd,"IntDen"]
}
Represent the data in a scatterplot, with the exposure time as the independent variable and the background corrected signal as the dependent one (Figure 3). The purpose of the graph is to determine visually the linear range for the increment of the signal.
ggplot(Results, aes(x = Exposure, y = BckgCorrect, color = Conditions))+
geom_point() + geom_line() + theme_classic()
Figure 3. Representation of band quantifications. The signal of each band (y-axis) with respect to the exposure time (x-axis) is shown. Note that in the samples with higher protein content (22.5, 30, and 37.5 µg) the linearity between exposure time and signal is lost at certain exposure time.
Optimize the model by manually selecting the first and last exposure time to be considered that stays within the linear range for all conditions of interest. The script will define a new data frame with the filtered data between these two points, named “Results_filtered.”
First_Expossure = 63
Last_Expossure= 828
Results %>% filter(between(Exposure, First_Expossure, Last_Expossure)) -> Results_filtered
Build the linear model using simple linear regression, with exposure time as predictor variable and corrected signal as response variable. The script will calculate the regression parameters for each condition of the experiment, obtaining a new data frame with the value of the slope of the regression line, the R-squared coefficient of determination to evaluate the goodness of fit, and the p-value to evaluate its statistical significance (Table 1). It will also generate a new scatterplot with the filtered data (Figure 4).
<- data.frame(t(mapply(unlist, by(Results_filtered, Results_filtered$Conditions, function (x) summary(lm(BckgCorrect~Exposure, x)))))) %>%
select(slope = coefficients2, adj.r.squared, p.value = coefficients8)
ggplot(Results_filtered, aes(x = Exposure, y = BckgCorrect, color = Conditions))+
geom_point() + geom_line() + theme_classic()
knitr::kable(Results_model)
Figure 4. Adjusted representation of band quantifications. After removing the samples with lower and/or higher exposure (in which linearity was lost), linearity for different amounts is kept and will allow to accurately calculate the slope for each concentration.
Table 1. Regression parameters for each condition of the experiment
Conditions Slope Adj. r. squared p. value
Background 0 NaN NaN
6 μg 1157.633 0.9988883 2.704757e-10
9 μg 3156.074 0.9993988 4.277239e-11
15 μg 16760.63 0.9986983 4.34215e-10
18 μg 26829.65 0.9994091 4.061036e-11
22.5 μg 57338.1 0.9993725 4.864208e-11
30 μg 121563.9 0.9989834 2.068317e-10
37.5 μg 223579.3 0.9998507 6.546559e-13
If the regression coefficient is not close enough to 1 and/or the scatterplot does not fit the linear range, go back to step C8, and adjust the exposure values again.
Export the analysis results. The script will overwrite the initial excel file, saving all the data in a sheet named “DATA,” the filtered data between exposure times in a sheet named “DATA_FILTERED,” and the coefficients of the linear model in a sheet named “MODEL.”
Note: Remember that these are chemiluminescent signal values. Western blotting is a semiquantitative technique, so the next step in interpreting the results should be to compare these values with a housekeeping or a reference protein (protein of interest/reference protein).
write.xlsx(list(DATA = Results, DATA_FILTERED = Results_filtered, MODEL = Results_model), file = "File_Name", row.names = T)
Example
As an example of the method described here, complementary to that published in Sánchez-Sánchez et al. (2023), we show the quantification of TrkA activation after stimulation with nerve growth factor (NGF) for different times. Briefly, we used primary cultures of sensory neurons enriched in TrkA-positive neurons selected with NGF for 5–6 days. We then deprived the culture of neurotrophin for 4 h and stimulated with NGF (50 ng/mL) for the corresponding time. We collected cell lysates and performed western blotting using pTrkA (for activated receptor) (Figure 5A, left panels) and TrkA (Figure 5B, left panels) antibodies, taking the top 140 kDa band as the reference protein. We quantified the signals from the pTrkA and TrkA bands as described in this protocol. We measured the signal intensity of different images for each sample (Figures 5A and 5B, exposure blots) and, together with the exposure time settings, defined the combined linear range for each protein (Figures 5A and 5B, right plots). As described above, we built a linear regression model. We used the slope as a representative value to calculate the ratio of pTrk to TrkA and plotted the results of independent experiments (Figure 5C).
Figure 5. Example of signal quantification. Images with different exposures showing pTrkA (panel A) and TrkA (panel B) signals were quantified in cultured sensory neurons after NGF stimulation for different stimulation times. The colored rectangles in both panels represent the membrane areas used for quantification. A rectangle of another region without signal was utilized as background (not shown in the images). Note that saturation can be detected at higher exposures of the TrkA blot in the pseudo color images (red color). On the right side of each panel there is a graph showing the signal increase curve for each time point. The range between dashed lines indicates the exposures taken to optimize the linear regression model and to perform the quantification within linearity. Note that the saturation in the pseudo color images matches the flattering of the curves in the plots. (C) Quantification of TrkA activation (pTrkA/TrkA ratio). Normalization was performed using the 5 min time point of NGF stimulation. Each colored line represents the results obtained in a single experiment (n = 3). Panels A and B correspond to experiment c in panel C.
Useful tips
It is important to include control and experimental/problem samples in the same membrane, to be able to compare since there is always some variability between membranes, even with those carried out in parallel for the whole experiment.
Total protein to be loaded in the gel (usually between 15 and 50 µg) will depend on the abundance of the protein of interest to be detected and the affinity of the antibodies to be used. Loading an excess of total protein can also saturate the membrane during the transfer, and differences between samples could be misleading.
Remember that western blot is a semiquantitative technique and a loading control, housekeeping protein, or reference protein are always needed to normalize the data.
The quality of the signal and the amount of background will depend in a great manner on the quality/affinity of the antibodies. If possible, use antibodies that have been previously tested by you or other laboratories.
When working with R software, do not forget to use quotation marks in what should be text.
Recipes
The following solutions/buffers are obtained using the quantities indicated, and the final concentration of each component is given in brackets.
Lysis buffer
2.42 g of Trizma base pH 8.0 (20 mM)
8 g of NaCl (137 mM)
10 mL of NP-40 (1%)
Ultrapure MilliQ water up to 1 L
Store at 4 °C
Just prior to use, add the corresponding volume to have a final concentration of 1 mM PMSF, 1 μg/mL aprotinin, 2 μg/mL leupeptin, 1 mM sodium orthovanadate, 10 mM NaF, and 20 mM β-glycerophosphate.
5× Laemmli buffer
30.29 g of Trizma base pH 6.8 (250 mM)
100 g of SDS (10%)
500 mL of glycerol (50%)
67 mg of bromophenol blue (0.01%)
39.07 g of β-mercaptoethanol (500 mM)
Ultrapure MilliQ water up to 1 L
Stored at -20 °C
Polyacrylamide gels
Acrylamide-bis solution up to the desired percentage, 350 mM Bis-Tris buffer pH 6.4, 0.1% SDS, ultrapure MilliQ water, APS (0.1%), and TEMED (6.6 mM). Mix the reagents in that order and avoid bubbles to prevent patchy polymerization. Prepare 4.5 mL of final volume per resolution gel, and 2 mL with lower acrylamide percentage for the stacking. The use of a thin layer of isopropanol to avoid contact with oxygen during polymerization of the resolving gel also helps to avoid bubbles and to have a clean separation with stacking. Homemade gels can be stored up to 3–4 days at 4 °C in a humid chamber.
Transfer buffer
5.8 g of Trizma base pH 8.3 (25 mM)
2.9 g of glycine (192 mM)
100 mL of methanol (10%)
Ultrapure MilliQ water up to 1 L and adjust pH
Store at 4 °C and reuse up to five times
TBST washing buffer
1.21 g of Trizma base pH 7.5 (10 mM)
8.8 g of NaCl (150 mM)
0.32 g of EDTA (1 mM)
10 mL of Tween-20 (0.1%)
Ultrapure MilliQ water up to 1 L and adjust pH
Primary antibody blocking solution
100 mL of TBST washing buffer
3 g of BSA (3%)
32.5 mg of sodium azide (0.05%)
Stored at 4 °C
Secondary antibody blocking solution
100 mL of TBST washing buffer
5 g of non-fat dry milk (5%)
Prepare just before use with gentle agitation to avoid foaming
Acknowledgments
We thank Deogracias’ and Arevalo’s lab for input into the manuscript. This work was supported by grant PID2020-113130RB-100 funded by MCIN/AEI/10.13039/501100011033 and by the European Union to J.C.A. D.C.G was granted by Consejería de Educación Junta de Castilla y León and the European Social Fund.
Competing interests
The authors declare no competing interests and mentioning of specific materials, reagents, and equipment does not imply endorsement by the funding agencies.
References
Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K. and Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27(1): 4-25.
Gassmann, M., Grenacher, B., Rohde, B. and Vogel, J. (2009). Quantifying Western blots: pitfalls of densitometry. Electrophoresis 30(11): 1845-1855.
Huang, Y. T., van der Hoorn, D., Ledahawsky, L. M., Motyl, A. A. L., Jordan, C. Y., Gillingwater, T. H. and Groen, E. J. N. (2019). Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting. J Vis Exp(146). doi: 10.3791/59438.
Janes, K. A. (2015). An analysis of critical factors for quantitative immunoblotting. Sci Signal 8(371): rs2.
McDonough, A. A., Veiras, L. C., Minas, J. N. and Ralph, D. L. (2015). Considerations when quantitating protein abundance by immunoblot. Am J Physiol Cell Physiolv 308(6): C426-33.
Moritz, C. P. (2020). 40 years Western blotting: A scientific birthday toast. J Proteomics 212: 103575.
Murphy, R. M. and Lamb, G. D. (2013). Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes. J Physiol 591(23): 5823-31.
Pillai-Kastoori, L., Schutz-Geschwender, A. R. and Harford, J. A. (2020). A systematic approach to quantitative Western blot analysis. Anal Biochem 593: 113608.
Sánchez-Sánchez, J., Vicente-García, C., Cañada-García, D., Martín-Zanca, D. and Arévalo, J. C. (2023). ARMS/Kidins220 regulates nociception by controlling brain-derived neurotrophic factor secretion. Pain 164(3): 563-576.
Taylor, S. C., Berkelman, T., Yadav, G. and Hammond, M. (2013). A defined methodology for reliable quantification of Western blot data. Mol Biotechnol 55(3): 217-226.
Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS 76: 4350-4354.
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© 2023 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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Peer-reviewed
Induction of Skeletal Muscle Injury by Intramuscular Injection of Cardiotoxin in Mouse
XF Xin Fu
SL Sheng Li
MJ Minzhi Jia
WY Wenjun Yang
PH Ping Hu
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4668 Views: 1624
Reviewed by: Nafisa M. JadavjiSuresh KumarSabine Le Saux
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Cited by
Original Research Article:
The authors used this protocol in Cell Research Dec 2020
Abstract
Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.
Keywords: Cardiotoxin Skeletal muscle injury Intramuscular injection
Background
Skeletal muscle regeneration is a series of highly regulated processes mainly executed by muscle stem cells (MuSCs) (Hawke and Garry, 2001), including necrosis of damaged myofibers, immune cell infiltration, clearance of the damaged myofibers, MuSC activation and proliferation, myofiber formation and maturation, and muscle reconstruction. MuSCs, also named satellite cells, were first discovered by Dr. Alexander Mauro in 1961 (Mauro 1961). In response to muscle injuries, the quiescent MuSCs are activated to enter the cell cycle, proliferate, and differentiate into new myofibers or fuse to the injured myofibers to repair muscle injury. After activation and proliferation, part of the MuSCs will return to quiescence and replenish the in vivo stem cell pool (Bischoff, 1975; Konigsberg et al., 1975; Fu et al., 2015 and 2022).
Induction of acute muscle injury is a critical step to study muscle regeneration in vivo. Several methods have been applied to induce consistent acute muscle injury, including freezing, barium chloride injection, glycerol injection, notexin injection, and cardiotoxin (CTX) injection (Mahdy et al., 2015; Hardy et al., 2016). Among the reagents to induce acute muscle injury, CTX is the most commonly used. CTX disrupts the membrane integrity of neuronal and skeletal muscle and cardiac muscle cells (Dufton and Hider 1988); it has been speculated to form pores on the cell membrane that result in membrane depolarization and influx of Ca2+, causing overwhelming muscle contraction, myofiber lysis, and acute muscle injury (Harvey et al., 1982). This protocol describes the step-by-step procedure to induce skeletal muscle injury via intramuscular injection of CTX. Following CTX injection, mice can be sacrificed at different time points for further analysis, according to variable experimental requirements.
Materials and Reagents
1 mL syringe (for intraperitoneal injection), needle 0.45 mm (Shanghai Kindly Group, 2012-3151233)
0.5 mL insulin syringe with 29 G ½ needle (for cardiotoxin injection) (EXELINT, catalog number: 26028)
0.2 µm filter membrane (Millipore, catalog number: SLGPR33RB)
Cardiotoxin (CTX) 5 mg, from Naja atra (BOYAO BioTechnology, catalog number: 9012-91-3)
Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number:10010023)
2,2,2-Tribromoethanol (TBE) (Sigma, catalog number: T48402)
Tert-amyl alcohol (Sigma, catalog number: 240486)
75% ethanol in liquid or in the form of pads (if using 95% or 99% ethanol, dilute with distilled water)
Equipment
Surgical scissors
Heating pad (Optional)
Procedure
Tools used for CTX injection are shown in Figure 1A. Video 1 shows how to inject CTX in mouse’s tibialis anterior (TA) muscle.
Video 1. Demonstration of cardiotoxin (CTX) injection in mouse’s tibialis anterior muscle
Figure 1. Illustration of cardiotoxin (CTX) injection in mouse’s TA muscle. A. Tools used to inject CTX; B–D. Key steps for CTX injection; E and F. Key steps for muscle dissection. TA: Tibialis anterior; EDL: Extensor digitorum longus.
Prepare 10 µM CTX by reconstituting CTX powder in PBS.
CTX PREPARATION: Calculate the volume of PBS that is needed for the reconstitution of CTX powder. CTX used for this protocol is from Naja atra and has a molecular weight of approximately 7,100 Da. Hence, the volume of PBS is 5 mg/(10 µM × 7,100 g/mol) = 70.4 mL. Filter the solution through a membrane with 0.2 μm or smaller pore size. Aliquot and store at -20 °C or lower temperature. Do not freeze and thaw frequently.
Anesthetize the mice by intraperitoneal injection of TBE (Avertin) or other equivalent anesthesia (Figure 1B). 200–250 mg of TBE per kilogram of mice body weight is recommended. For example, for a mouse weighing 30 g, 200–250 mg/kg × 30 g = 6–7.5 mg of TBE is needed. If using 2.5% working solution, injection volume will be 6–7.5 mg/2.5% (2.5% equals 2.5 g/100 mL) = 240–300 µL.
TBE PREPARATION: To prepare 100% TBE solution, mix 10 g of TBE with 10 mL tert-amyl alcohol. Make sure it is fully dissolved. To prepare working solution, dilute 100% stock to 1.25%–2.5%, v/v, in water or PBS. Stir vigorously until it is thoroughly dissolved. Filter the solution through a 0.2 μm filter membrane. Both the 100% stock and working solutions should be stored in the dark at 4 °C to prevent decomposition. The working solution is preferred to be prepared freshly from stock aliquots and is stable for at least a month.
To get a clear visualization of the TA muscle, either trim the hair or spray the hind limbs with 70% ethanol (Figure 1C and C’).
Determine the injection volume of CTX and aspirate a proper volume of the solution into the insulin syringe by pulling the plunger back slowly. To remove the air bubbles, hold the syringe with the needle upward and further gently pull the plunger down. Tap the side of the syringe to drive the air bubbles to top. Press the plunger until a few drops of liquid come out of the needle.
A different volume of CTX solution needs to be applied based on different purposes. For a muscular regeneration experiment, a total of 50–100 µL of CTX with 5–10 injection sites is recommended for each TA muscle. For induction of muscular damage in muscle stem cell transplantation experiments, a total of 10–20 µL of CTX with 1–2 injection spots is recommended.
Locate the center of the TA muscle, insert the needle at a 10°–30° angle, 2–3 mm deep, and perform the injection (Figure 1D). Approximately 10 µL of CTX solution is injected at each site. Leave the needle in the muscle for 2–3 s to prevent leakage of CTX. Slowly pull out the needle and continue the next injection until desired volume of injection is reached (Video 1).
After the procedure, place the cage on a heated pad until the mice are awake.
Perform muscle histology characterization using hematoxylin & eosin (H&E) staining. A detailed protocol of muscle dissection (Figure 1E and 1F) and H&E staining was published previously (Wang et al., 2017). H&E staining allows for the evaluation of the morphology of the regeneration process post CTX injury during skeletal muscle regeneration (Figure 2). Before injury, H&E–stained transverse sections show highly organized muscle structure with eosin-stained cytoplasm and hematoxylin-stained nuclei placed at the periphery of the fibers. At day 3 post CTX injury, the muscle structure is completely destroyed, and both necrotic myofibers and numerous mononucleated cells are observed. Along with the regeneration process, myofibers are newly regenerated by fusion and differentiation of myoblasts. Newly formed myofibers display centrally located nuclei and smaller size in the cross-sectional area compared to that of uninjured state (Day7 in Figure 2).
Figure 2. H&E staining of uninjured and injured tibialis anterior (TA) muscle induced by cardiotoxin (CTX). Scale bar, 50 µm.
Acknowledgments
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Science (XDA16020400 to PH), National Natural Science Foundation of China (32170804 to PH). This protocol is derived from the original research paper (Zhou et al., 2020).
Competing interests
All authors declare that there are no competing interests.
References
Bischoff, R. (1975). Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182(2): 215-235.
Dufton, M. J. and Hider, R. C. (1988). Structure and pharmacology of elapid cytotoxins. Pharmacol Ther 36(1): 1-40.
Fu, X., Wang, H. and Hu, P. (2015). Stem cell activation in skeletal muscle regeneration. Cell Mol Life Sci 72(9): 1663-1677.
Fu, X., Zhuang, C. L. and Hu, P. (2022). Regulation of muscle stem cell fate. Cell Regen 11(1): 40.
Hardy, D., Besnard, A., Latil, M., Jouvion, G., Briand, D., Thepenier, C., Pascal, Q., Guguin, A., Gayraud-Morel, B., Cavaillon, J. M., et al. (2016). Comparative Study of Injury Models for Studying Muscle Regeneration in Mice. PLoS One 11(1): e0147198.
Harvey, A. L., Marshall, R. J. and Karlsson, E. (1982). Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations. Toxicon 20(2): 379-396.
Hawke, T. J. and Garry, D. J. (2001). Myogenic satellite cells: physiology to molecular biology. J Appl Physiol (1985) 91(2): 534-551.
Konigsberg, U. R., Lipton, B. H. and Konigsberg, I. R. (1975). The regenerative response of single mature muscle fibers isolated in vitro. Dev Biol 45(2): 260-275.
Mahdy, M. A., Lei, H. Y., Wakamatsu, J., Hosaka, Y. Z. and Nishimura, T. (2015). Comparative study of muscle regeneration following cardiotoxin and glycerol injury. Ann Anat 202: 18-27.
Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9(2): 493-495.
Wang, C., Yue, F. and Kuang, S. (2017). Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining. Bio Protoc 7(10): e2279.
Zhou, S., Zhang, W., Cai, G., Ding, Y., Wei, C., Li, S., Yang, Y., Qin, J., Liu, D., Zhang, H., et al. (2020). Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration. Cell Res 30(12): 1063-1077.
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Murine Double Hit Model for Neonatal Cardiopulmonary Diseases: Bronchopulmonary Dysplasia (BPD) and Pulmonary Hypertension Associated with BPD
SG Steven P. Garrick
PB Philip J. Berger
MN Marcel F. Nold
CN Claudia A. Nold-Petry
Published: Vol 12, Iss 21, Nov 5, 2022
DOI: 10.21769/BioProtoc.4669 Views: 1140
Reviewed by: Vivien Jane Coulson-ThomasJaira Ferreira de VasconcellosAlberto Rissone
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Original Research Article:
The authors used this protocol in Science Translational Medicine Apr 2022
Abstract
Bronchopulmonary dysplasia (BPD) and pulmonary hypertension associated with BPD (BPD-PH) are of multifactorial origin and share common risk factors. Most murine models of BPD expose newborn pups to only one of these risk factors—more commonly postnatal hyperoxia—thereby mimicking the vital increased fraction of inspired oxygen (FiO2) that preterm infants in neonatal intensive care units often require. To improve representation of the multifactorial origins of BPD and BPD-PH, we established a double hit model, combining antenatal systemic inflammation followed by postnatal hyperoxia. On embryonic day 14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg of lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized and exposed to gas with either an FiO2 of 0.21 (room air) or 0.65 (hyperoxia 65%). In our BPD and BPD-PH double hit model, we can obtain multiple readouts from individual pups that include echocardiography, lung histology and immunohistochemistry, ex vivo X-ray micro computed tomography, and pulmonary and plasmatic immunity by RNA, protein, or flow cytometry.
Graphical abstract:
Figure 1. Murine double hit model of cardiopulmonary disease. On embryonic day (E)14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized to be exposed to gas with either a fraction of inspired oxygen (FiO2) of 0.21 (air; 21% O2) or 0.65 (hyperoxia; 65% O2) for a maximum of 28 days. According to the murine stage of lung development (Schittny, 2017), experimental endpoints include postnatal day (D)3, D5, D14, D28, and D60.
Keywords: Preclinical murine model Early life cardiopulmonary disease Bronchopulmonary dysplasia Pulmonary hypertension Clinical translation Neonatology Immunology Inflammation Antibody therapy
Background
Bronchopulmonary dysplasia (BPD) and pulmonary hypertension secondary to BPD (BPD-PH) are severe cardiopulmonary morbidities of premature infants that are underpinned by a surge in pulmonary inflammation. Pulmonary inflammation interrupts alveolar and vascular development, compromising gas exchange in infants with BPD (Thebaud et al., 2019). According to a systematic review and meta-analysis, approximately 17% of all infants with BPD develop BPD-PH, with a mortality rate of 14%–38% (Al-Ghanem et al., 2017). BPD-PH occurs due to a reduced cross-sectional area of the pulmonary vasculature that increases pulmonary vascular resistance and hence blood pressure (Parker and Abman, 2003; Khemani et al., 2007).
Pulmonary inflammation in the preterm infant can originate from many sources, with maternal inflammation being particularly prominent (Kemp, 2014). Environmental factors (stress, alcohol, smoking), autoimmune conditions (lupus erythematosus, inflammatory bowel disease, diabetes), and inflammatory pregnancy conditions (preeclampsia, chorioamnionitis) all contribute to a pro-inflammatory uterine environment in the mother. Infants with chorioamnionitis present with increased umbilical serum levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF (Dollner et al., 2002), which are important malefactors in BPD and BPD-PH (Sahni et al., 2020). Systemic maternal inflammation can be simulated in mice with an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), a bacterial endotoxin that binds to the toll-like receptor 4, a pattern recognition receptor of the innate immune system that promotes expression of pro-inflammatory cytokines, including IL-1β, TNF, and IL-6 (Palsson-McDermott and O'Neill, 2004). In pregnant rats, i.p. injection of 2.5 mg/kg LPS at gestational day 20 and 21 (full term = 22 days) resulted in prolonged pulmonary inflammation and delayed lung maturation in the offspring (Cao et al., 2009).
Unfortunately, early life inflammation is perpetuated postnatally by lifesaving interventions received in the neonatal intensive care unit. The most prevalent is the use of an increased fraction of inspired oxygen (FiO2) (hyperoxia) delivered to the preterm infant to counteract the reduced diffusing capacity of the immature lung. Although hyperoxia saves the life of the infant, it also comes with the unfortunate consequence of increased pulmonary inflammation via the production of reactive oxygen species (Torres-Cuevas et al., 2017).
Mimicking multifactorial diseases by including multiple pathophysiological drivers into in vivo models will lead to greater methodological rigor for faster translation of lifesaving therapies into clinics. However, to date, many murine BPD models only use one insult, namely hyperoxia. At the time we introduced our model, to the best of our knowledge, double hit strategies had been applied only twice to mimic BPD in newborn rodents (Choi et al., 2009; Velten et al., 2010), whereas there was none established to assess BPD-PH. Choi et al. (2009) first recognized the relevance of a double hit model, discovering that intra-amniotic LPS (0.5 or 1.0 mg) administration at day 20 of gestation (full term = 22.5) in neonatal rats amplified the lung injury induced by one week of exposure to hyperoxia (85% O2). One year later, Velten et al. (2010) injected 80 µg/kg LPS (i.p.) to pregnant mice at day 16 of gestation (full term = 19-21); upon birth, they placed pups in hyperoxia (85% O2) for 14 days followed by room air for another 14 days. These pups presented with a phenotype of arrested alveolarization, diffuse fibrosis, and impaired lung mechanics.
Expanding on these BPD models, we set out to establish a murine double hit model of early life cardiopulmonary disease representative for BPD and BPD-PH (Nold et al., 2013) (Figure 1). First, dams are injected (i.p.) with 150 µg/kg of lipopolysaccharide on day 14 of gestation, and upon birth pups are subsequently placed in hyperoxia (65% O2) or housed under room air control conditions. Pups subjected to this double hit develop alveolar and vascular changes representative of clinical features in BPD and BPD-PH. Our double hit model distinguishes itself from the earlier combined models by minimizing the FiO2 (65% compared to 85%) to better reflect clinical practice. Intuitively, we hypothesized that any improvement in BPD would most likely also improve BPD-PH, because of the inextricable link between alveolarization and angiogenesis.
Our team has established an experimental endpoint protocol so we could simultaneously investigate combinations of multiple individual readouts from every individual pup, including ex vivo (lung histology, immunohistochemistry, flow cytometry, x-ray micro computed tomography, precision cut lung slices, pulmonary/plasmatic immunity) and in vivo readouts (cine-angiography, echocardiography). Plethysmography, behavioral changes, and long-term neurodevelopmental outcomes could be considered as additional in vivo outcomes.
Our discovery that neonatal cardiopulmonary disease is underpinned by type 2 immunity (Lao et al., 2022) confirmed a pathogenetic similarity between BPD and asthma that has been speculated (de Kleer et al., 2016) but never fully proven. Proof of concept from our murine BPD/PH model (Figure 1) in a STAT6 deficient mouse, which lacks a full type 2 immune response, confirmed that the type 2 pathway is critical to the pathophysiology of BPD/PH and that blockade of type 2 pathways is highly effective in preventing the disease. This revelation might shift the current paradigm for treating BPD, which relies on gentle ventilation techniques and, ultimately, the use of corticosteroids as a rescue therapy. In the future, seeing BPD and BPD-PH as a type 2 disease will open up the use of well tolerated anti-type 2 drugs as a novel treatment strategy. In addition, our preclinical studies, in which we apply our model and other early life disease models, have shown that early and effectively blocking inflammation, specifically the potent pro-inflammatory interleukin (IL)-1, also holds great promise for preventing BPD and BPD-PH (Nold et al., 2013; Royce et al., 2016; Bui et al., 2019). Blocking IL-1 using its natural adversary IL-1 receptor antagonist (IL-1Ra, drug name anakinra) has an excellent safety and efficacy record, which has been established over two decades via anakinra’s clinical use in adults, children, and infants, as treatment for neonatal onset multi-system inflammatory disease (Sibley et al., 2012).
Materials and Reagents
Ultra-fine 31G insulin syringes 0.5 mL (Becton Dickinson, catalog number: 32281)
InsyteTM AutoguardTM winged, 24G × 0.75” (Becton Dickinson, catalog number: 381212)
MicrolanceTM hypodermic needle 30G × 0.5 (Becton Dickinson, catalog number: 304000)
10 µL gastight syringe, cemented needle, 26s gauge, 2 in., point style 2 (Hamilton, catalog number: 1701)
C57BL/6J mice
Lipopolysaccharide (LPS) from Escherichia coli O127:B8; purified by phenol extraction (Sigma, catalog number: L3129)
Sodium chloride intravenous infusion BP (water for injection) (Pfizer, catalog number: AUST R 49280)
Oxygen gas
Isoflurane (AbbVie, Forthane®, catalog number: AUST R 29656, store below 30 °C)
Paraformaldehyde (PFA) powder, 95% (Sigma-Aldrich, catalog number: 158127)
PBS (ThermoFisher Scientific, GibcoTM, catalog number: 14190250)
Cotton tip applicators (BSN Medical, catalog number: 7505-0)
Gauze
Liquid nitrogen (N2)
Wet ice
Polyester thread (sutures) (Birch, catalog number: 004601)
1.7 mL microtubules (Axygen, catalogue number: MCT-175-C)
Equipment
Custom plexiglass gas chamber [approximate dimensions: 40 × 125 × 30 (L × W × H) cm]
Bear cub BP2001 infant ventilator (Bear Medical Systems, Type #240)
Portable oxygen analyzer (Teledyne, model: AX300-I)
Oxygen sensor (Maxtec, model: R109P09)
Power lab/16SP (ADinstruments, model: ML795)
Heated respiratory humidifiers (Fisher and Paykel, model: MR730)
Autofill humification chamber (Fisher and Paykel, model: MR290)
Temperature pod (ADinstruments, model: ML312)
Pod expander (ADinstruments, model: ML305)
Compact scale, 510 g × 0.1 g (A&D Weighing, model: HT-500)
Gravity perfusion apparatus
Forceps, tip width 0.5 mm, length 10 cm (Fine Science Tools, catalog number: 11150-10)
Scissors (Precision Medical Specialties, model: E25-500)
Foam dissection tray
Liquid nitrogen container (Nalgene®, catalog number: 4150-2000)
-86 °C freezer 690 L (Froilabo, model: BMEVO69086G)
Software
Chart 5 Pro v5.5.1 (ADInstruments, www.ADInstruments.com)
Procedure
Murine double hit model of cardiopulmonary disease
Experimental conditions (Figure 2)
Figure 2. Setup of an experimental chamber. A custom plexiglass chamber is connected to a humidifier and ventilator. Humidified oxygen gas with a fraction of inspired oxygen (FiO2) of 0.65 is passed through the hyperoxia chamber at a flow rate of 10 L/min (air chamber has a FiO2 of 0.21). Power lab recording system records temperature, humidity, and oxygen levels in chambers. Humidity and temperature are kept constant at 50%–60% and 22 °C, respectively. Light is cycled in a 12 h day/night rhythm. Chamber setup included below the schematic for comparison.
Time mating
Use C57BL/6J mice.
Breeders are housed in enhanced specific pathogen free 2 conditions.
Preparation of female breeders:
Select 6–14-week-old females for consideration.
Younger females might not be sexually mature.
Older females mate less reliably.
One to two weeks prior to timed mating, house females together.
Group housed females to synchronize their estrous cycles. This phenomenon is known as the “Lee-Boot effect” (Van Der Lee and Boot, 1955).
Three days prior to timed mating, place material from a stud male cage into the female cages.
The exposure of group housed female mice to urine from a male mouse increases the chance of ovulation on the third night following exposure. This phenomenon is known as the “Whitten effect” (Whitten, 1956).
Preparation of male breeders:
Select 6–14-week-old males.
Preference proved stud males as sexual experience improves male reproductive performance (Swaney et al., 2012).
One to two weeks prior to timed mating, separate the stud males.
This allows the sperm count to increase and libido to recover from any previous mating.
Timed mating:
Place 1–2 females together with a stud male in a cage.
Check for viscous vaginal plugs in the mornings.
Components of the male ejaculate coagulate to form a hard plug that blocks the opening of the vagina and is an indicator of copulation. However, 37.1% of females that plug do not become pregnant (Heyne et al., 2015).
A positive plug is considered embryonic age (E)0.5 if pregnancy occurs.
After 48 h together, separate breeding pairs.
Early pregnancy detection
Monitor body weight gain of females.
Pregnant mice gain on average 3.49 g from E0 to E7–10 (Heyne et al., 2015).
Or
Palpate the pregnant dam’s abdomen with the thumb and forefinger at E12.
A positive pregnancy feels like a string of beads.
Antenatal inflammatory hit 1 at E14
Intraperitoneal injection of lipopolysaccharide (LPS) to the dam:
Determine the dam’s body weight.
Prepare LPS injection.
Reconstitute LPS powder in water for injection.
Final concentration injected is 150 µg/kg.
Final volume injected is 1 µL/g.
Inject LPS into the lower right quadrant of the dam at a 45° angle with an insulin syringe (Figure 3).
Adverse events associated with i.p. injection are related to errors in the placement of the injection. Ensure that the needle passes through the skin, muscle, and peritoneum of the abdomen without penetrating any abdominal organs.
Monitor dams closely to ensure welfare of animals.
If miscarriage occurs, humanely euthanize the dam and perform a postmortem.
Figure 3. Intraperitoneal injection site for LPS on day 14 of gestation in the pregnant dam
Postnatal inflammatory hit 2
Dams deliver pups naturally at E19–21 in the saccular stage of lung development (Backstrom et al., 2011).
The saccular stage is most susceptible to lung injury in mice (Backstrom et al., 2011), comparable to a premature human baby at 24–38 weeks gestation (Swaney et al., 2012).
Within 24 h of birth, randomly assign pups and dams to either:
Hyperoxia (FiO2 of 0.65)
Develop cardiopulmonary disease
Normal room air (FiO2 of 0.21)
Control animals
Set up foster pairs to avoid oxygen toxicity in the dam; pair one dam with pups in a hyperoxia cage with a counterpart dam and pups in an air cage.
In case of an odd number of deliveries:
Allocate the extra dam to an air group.
Or
Distribute the pups from the extra litter into the other litters under the experiment.
Choose experimental endpoints to determine different readouts.
Three days of 65% hyperoxia
Pulmonary immunity:
Postnatal exposure of pups to hyperoxia induces an immediate IL-1 and type 2 cytokine pulmonary immune response, which is most evident within the first three days of life. Accordingly, we see increased pulmonary IL-1β (20-fold), IL-1 receptor antagonist (2-fold) (Nold et al., 2013; Rudloff et al., 2017) type 2 cytokines (IL-4, IL-5, IL-13) (4- to 53-fold), and type 2 chemokines (CCL1, CCL17) (11- to 31-fold) (Lao et al., 2022).
Five days of 65% hyperoxia
Pulmonary immunity
Transcription factors that regulate the development of all innate lymphoid cells (ILCs) (Plzf and Id2) and ILC2s (Tcf7 and Bcl11b) are up to 23% lower after hyperoxia (Lao et al., 2022). There is a 2-fold increase in type 2 helper (TH2) cells after hyperoxia (Lao et al., 2022).
Pulmonary angiogenesis
Hyperoxia elevates vascular endothelial growth factor by 1.4-fold and endothelin-1 by 1.4-fold in murine lungs (Bui et al., 2019).
Fourteen days of 65% hyperoxia
Alveoli
Early changes in alveolar structure (Nold et al., 2013).
Twenty-eight days of 65% hyperoxia
Structural changes
1). Alveoli
Twenty-eight days marks the end of the alveolar stage of lung development in the mouse, similar to 2–3 years of age in humans (Backstrom et al., 2011). Hyperoxia reduces alveolar number (25%–44%), increases alveolar size (40%–230%), and reduces surface/volume ratio (18%–35%) (Figure 4) (Nold et al., 2013; Royce et al., 2016; Rudloff et al., 2017). These emphysematous changes in alveoli impair lung function by reducing the diffusing capacity of the lung (Pellegrino et al., 2005)
Figure 4. Emphysematous changes in lung morphology (H&E staining) at 28 days of life after antenatal LPS and postnatal exposure to 65% hyperoxia compared to air controls in C57BL/6J mice (sourced from Rudloff et al., 2017). Scale bars 100 µm, ×200 magnification.
2). Airways
Hyperoxia increases airway epithelial thickness (25%), subepithelial collagen deposition (200%), expression of α-smooth muscle actin (50%), and expression of the proliferation marker cyclin D1 mRNA (260%) (Royce et al., 2016). However, hyperoxia does not alter the airway goblet cell mucus production (Royce et al., 2016).
3). Pulmonary vascular bed
Hyperoxia reduces the number of small vessels (4–5 µm diameter) (84%) (Bui et al., 2019; Lao et al., 2022). No differences in the expression of α-smooth muscle actin and endothelin receptor type-A after hyperoxia (Bui et al., 2019).
4). Heart
Hyperoxia increases cardiac fibrosis (2.2-fold) and fibrotic/inflammatory markers galectin-3 and CCL2 (C-C motif) (Bui et al., 2019).
5). Brain
Hyperoxia does not cause apoptosis, cortical neuronal lamination, temporal cortical width (at hippocampus), white matter myelination, hemorrhage, loss of neurons, gliosis, inflammation, or any other significant morphologic abnormalities in C57Bl/6J mice (Rudloff et al., 2017).
Functional changes
1). In vivo
The inversely related surrogate marker time to peak velocity/right ventricular ejection time, as measured by echocardiography, increases in hyperoxic pups (0.27 compared to 0.32 in air controls) (Bui et al., 2019; Lao et al., 2022).
2). Ex vivo
Hyperoxia increases luminal contraction of airways to the muscarinic acetylcholine receptor agonist methacholine (10–3,000 nM) (89%) compared to air (44%) in precision cut lung slices (Royce et al., 2016). Pulmonary arterial contraction to the vasoconstrictors endothelin 1 and U46619 is not altered by hyperoxia in precision cut lung slices (Bui et al., 2019).
Cellular immunity of the lung
After hyperoxia, the overall number of immune cells (neutrophils, lymphocytes, monocytes/macrophages, natural killer cells, and dendritic cells) is approximately 3.1-fold lower. However, increased activation of macrophages and dendritic cells, as well as elevated IL-1β, is still evident in the lungs (Nold et al., 2013).
Sixty days (28 days hyperoxia, followed by 32 days room air)
Hyperoxia reduces vessel numbers at each branching generation in the murine lung (-18% in generation 2 and -21% in generation 3) (Bui et al., 2019).
Maintain comprehensive experimental records
Record oxygen application, temperature, and humidity with power lab instrument and Chart 5 Pro software.
Record assessments of animal welfare.
Fostering of pups (Figure 5 and Video 1)
Adult mice are more susceptible to hyperoxia-induced oxygen toxicity than neonatal mice (Frank et al., 1978). Therefore, dams need to be rotated between room air and hyperoxia pups in a three-day cycle to limit hyperoxia-induced toxicity. It is important to conduct this procedure promptly, so that pups are in the experimental conditions for as long as possible. Top up food and water during this step to minimize unnecessary opening of the hyperoxia chamber.
Remove cages from air and hyperoxia chambers.
Take one pair of one hyperoxia and one air cage at a time.
Swap the pups between the paired dams
Air pups will be moved into the dam's cage that was previously in hyperoxia.
Hyperoxia pups will be moved into the dam's cage that was previously in air.
Cover the pups in bedding material from the new cage
This procedure masks the scent of the other dam.
This reduces the likelihood of rejection from the foster dam.
Repeat for all paired cages
Return cages to air and hyperoxia chambers.
Pups are in the same conditions.
Dams previously in hyperoxia chamber are now housed in the air chamber; however, the pups stay under their allocated experimental condition.
Dams previously in air chamber are now housed in the hyperoxia chamber; however, the pups stay under their allocated experimental condition.
Figure 5. Fostering of pups. Hyperoxia animals (red), air animals (blue). Air dam rotates into the hyperoxic chamber every three days. Hyperoxia dam rotates into the air chamber every three days. Pups are cross fostered every three days between dams and, as a result, they are continuously housed under the same experimental condition from day 1.
Video 1. Fostering of pups
Subcutaneous injections of neonatal pups (Figure 6)
Gently scruff the pups.
Lift the skin above the neck.
Insert the syringe at a 45° angle.
Inject pups slowly.
Pull out the needle slowly to prevent leakage.
Figure 6. Subcutaneous injection of 11-day-old C57BL/6J pup
Sample preparation for lung histology and pulmonary immunity of a 28-day-old pup
Anesthetize the pup with isoflurane per open-drop method (Risling et al., 2012).
Euthanize the pup
Cervical dislocation
Place the thumb and forefinger in between the head and neck of the animal.
Note: The spinal column should be palpable.
Pinch the thumb and forefinger together and push down simultaneously until the cervical spine is cleanly separated.
1). Ensure that you do not apply too much pressure as the trachea needs to be intact for later inflation of the lung.
2). Pinch the foot with the forceps to ensure mouse is dead before dissecting.
Dissection (Figure 7, Figure 8, and Video 2)
Pin the mouse on a foam dissection board on its back with its four limbs spread out.
Hook the front teeth of the mouse with a suture; then, pull tight and pin to foam board.
This procedure helps to straighten the trachea to allow better inflation.
Dissect the mouse
Cut through the ventral surface of the mouse from the sternum up to the chin.
Open the chest cavity such that the entire lung is visible.
Expose the trachea.
1). The trachea is enclosed by the thyroid.
2). Grasp the thyroid with forceps and pull apart to remove.
3). Wipe away any excess blood with gauze soaked in PBS or use a cotton tip applicator.
4). Delicately cut away tissue around trachea so that the forceps can reach underneath.
5). Place two strings of suture underneath the trachea.
Samples for pulmonary immunity
Ensure that the left lobe is clearly visible.
Loop a piece of suture around the left lobe of the lung by holding the suture with your forceps and lift the lobe with a cotton tip applicator.
Organ will stick to the cotton without being ripped or damaged. Lungs exposed to hyperoxia are fragile.
Now loop the suture around the left bronchus and tie the left lobe off.
Ensure knots are tight so that no leakage occurs during the right lung inflation.
Cut off the left tied lobe, cut in two, and place in separate labeled 1.7 mL microtubules. Immediately snap freeze in liquid N2 (following all safety protocols of your institution) for later mRNA and protein analysis.
Store at -80 °C until ready for processing.
Inflation of the right lung lobes
Insert a 24G cannula into the trachea.
Tie the cannula in place with one of the sutures threaded earlier underneath the trachea.
Attach the cannula to a gravity perfusion apparatus.
Infuse the right lung lobes with 4% PFA (pH 7.4) under constant 20 cm H2O of hydrostatic pressure.
1). Reconstitute PFA powder with PBS.
2). Full inflation is determined by the tip of the post-caval lobe turning pointy.
Tie off the trachea with the second suture in front of the cannula to prevent backflow of PFA.
Place pup on ice for 10 min.
Samples for histology
Remove right lung lobes from thorax and place in tube with 4% PFA.
Keep in 4% PFA for a minimum of 6 h.
Process for paraffin embedding and sectioning.
Figure 7. Sample preparation of a 28-day-old mouse. Dissect the mouse and open the chest cavity to expose lungs and trachea (A). Tie off the left bronchus and remove the left lung for later analysis of pulmonary immunity (B). Inflate the right lung lobes with 4% paraformaldehyde for histology (C).
Figure 8. Pulmonary anatomy of a mouse. The left lobe (E) and the right lobes [superior lobe (A), middle lobe (B), inferior lobe (C), and post-caval lobe (D)]. At day 28, the left lobe is removed for analysis of pulmonary immunity (top third for RNA, bottom two thirds for protein). The right lobes are subsequently inflated with 4% paraformaldehyde and sections for histology are made from the inferior lobe.
Video 2. Sample preparation for lung histology and pulmonary immunity of a 28-day-old mouse
Acknowledgments
The authors acknowledge the original research papers that have applied this protocol (Nold et al., 2013; Royce et al., 2016; Rudloff et al., 2017; Bui et al., 2019; Lao et al., 2022). The authors thank Ina Rudloff, Christine Bui, and Elizabeth Skuza for their considerable work with the double hit model. Investigators were supported by a co-funded Monash graduate scholarship (S.P.G.); a National Heart Foundation of Australia Future Leader Fellowship (CF14/3517), the Fielding Fellowship 2019, and NHMRC Investigator Grant 1173584 (to C.A.N.-P.); and the Hudson Institute’s Star Recruitment Fellowship, Monash University’s Larkins Fellowship, and the Fielding Foundation Fellowship 2017 (to M.F.N.). The study was furthermore supported by grants from the Australian Synchrotron (EU9230, M8925, and M9422 to C.A.N.-P., and M.F.N.), from CSL Ltd. (to C.A.N.-P. and M.F.N.), from the Jack Brockhoff Foundation (JBF3105 to C.A.N.-P., and M.F.N.), from the Rebecca L. Cooper Foundation and Perpetual’s 2016 Impact Philanthropy Program (IPAP201600780 to C.A.N.-P.), and by the Victorian Government’s Operational Infrastructure Support Program.
Competing interests
The authors declare no competing interests.
Ethics
The double hit model protocol was approved by the animal review board MMCA of Monash University.
References
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Backstrom, E., Hogmalm, A., Lappalainen, U. and Bry, K. (2011). Developmental stage is a major determinant of lung injury in a murine model of bronchopulmonary dysplasia. Pediatr Res 69(4): 312-318.
Bui, C. B., Kolodziej, M., Lamanna, E., Elgass, K., Sehgal, A., Rudloff, I., Schwenke, D. O., Tsuchimochi, H., Kroon, M., Cho, S. X., et al. (2019). Interleukin-1 Receptor Antagonist Protects Newborn Mice Against Pulmonary Hypertension. Front Immunol 10: 1480.
Cao, L., Wang, J., Tseu, I., Luo, D. and Post, M. (2009). Maternal exposure to endotoxin delays alveolarization during postnatal rat lung development. Am J Physiol Lung Cell Mol Physiol 296(5): L726-737.
Choi, C. W., Kim, B. I., Hong, J. S., Kim, E. K., Kim, H. S. and Choi, J. H. (2009). Bronchopulmonary dysplasia in a rat model induced by intra-amniotic inflammation and postnatal hyperoxia: morphometric aspects. Pediatr Res 65(3): 323-327.
de Kleer, I. M., Kool, M., de Bruijn, M. J., Willart, M., van Moorleghem, J., Schuijs, M. J., Plantinga, M., Beyaert, R., Hams, E., Fallon, P. G., et al. (2016). Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung. Immunity 45(6): 1285-1298.
Dollner, H., Vatten, L., Halgunset, J., Rahimipoor, S. and Austgulen, R. (2002). Histologic chorioamnionitis and umbilical serum levels of pro-inflammatory cytokines and cytokine inhibitors. BJOG 109(5): 534-539.
Frank, L., Bucher, J. R. and Roberts, R. J. (1978). Oxygen toxicity in neonatal and adult animals of various species. J Appl Physiol Respir Environ Exerc Physiol 45(5): 699-704.
Heyne, G. W., Plisch, E. H., Melberg, C. G., Sandgren, E. P., Peter, J. A. and Lipinski, R. J. (2015). A Simple and Reliable Method for Early Pregnancy Detection in Inbred Mice. J Am Assoc Lab Anim Sci 54(4): 368-371.
Kemp, M. W. (2014). Preterm birth, intrauterine infection, and fetal inflammation. Front Immunol 5: 574.
Khemani, E., McElhinney, D. B., Rhein, L., Andrade, O., Lacro, R. V., Thomas, K. C. and Mullen, M. P. (2007). Pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era. Pediatrics 120(6): 1260-1269.
Lao, J. C., Bui, C. B., Pang, M. A., Cho, S. X., Rudloff, I., Elgass, K., Schroder, J., Maksimenko, A., Mangan, N. E., Starkey, M. R., et al. (2022). Type 2 immune polarization is associated with cardiopulmonary disease in preterm infants. Sci Transl Med 14(639): eaaz8454.
Nold, M. F., Mangan, N. E., Rudloff, I., Cho, S. X., Shariatian, N., Samarasinghe, T. D., Skuza, E. M., Pedersen, J., Veldman, A., Berger, P. J., et al. (2013). Interleukin-1 receptor antagonist prevents murine bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia. Proc Natl Acad Sci U S A 110(35): 14384-14389.
Palsson-McDermott, E. M. and O'Neill, L. A. (2004). Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 113(2): 153-162.
Parker, T. A. and Abman, S. H. (2003). The pulmonary circulation in bronchopulmonary dysplasia. Semin Neonatol 8(1): 51-61.
Pellegrino, R., Viegi, G., Brusasco, V., Crapo, R. O., Burgos, F., Casaburi, R., Coates, A., van der Grinten, C. P., Gustafsson, P., Hankinson, J., et al. (2005). Interpretative strategies for lung function tests. Eur Respir J 26(5): 948-968.
Risling, T. E., Caulkett, N. A. and Florence, D. (2012). Open-drop anesthesia for small laboratory animals. Can Vet J 53(3): 299-302.
Royce, S. G., Nold, M. F., Bui, C., Donovan, C., Lam, M., Lamanna, E., Rudloff, I., Bourke, J. E. and Nold-Petry, C. A. (2016). Airway Remodeling and Hyperreactivity in a Model of Bronchopulmonary Dysplasia and Their Modulation by IL-1 Receptor Antagonist. Am J Respir Cell Mol Biol 55(6): 858-868.
Rudloff, I., Cho, S. X., Bui, C. B., McLean, C., Veldman, A., Berger, P. J., Nold, M. F. and Nold-Petry, C. A. (2017). Refining anti-inflammatory therapy strategies for bronchopulmonary dysplasia. J Cell Mol Med 21(6): 1128-1138.
Sahni, M., Yeboah, B., Das, P., Shah, D., Ponnalagu, D., Singh, H., Nelin, L. D. and Bhandari, V. (2020). Novel biomarkers of bronchopulmonary dysplasia and bronchopulmonary dysplasia-associated pulmonary hypertension. J Perinatol 40(11): 1634-1643.
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Van Der Lee, S. and Boot, L. M. (1955). Spontaneous pseudopregnancy in mice. Acta Physiol Pharmacol Neerl 4(3): 442-444.
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4,670 | https://bio-protocol.org/en/bpdetail?id=4670&type=0 | # Bio-Protocol Content
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Peer-reviewed
A Novel Non-invasive Qualitative Assay Using Urinary Fluorescence Imaging to Assess Kidney Disease
TT Tomoaki Takata *
TI Takuji Iyama *
KY Kentaro Yamada
HI Hajime Isomoto
(*contributed equally to this work)
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4670 Views: 290
Reviewed by: Komuraiah Myakala Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in International Journal of Molecular Sciences Jul 2022
Abstract
In patients with chronic kidney disease, it is necessary to identify the etiology of glomerular disease. Renal biopsy is the gold standard for assessing the underlying pathology; however, it has the risk of potential complications. We have established a urinary fluorescence imaging technique to assess enzymatic activity using an activatable fluorescent probe targeting two enzymes: gamma-glutamyl transpeptidase and dipeptidyl-peptidase. The urinary fluorescence images can be easily obtained by adding an optical filter to the microscope with short incubation of the fluorescent probes. Urinary fluorescence imaging could help to assess underlying etiologies of kidney diseases and is a potential non-invasive qualitative assessment technique for kidney diseases in patients with diabetes.
Key features
• Non-invasive assessment of kidney disease.
• Urinary fluorescent imaging with enzyme-activatable fluorescent probes.
• Enables differentiation of diabetic kidney disease and glomerulonephritis.
Keywords: Activatable fluorescent probe Diabetic nephropathy Dipeptidyl-peptidase Gamma-glutamyl transpeptidase Glomerulonephritis Nephrosclerosis Photodynamic diagnosis
Background
Chronic kidney disease (CKD) is the major public health burden worldwide. Three major etiologies of CKD leading to end-stage renal disease include diabetic nephropathy, glomerulonephritis, and nephrosclerosis. Although these etiologies can be identified by clinical presentation and laboratory tests in typical cases, some cases require histological assessment to clearly differentiate these diseases, especially in patients with diabetes mellitus. Renal biopsy is the most important procedure to assess the underlying pathologies in CKD. However, it has potential complications such as bleeding, blood transfusion, and loss of renal function. Therefore, a convenient and noninvasive method is warranted for the assessment of kidney disease. Several urinary biomarkers have been suggested to be useful in assessing kidney diseases (Joachim et al., 2021; Takata et al., 2023); however, no biomarkers have been proved to be directly linked to the diagnosis of diabetic kidney disease. Recent advancements in the field of light-emitting substances have opened new approaches for the diagnosis of kidney disease (Huang et al., 2017; Yan et al., 2019). An enzyme-activatable fluorescent probe was recently developed for photodynamic diagnosis (Urano et al., 2011). This technique is based on the fluorescent emittance from hydroxymethyl rhodamine green (HMRG) upon enzymatic reaction with target aminopeptidases, such as gamma-glutamyl transpeptidase (GGTP) and dipeptidyl-peptidase (DPP). While this technique was originally developed for detecting cancer, it can be applied to renal biopsy specimens (Iyama et al., 2020; Takata et al., 2022). Our recent work has focused on investigating the feasibility of the activatable fluorescent probe for noninvasive assessment of the underlying kidney disease (Yamada et al., 2022). Urinary fluorescence was significantly stronger in patients with diabetic nephropathy than those with glomerulonephritis upon DPP-HMRG incubation. Besides, it was stronger in patients with nephrosclerosis compared to those with glomerulonephritis after GGTP-HMRG. Underlying kidney disease can be differentiated by urinary fluorescence imaging in combination with GGTP-HMRG and DPP-HMRG. This study has some limitations regarding the applicability to clinical settings: the results require further confirmation with larger studies, and a quantitative threshold needs to be determined. However, since urinary fluorescence imaging can be easily and quickly obtained, this technique would help to detect kidney diseases, particularly during health checks. We herein present a protocol for the induction of fluorescence by activatable fluorescent probe with some modifications for better clarity of the images.
Materials and Reagents
Pipettes (M&S Instruments, catalog numbers: F144059M, F144058, and F144055M) and pipette tips (VIOLAMO, catalog numbers: V-1000, V-200, and V-10)
1.5 mL Eppendorf centrifuge tubes
Phosphate buffer saline (PBS) (FUJIFILM, catalog number: 166-23555)
Dimethyl sulfoxide (FUJIFILM, catalog number: 041-29351)
EP-HMRG (GORYO Chemical, catalog number: GC811), which targets DPP. Dissolve in dimethyl sulfoxide at 1 mM and store at -20 °C before use (not necessary to filter)
ProteoGREENTM-gGlu (GORYO Chemical, catalog number: GC801), which targets GGTP. Dissolve in dimethyl sulfoxide at 1 mM and store at -20 °C before use (not necessary to filter)
Aqua-auto Kainos CRE-III plus (KAINOS, catalog number: TKA7500), for creatinine measurement
Equipment
Stereomicroscope (BioTools, catalog number: BS-3048BT)
Fluorescent unit (BioTools, catalog number: BT-ExSM)
Band pass filter (FUJIFILM, catalog number: BPB-45). This filter should be set at the light source of the fluorescent unit
Sharp cut filter (FUJIFILM, catalog number: SC-52). This filter should be set at the lens side of the fluorescent unit
Labnet C-1200 mini centrifuge (Marshall Scientific, product code: C-1200)
Procedure
Preparation of the fluorescent probe
Thaw EP-HMRG/ProteoGREENTM-gGlu stock solution at room temperature (protect from light).
Dilute EP-HMRG/ProteoGREENTM-gGlu stock solution into PBS to prepare working solution at a final concentration of 10 μM.
Scheme representing how the probe works is showed in Figure 1.
Figure 1. Scheme of the reaction of fluorescent probes. Figure adapted from Yamada et al. (2022).
Fluorescence imaging of the urine
Collect a urine sample from human subjects (1 mL is enough).
Briefly (a few seconds) spin down to remove sediment and transfer supernatant to another tube for testing.
Dilute urine sample in PBS to a creatinine concentration of 1 μg/μL.
Mix 10 μL of diluted urine sample and 10 μL of EP-HMRG/ProteoGREENTM-gGlu stock solution by pipetting to prepare fluorescent working solution. The final concentration of creatinine should be 0.5 μg/μL and fluorescent probes should be 5 μM (20 μL is enough for fluorescent imaging).
Incubate for one minute under dark conditions at room temperature.
Illuminate and observe the fluorescence. An excitation wavelength of 450 nm can pass through the band pass filter; then, the fluorescent working solution is excited and emits fluorescence. The sharp cut filter eliminates redundant wavelengths except for fluorescence (~520 nm).
Capture images of the samples at 2.0× magnification.
Scheme of the fluorescent imaging is showed in Figure 2.
Figure 2. Scheme of the fluorescent unit for the imaging
Notes
Prepare fluorescent working solution each time just before usage.
Acknowledgments
Protocol was based on “Fluorescence imaging using enzyme-activatable probes for detecting diabetic kidney disease and glomerular diseases”, in International Journal of Molecular Sciences (Yamada et al., 2022).
Competing interests
This research was founded by JSPS KAKENHI, grant number JP22K16242 (T.I.).
Ethics
This study was conducted in accordance with the Declaration of Helsinki and approved by the ethical committee of Tottori University Hospital (approval number: 18A135 and 20A001). Informed consent was obtained from all subjects.
References
Joachim, H. and Shilpak, M. G. (2021). The promise of tubule biomarkers in kidney disease: A review. Am J Kidney Dis 78(5): 719-727.
Takata, T. and Isomoto, H. (2023). The versatile role of uromodulin in renal homeostasis and its relevance in chronic kidney disease. Intern Med doi: 10.2169/internalmedicine.1342-22.
Yan, F., Tian, X., Luan, Z., Feng, L., Ma, X. and James, T. D. (2019). NAG-targeting fluorescence based probe for precision diagnosis of kidney injury. Chem Commun (Camb) 55(13): 1955-1958.
Huang, J., and Gretz, N. (2017). Light-emitting agents for noninvasive assessment of kidney function. ChemistryOpen 6(4); 456-471.
Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L. and Kobayashi, H. (2011). Rapid cancer detection by topically spraying a gamma-glutamyltranspeptidase-activated fluorescent probe. Sci Transl Med 3(110): 110ra119.
Iyama, T., Takata, T., Yamada, K., Mae, Y., Taniguchi, S., Ida, A., Ogawa, M., Yamamoto, M., Hamada, S., Fukuda, S., Kanda, T., Sugihara, T., Isomoto, H. and Urano, Y. (2020). A novel method for assessing the renal biopsy specimens using an activatable fluorescent probe. Sci Rep 10(1): 12094.
Takata, T., Isomoto, H., Iyama, T. and Yamada, K. (2022). A novel imaging technique for the on-site assessment of renal biopsy specimens. Bio Protoc 12: e4517.
Yamada, K., Takata, T., Iyama, T., Hamada, S., Mae, Y., Sugihara, T. and Isomoto, H. (2022). Fluorescence imaging using enzyme-activatable probes for detecting diabetic kidney disease and glomerular diseases. Int J Mol Sci 23: 8150.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Medicine
Medicine
Biological Sciences > Biological techniques
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4,671 | https://bio-protocol.org/en/bpdetail?id=4671&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Novel Antibody-independent Method to Measure Complement Deposition on Bacteria
TW Toska Wonfor
SL Shuxian Li
ML Maisem Laabei
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4671 Views: 460
Reviewed by: Alessandro Didonna Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Scientific Reports Sep 2022
Abstract
During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such as Staphylococcus aureus to overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such as S. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and both Lactococcus lactis and S. aureus.
Keywords: Complement deposition Staphylococcus aureus Bacterial pathogenicity Immune evasion Flow cytometry
Background
Staphylococcus aureus has become a major global burden due to widespread infection within hospitals and communities (Murray et al., 2022). Due to the prevalence of antibiotic resistance, chronic-infections, and surgical complications, the demand to understand this pathogen and its relationship with the host has become critical (Tong et al., 2015). For a successful infection, pathogens must combat the first line of innate defence, the complement system. Complement is quick to identify pathogens, label them for efficient phagocytosis, and raise the alarm to other immune effectors of both the innate and adaptive systems (Noris and Remuzzi, 2013). For successful infection, pathogens must disable this pathway. S. aureus has a surprisingly large arsenal of complement-disabling proteins; however, due to restrictions in phenotypic assays, the details of virulence factors involved are not fully understood (Thammavongsa et al., 2015). The common method for observing complement activity/deposition utilises labelled antibodies that are detected by methods such as flow cytometry. In the case of S. aureus, this method is not possible due to the expression of two immunoglobulin binding proteins, Protein A (Spa) and Sbi, which bind indiscriminately to the Fc region of antibodies (Yang et al., 2018; Cruz et al., 2021). Previous attempts to bypass this issue used mutants unable to express IgG binding proteins (∆spa∆sbi); however, both proteins are important virulence factors in the defence against complement activation and are known to be expressed in >95% of clinical isolates.
In our study, we designed a novel probe utilising the C3 binding domain of Sbi (domain IV, see Figure 1) and biotinylated it to allow recognition by fluorophore-labelled streptavidin (Clark et al., 2011; Wonfor et al., 2022). We show that it is able to bypass Spa/Sbi interference, and binding is specific to C3 deposition. This probe opens the door to analysing the immune evasion capacity of large collections of clinical isolates, allowing for a greater understanding of complement evasion at a population level. Staphylococci are not the only pathogens to bind IgG: Streptococcus has protein G, and Peptostreptococcus has protein L; therefore, this method may also enlighten the role of complement in other pathogen invasions (Sidorin and Solov'eva, 2011). Finally, Sbi-IV probe has the advantage of being very small in size. As a 11 kDa protein, in comparison to 150 kDa IgG antibody, the probe may have advantages outside immunoglobulin binding pathogens, and we envisage a role in in vivo diagnostics, such as detecting tissue inflammation.
Figure 1. Scheme showing domains within Sbi protein. Residue numbers are shown below. Domains I and II bind IgG. Domains III and IV bind C3. Domain IV consists of residues 196–253. Additional residues added ensure stability and correct folding (Smith et al., 2012).
Materials and Reagents
General lab reagents
Beakers, 250 mL
Eppendorf: 1.5 mL, 2 mL
Falcons: 15 mL, 50 mL
Flasks 2 L
Flea (magnetic stirring bar)
Foil
Petri dish/staining box
Syringe: 5 mL, 10 mL, 50 mL
Syringe needles
Centrifuge bottles compatible with specific floor centrifuge rotor (1 L, 50 mL)
Filter 0.45 μm (Thermo Fisher, Millipore, catalog number: SLHA033SS)
His-Trap column 1 mL (Fisher Scientific, Cytiva, catalog number: 10431065)
Microcuvettes 1.6 mL (Fisherbrand, catalog number: FB55147)
Size Exclusion Column HiLoad 16/600 Superdex 200 prep grade, 120 mL (Fisher Scientific, Cytivia, catalog number: 45002490)
Round bottom 96-well plate (Corning, catalog number: 3788)
Transfer pipette 5 mL sterile (Sigma, catalog number: HS206371C)
TurboBlot transfer pack (nitrocellulose membrane 0.2 μm) (Bio-Rad, catalog number: 1704158)
V-bottomed 96-well plate (Thermo Fisher, Nunc, catalog number: 249570)
BamHI-HF (NEB, catalog number: R3136S)
BL21 DE3 competent cells (NEB, catalog number: C2527I). Alternatively, make your own competent cells using CaCl2
GeneJET Miniprep kit (Thermo Fisher, catalog number: K0502)
HindIII-HF (NEB, catalog number: R3104S)
NovaBlue competent cells (Sigma, catalog number: 70181-3)
Phusion High-Fidelity PCR master mix (Thermo Fisher, catalog number: F351S)
T4 DNA ligase (NEB, catalog number: M0202S)
Wizard SV Gel and PCR clean up kit (Promega, catalog number: A9281)
Ampicillin (Fisher Scientific, catalog number: BP176025). Dilute powder in ddH2O to a final concentration of 100 mg/mL stock (store dilution at -20 °C)
Coomassie Brilliant Blue R-250 destain (Bio-Rad, catalog number: 1610438)
Coomassie Brilliant Blue R-250 stain (Bio-Rad, catalog number: 1610436)
Ethanol absolute (VWR, catalog number: 20821.330). Dilute to 20% with water
IPTG (Sigma, catalog number: 16758). Dilute powder in ddH2O to a final concentration of 0.5 M (store dilution at -20 °C)
Imidazole (Sigma, catalog number: I2399)
LB broth (Sigma, catalog number: L3022)
Laemmli sample buffer 4× (Bio-Rad, catalog number: 1610747)
mPAGE SDS running buffer powder (Sigma, catalog number: 20347927)
NaCl (Sigma, catalog number: S7653)
PageRuler Plus Prestained protein ladder (Thermo Fisher, catalog number: 26619)
Protease Inhibitor Cocktail Set VII (Sigma, Millipore Corp, catalog number: 539138)
SDS-PAGE precast gel 12% (Bio-Rad, catalog number: 4561043)
Trizma HCl (Sigma, catalog number: T3253)
Vivaspin 20 (5 kDa MWCO) (Sigma, catalog number: Z614580)
Vivaspin 500 (5 kDa MWCO) (Sigma, catalog number: Z614009)
BCA Protein Assay kit (Thermo Fisher, Pierce, catalog number: 23225)
BD Vacutainer® Clot Activator Tube (BD, catalog number: 367895)
ECL detection reagent (Cytivia, Amersham, catalog number: RPN2235)
Mini-PROTEAN TGX precast gel 4%–20% 10 wells (Bio-Rad, catalog number: 4561094)
Phosphate buffered saline (PBS) tablets (Thermo Fisher, Oxoid, catalog number: BR0014G)
Expression plasmid pQE30-Sbi-IV, available upon request
EZ-LinkTM Sulfo-NHS-Biotinylation kit (Thermo Fisher, catalog number: 21425)
Skim milk powder (BD, Difco catalog number: 232100)
TBST powder (Sigma, catalog number: T9039)
Ultra-streptavidin-HRP (Thermo Scientific, catalog number: N504)
Barbituric acid (Sigma, catalog number: 185698)
Bovine serum albumin (BSA) (Sigma, Roche, catalog number: 03117332001)
Calcium chloride (CaCl2) (Sigma, catalog number: 223506)
Cell trace Far Red (Thermo Fisher, Invitrogen, catalog number: C34564)
FC loading tubes (5 mL polystyrene round-bottom test tube) (Fisher Scientific, Falcon, catalog number: 352052)
Fetal calf serum (FCS) (Thermo Fisher, Gibco, catalog number: A5256701)
Gelatin (from porcine skin) (Sigma, catalog number: G1890)
Compstatin (AMY-101) (MedChemExpress, catalog number HY-P1717)
Glucose (Sigma, catalog number: G8270)
Glass universal (for growing S. aureus) (Fisher Scientific, catalog number: 14823562)
M17 broth (Sigma, catalog number: 56156)
Magnesium chloride (MgCl2) (Sigma, catalog number: M8266)
Na-barbital (sodium 5,5-diethylbarbiturate) (Sigma, catalog number: B0500)
Streptavdidin-488 (Thermo Fisher, Invitrogen, catalog number: S32354)
Tryptic soy broth (Sigma, catalog number: T8907)
His A buffer (see Recipes)
His B buffer (see Recipes)
SEC buffer (see Recipes)
Milk block (see Recipes)
M17-G broth (see Recipes)
10% FCS/PBS (see Recipes)
BSA/PBS (see Recipes)
Veronal buffer stock (VBS) (25 mM) (see Recipes)
GVB++ buffer (see Recipes)
Equipment
Forceps (Fisher Scientific, catalog number: 15281209)
AKTA purifier 10 (Amersham Pharmacia Biotech)
Belly dancer/rocker (Fisherbrand, 3D Platform Rotator)
Bench centrifuge with compatible rotor for Eppendorfs capable of reaching speeds of 16,000× g
Centrifuge capable of speeds of at least 60,000× g and compatible with rotors for 50 mL and 1 L bottles (e.g., Beckman Avanti)
Centrifuge with buckets compatible for 96-well plates capable of reaching 3,400× g (Eppendorf, model: 5810 R)
Electrophoresis Power Supply (Cleaver Scientific, model: PowerPRO 300)
FACS CANTO Flow Cytometer (BD)
Falcon centrifuge capable of reaching 3,400× g (Eppendorf, model: 5810 R)
Freezer (-80 °C) for storage of serum and Sbi-IV protein
Gel imaging system (Azure Biosystem 400) for viewing chemiluminescence
Heat block reaching temperatures of 95 and 56 °C (Fisherbrand, Isotemp)
Magnetic stirrer (Stuart, model: US152)
NanoDrop/spectrophotometer (absorbance at 600 nm) (DeNovix, model: DS-11)
Protein electrophoresis tank (Bio-Rad, model: Mini-PROTEAN Tetra Cell)
Shaking incubator (capable of holding 2 L flasks and shaking at 200 rpm) set to 37 °C (e.g., Stuart SI500)
Sonicator (Soniprep 150 plus)
Spectrophotometer (absorbance at 562 nm) (Tecan, Sunrise)
Static incubator, set to 37 °C (Fisher Scientific, HERATherm, catalog number: 10744262)
Thermocycler (Applied Biosystem, SimpliAmp)
Trans-Blot Turbo Transfer System (Bio-Rad)
Western Blot Roller (Thermo Fisher, catalog number: 84747)
Software
BD FACSDiva (BD) for flow cytometer
FlowJoTM v10 software (BD Life Sciences) (www.flowjo.com)
GraphPad Prism 8 (Dotmatics) (www.graphpad.com)
Unicorn 5.32 (Cytiva) for AKTA
Procedure
Cloning, protein expression, and purification of Sbi-IV
Using the Phusion High-Fidelity PCR master mix kit, amplify the IV domain of Sbi protein (amino acids V198-A266) with S. aureus Mu50 genomic DNA and primers: Sbi-IV forward (CGGGATCCGTTTCAATTGAAAAAGCAATC) and Sbi-IV reverse (CCCAAGCTTTCATTACGCCACTTTCTTTTCAGC), using 60 °C annealing temperature and 15 s extension time.
Thermocycling conditions following manufacturer’s protocol: initial denaturation: 98 °C, 30 s; 30 cycles of (denaturation: 98 °C, 5 s; annealing: 60 °C, 30 s; extension 72 °C, 15 s); final extension: 72 °C, 5 min; 4 °C ∞.
Notes:
Our pQE30-Sbi-IV plasmid is available upon request.
pQE30 contains a His-tag. If using an alternative expression plasmid, please include a His-tag to the cloned region during PCR.
Digest 1 μg of both the PCR product and pQE30 plasmid with restriction enzyme BamHI-HF and HindIII-HF. Incubate for 4 h at 37 °C using a thermocycler.
Note: We recommend performing the plasmid digest in quadruplicate to improve yield after gel extraction.
Gel-extract the digested plasmid and clean up digested PCR product following the Wizard SV Gel and PCR clean up kit protocol. Quantify the product using a NanoDrop.
Ligate the plasmid and PCR products using a molar ratio of 3:1 with NEB T4 DNA ligase enzyme. Incubate at 16 °C overnight using a thermocycler.
The next morning, transform competent cells (NovaBlue) with 1 μL of ligation following the suggested heat-shock protocol. Grow on LB agar containing ampicillin (100 μg/mL).
Miniprep the plasmid using GeneJET miniprep kit.
Transform 10 ng of plasmid into BL21 DE3 competent cells.
Note: Always use freshly transformed cells for protein expression for maximum efficiency.
Inoculate 2 × 20 mL of LB broth with a colony of BL21 DE3 E. coli containing pQE30-Sbi-IV plasmid. Add 100 μg/mL ampicillin. Grow cultures overnight at 37 °C with shaking at 180 rpm.
The next morning, pour the overnight culture into two 2 L flasks containing 500 mL of LB. Add 100 μg/mL ampicillin and grow culture at 37 °C with shaking until it reaches an OD600nm between 0.5 and 0.7.
Add 0.5 mM IPTG (500 μL of 0.5 M stock) to each flask and grow for a further 3 h.
Pour the cultures into four 500 mL centrifuge bottles and centrifuge at 4,000× g for 10 min at 4 °C.
Resuspend the pellet in 40 mL of 4 °C His A buffer.
Resuspend the first pellet in 40 mL of buffer, then move that resuspended solution into the next bottle. All four pellets should be resuspended in the same 40 mL solution.
Move solution into a 50 mL Falcon.
Repeat the centrifugation step and discard the supernatant.
Notes:
At this step, the pellet can be frozen and stored at -20 °C (or -80 °C long term).
To continue, thaw the pellet on ice for 30 min.
Resuspend the pellet in 30 mL of fresh His A buffer and add 300 μL of Protease Inhibitor Cocktail Set VII.
Lyse the cells by sonicating in 10 s bursts for 10 min at 80% amplitude.
Notes:
Fill a beaker with ice and place the falcon within. Ensure that the pellet solution is fully surrounded by ice. Refill ice between sonication bursts if it melts away.
Use a large sonicator tip.
Always use ear protection.
Pour solution into a 50 mL centrifuge bottle (use a second tube with the same weight of water for balancing) and centrifuge the lysed solution at 60,000× g for 30 min at 4 °C.
Notes:
Balance with equivalent weight water.
DO NOT discard the supernatant!
Filter the supernatant through a 0.45 μm filter using a 50 mL syringe. Filter solution into a clean 50 mL Falcon.
Prepare the AKTA by washing the lines with water, followed by attaching the His-column.
Notes:
Reduce the flow speed to 1 mL/min.
Loosen the storage caps on either end of the column. Remove top cap and attach the AKTA line. Ensure liquid fills the inlet valve of the column before screwing in. This prevents bubbles from forming within the column. Release the bottom screw and wait for a drop of liquid to form at the bottom of the column before attaching to the AKTA.
Once column is attached, ensure flow speed is limited to column capacity, i.e., 1 mL/min. Exceeding this speed could damage the column.
Wash the column with five column volumes of water.
Note: Run for 5 min at 1 mL/min.
Place the intake line A1 into His A buffer and line B1 into His B buffer and wash the column with buffer A for 10 column volumes (10 min).
Note: Line numbers may differ between AKTAs.
Load the cassette with thirty 2 mL collection tubes ready for elution.
Note: The required number of tubes may also vary.
Once the AKTA is prepared, load the filter-sterilised sample onto the AKTA via the injection valve (you may require a large loading line).
Run a pre-set program for His-Tag purification using the Unicorn software.
Notes:
This will wash the sample through the column with His A buffer allowing His-tag binding. Once the sample is washed through, it will incrementally switch from buffer A to buffer B containing a high concentration of imidazole. This will displace the His-tag and elute the bound proteins.
Always double-check the flow speed and elution volume.
Label collected fractions by number and store in the fridge.
Note: Note which fraction numbers showed a high peak at 280 nm. This can be used to predict which fractions contain your protein.
Wash the His-column with 10 column volumes of water, followed by 5 column volumes of 20% ethanol.
Remove the column and store in the fridge for future use.
Continue running the AKTA at 1 mL/min.
While it is running, remove column carefully by first disconnecting the bottom screw. Replace with the storage cap. When disconnecting the top, allow a drop of liquid to fill the top of the inlet value before attaching the cap to prevent air from getting trapped within the column.
Run alternating fractions on an SDS-PAGE gel.
Take 15 μL from each even number fraction. Alternatively, run the fractions indicated by a 280 nm peak.
Add 5 μL of 4× Laemmli sample buffer and heat samples at 95 °C for 5 min using a pre-heated heat block.
Load the samples onto the precast gel. Include 5 μL of protein ladder in the end well.
Run gel at 200 V for 60 min (or until loading dye reaches bottom of the gel).
Stain the gel by placing into a Petri dish and pouring over Coomassie Brilliant Blue R-250 stain (enough to cover the gel). Place on a rocker for 1 h.
To destain, pour out the used Coomassie and pour in a similar volume of Coomassie Brilliant Blue R-250 destain. Incubate for 15 min on a rocker. Pour out and replace with fresh destain and incubate overnight.
Note: Coomassie stain contains hazardous chemicals and must be disposed of carefully.
View gel on a light box or gel dock.
Identify the recombinant protein using the protein ladder and the protein predicted molecular weight. It should be a very thick and prominent band. Pool together all fractions containing target protein, as indicated by the gel.
Note: Include those with visible contaminant proteins, if continuing with a second round of purification.
Concentrate the pooled fractions using Vivaspin 20 centrifugal concentrator. Note the MWCO size to ensure no loss of protein (for Sbi-IV, use MWCO 5 kDa).
Notes:
Load sample into the top of column.
Centrifuge at 3,400× g until sample volume drops below the 5 mL line (approximately 10 min).
Collect sample from the top of the spin column and discard the flowthrough.
To remove aggregate proteins from sample, load into a 15 mL Falcon and centrifuge at 10,000× g for 10 min.
Note: Collect supernatant using a needle attachment on syringe. This ensures that you collect every drop.
Prepare AKTA and attach Size Exclusion Column (SEC). For further details on the delicate procedure of attaching and detaching columns, follow the manufacturer’s protocol.
Place line A1 in SEC buffer and line B in ddH2O water.
Set AKTA flow (with water) to 1 mL/min.
Attach SEC adaptor line and wait for it to fill with solution.
When a drop of liquid is released on the end, carefully screw into top of the SEC column. Always allow the inlet valve to fill with solution before attaching.
Detach the buffer plug and screw in the bottom column line.
Wash with water for 1.5 column volumes, followed by the same volume of SEC buffer.
Wash the 5 mL loading line by injecting solution using a 25 mL syringe. Wash with ethanol, then water, followed by SEC buffer before loading sample.
Load sample onto AKTA and run prepared SEC program.
Use the 280 nm absorbance to predict fractions containing the recombinant protein, which should show a clear peak.
Note: The elution fraction can also be predicted by the protein size and capacity of the column. As our protein was very small, we can assume the protein will be eluted in the final fractions.
Run samples on gel that showed a peak on 280 nm graph (or where the protein was predicted to elute) to confirm protein location (see Figure 2).
Figure 2. SDS-PAGE gel showing collected aliquots following purification. Here, aliquots C4–C12 were pooled together and concentrated. Store collected fractions at -20 °C after confirming fractions on gel. Thaw samples slowly on ice, and pool protein fractions together. Be sure to only combine those containing pure protein with no contaminant bands. Concentrate using Vivaspin 20 & 500 concentration columns (centrifuge at 4,000× g and 4 °C). Concentrate until sample reaches a volume between 250 and 500 μL.
Protein concentration determination
Perform BCA using PierceTM BCA Protein Assay kit.
Follow the manufacturer’s protocol. All reagents required are included in the kit. In addition, a spectrophotometer/microplate reader capable of reading absorbance at 562 nm is needed.
Compare sample absorbance to standard curve to calculate concentration of sample. You may have to dilute the sample 1:10 or more to fit the curve.
Note: We collected 0.5 mL of protein at 5.2 mg/mL.
Biotinylation
Perform biotinylation using EZ-LinkTM Sulfo-NHS-Biotinylation kit following the manufacturer’s detailed protocol.
Note: This kit biotinylates the protein using lysine residues. This can impact protein function if lysines are exposed within functional sites.
Defrost the recombinant protein on ice. Using the concentration calculated by BCA and protein molecular weight in Da, calculate the required concentration of biotin. Make sure the sample volume falls between 0.5 and 2.0 mL.
Calculate millimoles of Sulfo-NHS-LC-Biotin to add to the reaction for a 20-fold molar excess:
Calculate microliters of 10 mM Sulfo-NHS-LC-Biotin to add to the reaction:
Perform buffer exchange of protein sample using one of the desalting columns included in the kit. This is to replace the SEC buffer with PBS.
Dilute 2.2 mg of Sulfo-NHS-LC-Biotin in 400 μL of ultrapure water and add thecalculatedvolume of biotin solution to your protein. Incubate on ice for 2 h.
Repeat buffer exchange to remove excess biotin. Aliquot the sample into small volumes (10 μL) to reduce freeze–thawing cycles.
Western blot
Western blots are useful tools to confirm if the biotinylation of the protein was successful.
Begin by running 100 ng of biotinylated and non-biotinylated protein on an SDS-PAGE gel.
Prepare 15 μL of 100 ng biotinylated and non-biotinylated protein. Dilute to required concentration using PBS.
Add 5 μL of 4× Laemmli sample buffer to each sample.
Heat sample at 95 °C for 5 min using a heat block.
Prepare Mini-PROTEAN TGX precast gel (4%–20%) by removing the tab at the bottom and fixing the gel in the gasket. Fill buffer chamber with mPAGE SDS running buffer.
Load the full 20 μL of sample on the precast gel.
Add 5 μL of protein ladder to an end well.
Perform electrophoresis with 150 V for approximately 1 h (or until the blue loading dye is visible at the very bottom of the gel).
Transfer proteins onto a 0.2 μm nitrocellulose membrane using Trans-Blot Turbo Transfer System (any other transfer system will also work).
Open the Trans-blot turbo cassette and assemble the blotting sandwich (use Bio-Rad’s Quick-start guide to assemble correctly).
On the base of the cassette, place the bottom membrane using forceps. Place your gel carefully over the membrane (we recommend trimming off the comb and any excess/unused wells before placing). Once the gel touches the membrane, do not adjust it.
Place the top membrane over the gel and close the cassette.
Place the cassette into the Trans-blot machine and run on turbo mini gel 4 pre-set program. This will only take 7 min.
Using forceps, remove the upper transfer layers and SDS-PAGE gel to reveal the membrane.
Note: If transferred correctly, the dye from the ladder should be visible on the membrane.
Carefully, with forceps, place the membrane in a clean Petri dish and immediately pour over 10% milk block.
Note: Be careful to never let the membrane dry. Have the milk block prepared before transfer.
Place dish on a rocker for 1 h (at room temperature).
Wash the membrane three times with TBST.
Pour off the milk block and pour on enough TBST to cover the membrane. Leave on rocker for 5 min and pour out. Repeat three times.
Pour on 5% milk block with 1:20,000 dilution of ultra-streptavidin-HRP. Incubate on the rocker for 1 h (at room temperature).
Repeat the membrane wash three times.
Add ECL detection reagent and image the gel.
In a Falcon, mix 1 mL of ECL reagent A with 1 mL of ECL reagent B.
With forceps, move the membrane into a clean Petri dish.
Using a pipette, cover the membrane with the mixed solution. Cover with foil and incubate for 30 s.
Move the membrane onto some clear film and use a roller to push away excess ECL solution.
Place on a gel imager and read chemiluminescence. Use the auto image function ~30 s.
Note: This can be optimised with different protein and antibody dilutions and exposure times. If biotinylation has been successful, a visible band should only be visible on the biotinylated protein, as shown in Figure 3.
Figure 3. SDS-PAGE and western blot analysis of biotinylated and nonbiotinylated Sbi-IV. Figure taken from Wonfor et al. (2022).
Serum collection
Note: Collection and experimental use of serum requires ethical approval by your institution.
Using a qualified phlebotomist, collect blood from at least eight people (5 × 10 mL from each individual). Blood must be collected in Clot Activator tubes to ensure proper clotting.
Collect written consent forms for each donor.
Incubate the blood collection tubes containing blood for 30 min at room temperature so that it is well clotted.
Write down the time of blood collection for each individual so that all tubes are left to clot for the same amount of time.
After clotting, place the tubes on ice.
Continue all remaining steps on ice or at 4 °C.
Centrifuge clotted tubes at 700× g for 7 min at 4 °C.
Using a transfer pipette, move all the sera (clear yellow liquid) from the blood tubes into a clean 50 mL Falcon (on ice). Be careful not to lift any of the clotted blood (red pellet). Pool all the sera from one individual into the same Falcon.
Spin down the individual sera at 700× g for 7 min at 4 °C.
Take the supernatant and pool into one autoclaved 100 mL beaker on ice.
Notes:
It is essential to avoid picking up any blood (red pellet). Leave a little serum behind to make this easier.
Be sure to autoclave the beaker with a flea (magnetic stirring bar).
In a 4 °C room, set up an aliquoting station. Place tube storage boxes open on ice and fill with open Eppendorf tubes.
Briefly mix the pooled serum from all donors on a magnetic stirrer.
Note: Proper mixing is essential to ensure all aliquots are uniform in composition.
In the 4 °C room, begin aliquoting the serum into tubes. We recommend aliquoting in 50, 100, and 250 μL aliquots, working as quickly as possible.
Immediately after aliquoting, store them at -80 °C.
Note: The protocol below shows the conditions for observing complement deposition upon L. lactis. L. lactis has no virulence factors to evade complement, which makes it a useful tool for optimizing the conditions for the new probe. This protocol was optimized with L. lactis strain MG1363.
Complement deposition and labelling upon L. lactis with Sbi-IV
Set up an overnight culture of L. lactis. Inoculate 5 mL of M17-G with a single colony of L. lactis and incubate at 30 °C overnight (static).
The following morning, thaw an aliquot of NHS slowly in a bucket of ice. Thaw a 5 mL aliquot of GVB++ buffer in a beaker of hot water for 30 min, and then place on ice.
Prepare a 96-well V-bottomed plate by adding 200 μL of 10% FCS/PBS to all wells. Incubate for 30 min on the bench.
Wash the wells three times by filling with 200 μL of PBS. Tap the plate face down on blue roll to dry.
Centrifuge 1 mL of overnight L. lactis culture at 16,000× g for 5 min. Remove the supernatant and resuspend in 1 mL of PBS buffer. Repeat the centrifugation to wash away any residual culture media and discard supernatant. Finally, resuspend in 1 mL of fresh PBS.
Normalise the bacteria to an OD600nm = 1.
Read absorbance of 1 mL by diluting 1:10 in a cuvette.
Calculate the volume of cells required for OD600nm = 1. The target volume will depend on how many samples you wish to run. 50 μL is required per sample (add ~100 μL excess for gating).
Using the calculation above, add the volume needed into a fresh Eppendorf.
Keep an aliquot of unstained bacteria on ice for initial gating.
To stain with cell trace Far Red, add 1 μL of cell trace per 1 mL of OD600nm = 1 bacteria (to a final concentration of 1 μM). Cover with foil and incubate for 20 min at 37 °C with shaking at 180 rpm. From this point on, keep stained bacteria covered with foil.
To remove excess stain, either:
(For small volumes, <500 μL) add 3× volume 3% BSA/PBS and incubate on bench for 10 min.
(For large volumes, >500 μL) centrifuge for 5 min. Discard the supernatant. Resuspend the pellet in 1 mL of 3% BSA/PBS. Incubate on the bench for 10 min.
Centrifuge bacteria for 5 min at 16,000× g and resuspend pellet in target volume of GVB++. Bacteria are now stained and normalised to OD = 1.
Make a 2× stock of serum (NHS) by diluting in GVB++ buffer. As before, the volume required is dependent on the number of samples, where 50 μL is required per sample.
Notes:
NHS concentration is generally described as % and the concentration required will need optimizing. We recommend beginning with a gradient of 2% to 20%. For ano serum controljust add GVB++ buffer.
Only dilute NHS when you are ready to add to the plate. Once diluted, use immediately.
Add 50 μL of diluted NHS and 50 μL of stained bacteria to the coated V-bottomed well plate. Cover with foil and incubate for 30 min at 37 °C (static).
Note: Keep plate covered with foil for all remaining incubation steps.
Centrifuge the plate at 3,400× g for 7 min (at room temperature) (if possible, set the deceleration speed to 6). Discard the supernatant gently (careful not to disturb the pellet) and resuspend each well in 100 μL of 1% BSA/PBS. Repeat the centrifugation and gently discard the supernatant.
Dilute primary probe in 1% BSA/PBS. Prepare enough for 100 μL per sample.
Note: Dilutions vary depending on the probe/antibody. For Sbi-IV, we used a final concentration of 175 μM, but we would recommend performing your own optimization using various dilutions of your purified stock (i.e., 1:250, 1:500, 1:1,000, and 1:2,000). Determine which concentration gives a high geometric mean with minimal background on negative controls.
Resuspend the cells in 100 μL of probe solution and incubate on the bench for 45 min.
Repeat centrifugation and wash as described in step 12.
Dilute secondary probe (with conjugated fluorophore) in 1% BSA/PBS. Make enough for 100 μL per sample (for streptavidin-488, dilute 1:1,000). Keep diluted stock covered with foil until ready to use.
Resuspend bacteria in 100 μL of secondary probe solution and incubate on the bench for 45 min.
Repeat centrifugation and wash from step 12, then resuspend for a final time in 100 μL of PBS. Keep plate covered with foil and carry over to the flow cytometer.
Complement deposition and labelling upon S. aureus with Sbi-IV
Note: Follow the protocol as described above with the adaptations listed below. This protocol was optimized with S. aureus strain JE2).
Set up an overnight culture of S. aureus. Inoculate 2 mL of tryptic soy broth with a single colony of S. aureus and incubate at 37 °C with shaking at 180 rpm overnight.
The following morning, subculture the overnight culture 1:200, either in 5 mL (25 μL of culture) or 50 mL (250 μL), and grow with shaking at 37 °C to OD600nm = 0.5–0.6.
Notes:
Two hours is enough for S. aureus strain JE2. The time will vary between different strains, but usually takes 2–3 h. For slower strains (such as TW20), we recommended changing the subculture to 1:100 or 1:50.
We recommend growing S. aureus in either 25 mL glass universals or glass flasks.
When normalising (step F6 above), for S. aureus we recommend normalising to OD600nm = 2.
To stain (step F7 above), add 2 μL of 1 mM cell trace per 1 mL of OD600nm = 2 S. aureus (2 μM final concentration).
Controls that can be used in optimisation and analysis of complement deposition on bacteria using Sbi-IV to identify any non-specific binding in the absence of C3 deposition:
No NHS = GVB buffer only
CP40-NHS = Incubate 2× NHS stock with 50 μM compstatin (AMY-101). Incubate on ice for 30 min before mixing with bacteria.
HI-NHS = Heat 2× NHS stock at 56 °C using a heat block for 30 min. Cool briefly on ice before mixing with bacteria.
C3 depleted NHS (purchased) = dilute in GVB to 20% (= 2× stock). Add as normal.
Note: The flow cytometry protocol written here can also be used with commercial antibodies to study complement deposition on non-immunoglobulin binding pathogens (such as L. lactis). This can include antibodies targeting both C9 as well as C3. i.e., rabbit anti-human C3d (Dako), or mouse anti-human C9 (aE11, Abcam). Note that you will need compatible fluorophore-labelled secondary antibody.
Setting up FACS CANTO
Read sample using FACS CANTO (BD) with lasers capable of reading wavelengths of 488 nm and 633 nm. Count 20,000 events.
Prepare the flow cytometer for reading samples.
Switch on FACS Canto.
Launch FACSDiva software.
Run the program Fluidics Start Up.
Note: This will wash the system and allow time for the lasers to warm up.
Acquire data from samples.
Transfer 100 μL of sample from a 96-well plate to an FC loading tube.
Push lever to the left and insert tube onto the probe (Figure 2). Release the lever returning it to its starting position.
Note: The lever base should sit comfortably under the tube; if not, push it on a little further (as shown in Figure 4 below).
Figure 4. Flow cytometry sample insertion
Click Acquire Data and wait approximately 10 s (this allows the number of events per second to settle), before clicking Record Data. This will automatically stop acquisition when the threshold events to record is reached (set this to 20,000 events, as shown in Figure 5 below).
Figure 5. Flow cytometry data acquisition
Click Remove tube and wait for the progress box to pop up. Push the lever all the way to the left, remove the tube, and then release the lever.
Note: This automatically triggers the FACS CANTO to wash the probe. If you do not move the lever properly, an error will pop up.
Click Next tube when ready to acquire the next sample.
Prepare a worksheet for collecting samples. On the right-hand side, open a Global Worksheet.
Note: Setting up workbooks and gating for flow cytometry can be complicated and very easy to manipulate incorrectly. We highly recommend setting this up with an experienced technician, to ensure the gating is performed accurately. These are the graphs we recommend:
SSC vs. FSC (side vs. forward scatter, to gate bacteria).
FSC-H vs. FSC-A (forward area vs. height).
Histogram for APC-A (633 nm) to gate stained bacteria.
Histogram for Alexa Fluor 488-A (488 nm) to observe probe.
APC-A vs. Alexa Fluor 488-A.
Initial setup requires adjusting the cytometer parameters under Cytometer (Figure 6). Select FSC-A, FSC-H, SSC-A, SSC-H, 488-A, and APC-A. Make sure to select Log for all four parameters.
Figure 6. Adjusting cytometer parameters
Begin gating by loading 100 μL of unstained bacteria into an FC loading tube (Figure 7). Click Acquire Data.
Note: You do not need to click Record data while adjusting the voltages and gating. Only Record experiment samples.
View particles on the FSC vs. SSC. Adjust the voltages of FSC and SSC until the bacterial population is visible and close to centre of the graph.
FSC shifts the particles right or left. Increasing FSC voltage shifts to the right, and lowering it shifts the scatter to the left.
SSC voltage shifts the particles up or down. Increasing SSC voltage shifts the scatter up, and lowering the voltage shifts the scatter down.
Apply a box gate around the cluster of cells. This will be gate P1.
Figure 7. Gating of bacterial sample
Perform doublet discrimination: examining the FSC-A vs. FSC-H, apply a second gate (P2) that fits tightly to the bottom-right edge of the cluster (Figure 8). This will remove doublets.
Note: When two particles pass the laser at the same time, the flow cytometer designates this as a single event; performing doublet discrimination increases accuracy of your analysis.
Figure 8. Removing doublet events
Remove the sample and load 100 μL of stained bacteria (Figure 9).
View the histogram for APC-A and adjust the APC voltage until the peak for stained bacteria is visible.
Increasing the APC voltage shifts the visible peak to the right. Lowering the voltage shifts to the left.
On the histogram for APC, add a gate around the visible peak with stained bacteria (Gate P3). Ensure there is no peak within this gate when running a sample of unstained bacteria.
Finally, run a positive control sample (i.e., L. lactis + 10% serum) to adjust the peak for 488. Biotinylated Sbi-IV will be used to detect complement deposition.
Figure 9. Screenshots showing histograms for 488-A (top; biotinylated Sbi-IV) and APC-A (bottom; bacteria). A) Unstained bacteria, 0% serum. B) Stained bacteria, no serum. C) Stained bacteria + 5% serum. P3 gate is essential to ensure counting only occurs on stained bacteria.
The final gate is applied to APC-A vs. 488-A. This separates the graph into quadrants based upon the histograms viewed above (Figure 10).
Q1: Particles visible here are stained with APC but have no labelled Sbi-IV or antibodies bound (no 488 signal).
Q2: Particles here are both stained AND have labelled Sbi-IV or antibodies deposited.
Q3: Particles here are unstained and have no labelled Sbi-IV or antibodies (possible contamination).
Q4: Particles here are not stained but do have labelled Sbi-IV or antibodies deposited upon them.
Note: For Q4, this result would strongly suggest improper staining of cells.
Figure 10. Final gating of workbook. P3 may be above Q’s. Ensure the stopping and storage gates are set to P3.
These settings and gates may have to be adjusted with each new experiment; therefore, always begin by running an unstained and stained control before collecting data.
Save the data by clicking on the active workbook and click Export, followed by FCS files. Select FCS 3.0 and continue. This will save the samples in the correct format for analysis in FlowJo.
Clean the cytometer before shutting down. Follow the routine recommended by your technicians.
Wash probe by running 10% bleach for 5 min (click Acquire, do not click Record). Repeat with water for 5 min. Make sure the FC sample tube is at least half full with water before running Fluidics Shutdown.
Close the FACS Diva software and turn of the machine by pushing the green button.
Analysing the data in FlowJo v10 Software (Figure 11):
Open FlowJo software and drag and drop FCS files into the open box.
As all the samples were gated, and counted events were restricted to only those within the P1, P2, and P3 gate, you do not need to gate within FlowJo. However, we recommend adding the Q1–Q4 gate to assist in visualising the results.
Double-click on a sample. Adjust the x- and y-axis to APC-A vs. 488-A.
Add the gate as close to the position originally placed in FACS DIVA.
Note: This gate is not used for quantitative analysis; only observation.
To analyse geometric mean of 488, click on the Statistics box on the bottom of the pop-up box. Click the Sigma+, which will open another box. Select Geometric mean on the left, and Alexa Fluor 488-A on the right, followed by Add. Close both boxes.
Highlight all four Q gates + geometric mean (shift click), then drag and drop onto All samples in the box above. This will apply the same gate to all samples.
Figure 11. Data acquisition using FlowJo
The geometric mean value observed under Statistic is the number used to plot geometric mean in a graph. We performed this in GraphPad.
Scatters/histograms can also be observed by dragging and dropping into the Layout editor.
Recipes
His A buffer (500 mL)
50 mM Tris (Trizma HCl)
150 mM NaCl
20 mM imidazole
ddH2O to final volume
Adjust to pH 7.4
Filter-sterilise with 0.22 μm filter and autoclave
Store at 4 °C
His B buffer (500 mL)
50 mM Tris (Trizma HCl)
150 mM NaCl
500 mM imidazole
ddH2O to final volume
Adjust to pH 7.4
Filter-sterilise with 0.22 μm filter and autoclave
Store at 4 °C
SEC buffer (500 mL)
20 mM Tris (Trizma HCl)
150 mM NaCl
ddH2O to final volume
Adjust to pH 7.4
Filter-sterilise with 0.22 μm filter and autoclave
Store at 4 °C
Milk block
For 10% block, dilute 1 g of skim milk powder in 10 mL of TBST buffer
For 5% block, dilute 0.5 g of skim milk powder in 10 mL of TBST buffer
Shake well to dissolve
M17-G broth
Prepare M17 broth by dissolving in ddH2O and send to autoclave.
Make 10% glucose by diluting in ddH2O, and filter-sterilise with 0.45 μm filter.
Add 10% glucose stock to a final concentration of 1%.
10% FCS/PBS
Dilute FCS in PBS to a final concentration of 10% FCS.
Filter-sterilise using 0.45 μm filter.
Store at 4 °C.
BSA/PBS
Add BSA powder to PBS to a final concentration of 1% or 3% (1 g per 100 mL of PBS for 1%, 3 g per 100 mL of PBS for 3%).
Filter-sterilise using 0.45 μm filter.
Store at 4 °C.
Veronal buffer stock (VBS) (25 mM) (500 mL)
NaCl (720 mM)
Na-barbital (9.0 mM)
Barbituric acid (15.5 mM). Dissolve powder in hot water first
ddH2O to final volume
Adjust to pH 7.35
Filter-sterilise using 0.45 μm filter
Aliquot in 10 mL/50 mL batches
Store at -20 °C
GVB++ buffer (500 mL)
5 mM veronal buffer (1:5 dilution of VBS); thaw in hot water for 20 min
0.1% (w/v) gelatin. Dissolve powder in hot water first
1 mM MgCl2
0.15 mM CaCl2
ddH2O to final volume
Filter-sterilise using 0.45 μm filter
Aliquot in 5 mL batches
Store at -20 °C
Acknowledgments
M.L. was supported by the Academy of Medical Sciences Springboard Grant (SBF006/1023) and Royal Society (192103). T.W. was supported by an URSA PhD studentship from the University of Bath. This protocol was modified from our previous work (Wonfor et al., 2022).
Competing interests
Authors declare no competing interests.
References
Clark, E. A., Crennell, S., Upadhyay, A., Zozulya, A. V., Mackay, J. D., Svergun, D. I., Bagby, S. and Van Den Elsen, J. M. H. (2011). A structural basis for Staphylococcal complement subversion: X-ray structure of the complement-binding domain of Staphylococcus aureus protein Sbi in complex with ligand C3d. Mol Immunol 48(4): 452-462.
Cruz, A. R., Boer, M. A. D., Strasser, J., Zwarthoff, S. A., Beurskens, F. J., de Haas, C. J. C., Aerts, P. C., Wang, G., de Jong, R. N., Bagnoli, F., et al. (2021). Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation. Proc Natl Acad Sci U S A 118(7): e2016772118.
Murray, C. J., Ikuta, K. S., Sharara, F., Swetschinski, L., Robles Aguilar, G., Gray, A., Han, C., Bisignano, C., Rao, P., Wool, E., et al. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet 399(10325): 629-655.
Noris, M. and Remuzzi, G. (2013). Overview of complement activation and regulation. Semin Nephrol 33(6): 479-492.
Sidorin, E. V. and Solov'eva, T. F. (2011). IgG-binding proteins of bacteria. Biochemistry (Mosc) 76(3): 295-308.
Smith, E. J., Corrigan, R. M., van der Sluis, T., Grundling, A., Speziale, P., Geoghegan, J. A. and Foster, T. J. (2012). The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid. Mol Microbiol 83(4): 789-804.
Thammavongsa, V., Kim, H. K., Missiakas, D. and Schneewind, O. (2015). Staphylococcal manipulation of host immune responses. Nat Rev Microbiol 13(9): 529-543.
Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L. and Fowler, V. G., Jr. (2015). Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28(3): 603-661.
Wonfor, T., Li, S., Dunphy, R. W., Macpherson, A., van den Elsen, J. and Laabei, M. (2022). Novel method for detecting complement C3 deposition on Staphylococcus aureus. Sci Rep 12(1): 15766.
Yang, Y., Back, C. R., Grawert, M. A., Wahid, A. A., Denton, H., Kildani, R., Paulin, J., Worner, K., Kaiser, W., Svergun, D. I., et al. (2018). Utilization of Staphylococcal Immune Evasion Protein Sbi as a Novel Vaccine Adjuvant. Front Immunol 9: 3139.
Article Information
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Microbiology > Microbe-host interactions > Bacterium
Immunology > Complement analysis
Molecular Biology > Protein > Flow Cytometry
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Peer-reviewed
Modified Pseudo-Schiff Propidium Iodide for Staining the Shoot Apical Meristem in Arabidopsis
RL Ruiqi Li
QL Qi Li
LM Ligeng Ma
Published: Vol 13, Iss 9, May 5, 2023
DOI: 10.21769/BioProtoc.4672 Views: 985
Reviewed by: Ansul LokdarshiRicardo Urquidi CamachoPablo Bolanos-Villegas
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Original Research Article:
The authors used this protocol in Plant Physiology Aug 2022
Abstract
Visualization of cell structure with fluorescent dye for characterizing cell size, shape, and arrangement is a common method to study tissue morphology and morphogenesis. In order to observe shoot apical meristem (SAM) in Arabidopsis thaliana by laser scanning confocal microscopy, we modified the pseudo-Schiff propidium iodide staining method by adding a series solution treatment to stain the deep cells. The advantage of this method is mainly reflected by the direct observation of the clearly bounded cell arrangement and the typical three-layer cells in SAM without the traditional tissue slicing.
Keywords: Shoot apical meristem (SAM) Cell arrangement Arabidopsis thaliana Laser scanning confocal microscopy (LSCM) Propidium iodide
Background
Most of the above-ground plant organs are derived from the shoot apical meristem (SAM) (Steeves and Sussex, 1989; Dinneny and Benfey, 2008). As a key tissue involved in plant growth and development, it plays a role in maintaining the ability of stem cells to divide continuously (Fletcher, 2018; Hu et al., 2018), controlling the initiation of organs development (Spinelli et al., 2011; Balkunde et al., 2017), accurately regulating the spatio-temporal specific gene expression (Mayer et al., 1998; Long et al., 1996; Williams, 2021), and integrating the signals from various cell types (Zhao et al., 2010; Azizi et al., 2015; Chung et al., 2019). Thus, exploring the mechanism of complex regulation patterns in the meristem is inseparable from observation of meristem at the cellular level.
Unlike the inflorescence meristem, the SAM of Arabidopsis is tightly wrapped by the surrounding leaf primordia and leaves, which makes it difficult to observe its cellular structure in its deeper part (Moreno et al., 2006). Scientists usually observe the cell structure and morphogenesis of SAM by semithin sections or tissue clearing, but only one cell layer or the surface layer of the SAM is observed (Koi and Kato, 2007; Zhang et al., 2021; Wang et al., 2022). To visualize three-dimensional structure of plant tissues, scientists prefer to use fluorescent dyes, such as propidium iodide, to stain tissues for optical section for laser scanning confocal microscopy (Haseloff, 2003; Truernit et al., 2008; Prunet et al., 2016; Shi et al., 2016).
Previously, we observed the cell structure of SAM and inflorescence meristem in Arabidopsis by modifying the pseudo-Schiff propidium iodide staining method (Li et al., 2022). Compared with traditional methods, this method presents several advantages. First, it allows the observation of the different cell layers in the whole SAM, which is a continuous and complete structure. Second, the cellular structure of the initial leaf primordium is observable except for the SAM. Third, unlike the traditional method that takes seven days, this method is simple in terms of procedure and time saving, taking only three days to complete. It can be foreseen that this method will likely be applicable to the observation of other tissues or SAM of other plant species. The operation details are described in the following section.
Materials and Reagents
1.5 mL centrifuge tubes (Chenglin, catalog number: CL22091809)
Microscope slides (CITOTEST, catalog number: 10127101P-G)
Microscope cover glass (CITOTEST, catalog number: 10212222C)
Qualitative filter paper (Beimu, catalog number: 101)
60 mm round culture dishes (Jindian, catalog number: 16021-1)
Aluminum foil for experiment (Zaowufang, catalog number: 615)
1 mL syringe (Zhi Yu, catalog number: V500111)
Single edge blades (Personna, catalog number: 84-0701)
Sodium hypochlorite solution (Aladdin, catalog number: S101636)
Murashige and Skoog basal medium (Sigma, catalog number: M5519)
Sucrose (Hushi, catalog number: 10021418)
Potassium hydroxide (Hushi, catalog number: 10017008)
Methanol (Aladdin, catalog number: M116115)
Acetate (Aladdin, catalog number: A116172)
Ethanol (Aladdin, catalog number: E130059)
Hydrochloric acid (Aladdin, catalog number: H399657)
Sodium hydrogen bisulfite (Hushi, catalog number: 10019018)
Phytagel (Sigma, catalog number: P8169)
Periodic acid (Sigma, catalog number: P0430)
Propidium iodide (Sigma-Aldrich, catalog number: P4170)
Chloral hydrate C-IVN (Sigma-Aldrich, catalog number: 15307)
Glycerol (Sigma-Aldrich, catalog number: G7893)
Gum arabic from acacia tree (Sigma, catalog number: G9752)
Solid MS medium (see Recipes)
Fixative solution (see Recipes)
1% periodic acid solution (see Recipes)
Pseudo-Schiff propidium iodide solution (see Recipes)
Chloral hydrate solution (see Recipes)
Hoyer′s solution (see Recipes)
Ethanol solutions (see Recipes)
Equipment
Tweezers (Venus, #5)
Dissecting microscope (Zeiss, model: Axio Zoom V16)
Laser scanning confocal microscope (Zeiss, model: 780)
Growth chamber (Percival, model: CU36L5)
Fume hood
Clean bench
Procedure
Arabidopsis seedlings growth
Soak the Arabidopsis seeds with 1 mL of ddH2O in a 1.5 mL centrifuge tube and place the tube at room temperature for 1 h.
Pour out all the liquid and sterilize the Arabidopsis seeds with 15% sodium hypochlorite solution. Shake the tube by hand and let it soak thoroughly for 10 min.
On a sterile clean bench, pour out the sodium hypochlorite solution from the tube and wash the seeds 3–5 times with 1 mL of sterile ddH2O. Then, place the seeds at 4 °C for three days to break dormancy.
On a sterile clean bench, place the seeds onto round culture dishes with solid MS medium (see Recipes), and seal plates with micropore tape. Preparing 50 seedlings per condition is recommended for subsequent experiments.
Place the solid MS medium plate in the growth chamber under 16 h/22 °C light and 8 h/18 °C darkness for nine days (or any desired growth conditions). Use 9-day-old seedlings for subsequent experiments.
SAM dissection
Using a single edge blade, cut the cotyledons and root to separate the other tissue from the shoot and leave the hypocotyl connected to the shoot tips (Figure 1A and 1B).
Add 30 shoot tips to the centrifuge tube containing freshly prepared fixative solution (see Recipes) and leave at 4 °C overnight (Figure 1C).
Pour out the fixative solution and wash the shoot tips with ddH2O twice.
Pour out the water and add 30% ethanol solution for 15 min. Repeat this step with 50%, 70%, 80%, 90%, 95%, and 100% ethanol series (see Recipes).
Note: At this dehydrated stage, the shoot tips will turn white from the ethanol treatment (Figure 1D).
Figure 1. Preparation of shoot tips. A. Dissecting microscope images of the 9-day-old plant. Scale bar: 1,000 μm. B. Dissecting microscope images of the shoot tips with hypocotyl. Scale bar: 1,000 μm. White dashed lines mark the base of petiole. Cut the cotyledons and root along the dashed lines using a single edge blade. C. Centrifuge tube containing shoot tips (green) in fixative solution. D. Centrifuge tube containing shoot tips (white) in 100% ethanol.
Transfer the shoot tips to new 100% ethanol overnight at room temperature.
Note: At this stage, you can keep the shoot tips in ethanol at -20 °C for several weeks.
Transfer the shoot tips to the 60 mm round culture dishes with 100% ethanol and place the culture dishes under the dissecting microscope (Figure 2A). Then, grip the hypocotyl with tweezers (Figure 2B) and remove the tiny true leaves from the shoot tip using a syringe needle under the dissecting microscope (Figure 2C–2E). Subsequently, transfer the shoot tips to the 1.5 mL centrifuge tube with 100% ethanol.
Note: This sample is very small and easily broken; be sure to gently wash the sample with solution in the subsequent experiment.
Figure 2. Dissection of shoot tips. A. Round culture dish containing shoot tips in 100% ethanol. B. Dissection of shoot tips using a syringe needle or tweezers under the dissecting microscope. C. Dissecting microscope images of the shoot tips before dissection. The arrow indicates the hypocotyl; white dashed lines mark the base of petiole. Scale bar: 500 μm. D. Images of the shoot tips from the top before dissection. White dashed lines mark the base of petiole. Scale bar: 500 μm. Grip the hypocotyl of shoot tips with tweezers and remove the leaves along the dashed line using a syringe needle or tweezers. E. Dissecting microscope images of the shoot tips after dissection. Scale bar: 500 μm.
Staining of SAM
Remove the 100% ethanol carefully and add 95% ethanol solution for 15 min. Repeat this step with 90%, 80%, 70%, 50%, 30%, and 15% ethanol series and ddH2O. Then, wash the shoot tips with ddH2O once.
Remove the water carefully and add 1 mL of 1% periodic acid solution (see Recipes) for 40 min. Then, wash the shoot tips with ddH2O twice.
Remove the water carefully and add 1 mL of pseudo-Schiff propidium iodide solution (see Recipes) for 2 h. Wrap the centrifuge tube with aluminum foil to protect the shoot tips from light.
Note: At this stage, the shoot tips will turn pink from the pseudo-Schiff propidium iodide solution. Protect the shoot tips from direct light in subsequent experiments (Figure 3A).
Carefully remove the pseudo-Schiff propidium iodide solution and wash the shoot tips with ddH2O twice.
Note: Because propidium iodide is toxic, discarded pseudo-Schiff propidium iodide solution requires a special waste container.
Transfer the shoot tips to the microscope slide and blot dry the liquid around the sample with qualitative filter paper. Then, drop 10–20 μL of chloral hydrate solution (see Recipes) and leave for 20–30 min at room temperature, away from light (Figure 3C).
Note: At this stage, the shoot tips will become transparent from chloral hydrate solution treatment (Figure 3B).
Remove the chloral hydrate solution with qualitative filter paper and drop 100 μL of Hoyer′s solution (see Recipes) to cover the shoot tips. Then, carefully lay down a microscope cover glass, avoiding air bubbles (Figure 3D).
Place the slides for three days at room temperature away from light. You can keep the slides at room temperature for several weeks.
Figure 3. Staining of shoot tips. A. Centrifuge tube containing shoot tips in pseudo-Schiff propidium iodide solution. The shoot tips are pink. B. Dissecting microscope images of the shoot tips during chloral hydrate solution treatment. Scale bar: 500 μm. C. Microscope slide with shoot tips in chloral hydrate solution. The arrow indicates the shoot tip. D. Microscope slide with shoot tips in Hoyer′s solution. The arrow indicates the shoot tip.
Imaging of SAM
Observe the shoot tips under a Zeiss 780 laser scanning confocal microscope with a microscope objective (Plan-apochromat 40×/1.3 Oil DIC M27).
Set channel parameters as: excitation wavelength 514 nm; emission wavelength 566–658 nm; intensity of laser approximately 20%.
Set the zoom 5× to magnify the image; resolution is 1,024 × 1,024. Set speed of scanning parameter as 7. Observe and image the different cell layers in the whole SAM by adjusting the z-axis. If you want a layer of cells, set the scan mode to x-y recording; if you want images of a complete SAM, set the scan mode to z-stack collection. In the x, y, z collection mode, set the parameter “line averaging” to achieve nice quality. In the central layer of SAM (Z10), you can observe typical three-layer cells (Figure 4).
Figure 4. Imaging of shoot tips. Series of SAM images with different z-axis obtained using a laser scanning confocal microscope. The arrow indicates the SAM; P indicates the leaf primordia; Z indicates different z-axis; white dashed lines in Z10 mark the cell layers. Scale bar: 50 μm.
Recipes
Solid MS medium
Dissolve 4.4 g of Murashige and Skoog basal medium and 10 g of sucrose in 1 L of ddH2O. Adjust the pH to 5.8 by adding potassium hydroxide. Then, add 3 g of phytagel and autoclave.
Fixative solution
Add 5 mL of methanol and 1 mL of acetate in 4 mL of ddH2O. The solution should be freshly prepared just before use.
1% periodic acid solution
Dissolve 0.1 g of periodic acid in 10 mL of ddH2O. The solution should be freshly prepared just before use.
Pseudo-Schiff propidium iodide solution
Dissolve 0.1 g of sodium hydrogen bisulfite and 100 μg of propidium iodide in 4.9 mL of ddH2O. Add 62.5 μL of hydrochloric acid.
This solution should be stored away from light and freshly prepared just before use.
Chloral hydrate solution
Dissolve 8 g of chloral hydrate in 2 mL of ddH2O and add 1 mL of glycerol.
This solution can be stored at room temperature for a long time.
Hoyer’s solution
Dissolve 6 g of gum arabic in 10 mL of ddH2O for one day; then, add 5 mL of glycerol and 40 g of chloral hydrate.
Wait 10 days before using; the solution can be stored away from light for a long time.
Ethanol solutions
All ethanol solutions are prepared with 100% ethanol and ddH2O by volume ratio. The 100% ethanol refers to absolute ethyl alcohol.
Acknowledgments
This work was funded by grants from the National Natural Science Foundation of China. This protocol was modified based on the previous publication (Li et al., 2022). The authors thank the Imaging Center, College of Life Sciences, Capital Normal University, Beijing, China.
Competing interests
The author declares no competing financial interests.
References
Azizi, P., Rafii, M. Y., Maziah, M., Abdullah, S. N., Hanafi, M. M., Latif, M. A., Rashid, A. A. and Sahebi, M. (2015). Understanding the shoot apical meristem regulation: a study of the phytohormones, auxin and cytokinin, in rice. Mech Dev 135: 1-15.
Balkunde, R., Kitagawa, M., Xu, X. M., Wang, J. and Jackson, D. (2017). SHOOT MERISTEMLESS trafficking controls axillary meristem formation, meristem size and organ boundaries in Arabidopsis. Plant J 90: 435-446.
Chung, Y., Zhu, Y., Wu, M. F., Simonini, S., Kuhn, A., Armenta-Medina, A., Jin, R., Ostergaard, L., Gillmor, C. S. and Wagner, D. (2019). Auxin response factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS. Nat Commun 10: 886.
Dinneny, J. R. and Benfey, P. (2008). Plant stem cell niches: standing the test of time. Cell 132: 553-557.
Fletcher, J. C. (2018). The CLV-WUS stem cell signaling pathway: a roadmap to crop yield optimization. Plants (Basel) 7(4):87.
Haseloff, J. (2003). Old botanical techniques for new microscopes. Biotechniques 34: 1174-1178, 1180, 1182.
Hu, C., Zhu, Y., Cui, Y., Cheng, K., Liang, W., Wei, Z., Zhu, M., Yin, H., Zeng, L., Xiao, Y., et al. (2018). A group of receptor kinases are essential for CLAVATA signalling to maintain stem cell homeostasis. Nat Plants 4: 205-211.
Koi, S. and Kato, M. (2007). Developmental morphology of the shoot in Weddellina squamulosa and implications for shoot evolution in the Podostemaceae.Ann Bot 99(6):1121-1130.
Li, R., Wei, Z., Li, Y., Shang, X., Cao, Y., Duan, L. and Ma, L. (2022). SKI-INTERACTING PROTEIN interacts with SHOOT MERISTEMLESS to regulate shoot apical meristem formation. Plant Physiol 189(4):2193-2209.
Long, J. A., Moan, E. I., Medford, J. I. and Barton, M. K. (1996). A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379: 66-69.
Mayer, K.F., Schoof, H., Haecker, A., Lenhard, M., Jürgens, G. and Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95: 805-815.
Moreno, N., Bougourd, S., Haseloff, J. and Feijo, J. (2006). Imaging plant cells. In: Pawley, J. (Ed.). Handbook of Biological Confocal Microscopy (pp. 769-787). New York: SpringerScience and Business Media.
Prunet, N., Jack, T. P. and Meyerowitz, E. M. (2016). Live confocal imaging of Arabidopsis flower buds. Dev Biol 419(1): 114-120.
Shi, B., Zhang, C., Tian, C., Wang, J., Wang, Q., Xu, T., Xu, Y., Ohno, C., Sablowski, R., Heisler, M. G., et al. (2016). Two-step regulation of a meristematic cell population acting in shoot branching in Arabidopsis. PLoS Genet 12(7): e1006168.
Spinelli, S. V., Martin, A. P., Viola, I. L., Gonzalez, D. H. and Palatnik, J. F. (2011). A mechanistic link between STM and CUC1 during Arabidopsis development. Plant Physiol 156: 1894-1904.
Steeves, T. A. and Sussex, I. M. (1989). Patterns in plant development (New York: Cambridge University Press).
Truernit, E., Bauby, H., Dubreucq, B., Grandjean, O., Runions, J., Barthelemy, J. and Palauqui J. C. (2008). High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20: 1494-1503.
Wang, W., Hu, C., Li, X. N., Zhu, Y. F., Tao, L., Cui, Y. W., Deng, D. Q., Fan, X. X., Zhang, H., Li, J., Gou, X. P. and Yi, J. (2022). Receptor-like cytoplasmic kinases PBL34/35/36 are required for CLE peptide-mediated signaling to maintain shoot apical meristem and root apical meristem homeostasis in Arabidopsis.Plant Cell 34(4):1289-1307.
Williams, L.E. (2021). Genetics of shoot meristem and shoot regeneration. Annu Rev Genet 55: 661-681.
Zhang, L., DeGennaro, D., Lin, G., Chai, J. and Shpak, E.D. (2021). ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem.Development 148(5):dev189753.
Zhao, Z., Andersen, S. U., Ljung, K., Dolezal, K., Miotk, A., Schultheiss, S. J. and Lohmann, J. U. (2010). Hormonal control of the shoot stem-cell niche. Nature 465: 1089-1092.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Plant Science > Plant developmental biology > Morphogenesis
Developmental Biology > Morphogenesis > Cell structure
Cell Biology > Cell structure > Nucleus
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In vivo Drug Screening to Identify Anti-metastatic Drugs in Twist1a-ERT2 Transgenic Zebrafish
JN Joji Nakayama
HM Hideki Makinoshima
ZG Zhiyuan Gong
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4673 Views: 551
Reviewed by: Xi FengAmr Galal Abdelraheem IbrahimAlberto Rissone
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Original Research Article:
The authors used this protocol in Molecular cancer research Mar 2020
Abstract
Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.
Graphical overview
Timing
Day 0: Zebrafish spawning
Day 8: Primary tumor induction
Day 11: Chemical treatment
Day 11.5: Metastatic dissemination induction in the presence of a test chemical
Day 16: Data analysis
Keywords: In vivo drug screen Metastasis EMT Twist1 Zebrafish
Background
Metastasis is responsible for approximately 90% of cancer-associated mortality. It proceeds through multiple steps: invasion, intravasation, survival in the circulatory system, extravasation, colonization, and metastatic tumor formation in secondary organs with angiogenesis (Nguyen et al., 2009; Chaffer and Weinberg, 2011; Welch and Hurst, 2019). The dissemination of cancer cells is an initial step of metastasis, and its molecular mechanism involves a local breakdown of basement membrane, loss of cell polarity, and induction of epithelial to mesenchymal transition (EMT). EMT plays a central role in early embryonic morphogenesis; its process enables various types of epithelial cells to convert into mesenchymal cells, through a downregulation of epithelial markers such as E-cadherin and an upregulation of mesenchymal markers such as vimentin. Twist, a basic helix-loop-helix transcription factor, plays a critical role in inducing the EMT program (Tsai and Yang, 2013; Lu and Kang, 2019). Past studies showed that elevated expression of Twist is associated with poor survival rates in patients with cancer; also, ectopic expression of Twist confers metastatic properties on cancer cells through induction of EMT (Yang et al., 2004; Tsai et al., 2012).
Cancer research using zebrafish as a model has attracted attention because this model offers many unique advantages that are not readily provided by other animal models (White et al., 2013; Osmani and Goetz, 2019). Furthermore, the zebrafish system has also been increasingly recognized as a chemical screening platform because it provides the advantage of high-throughput screening in an in vivo vertebrate setting with physiologic relevance to humans (Zon and Peterson, 2005; Letrado et al., 2018; Nakayama and Gong, 2020; Nakayama and Makinoshima, 2020; Nakayama et al., 2021b, 2022a and 2022b). Our study previously established a tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line that serves as an in vivo drug screening platform for the identification of anti-metastasis drugs targeting metastatic dissemination of cancer cells. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish showed spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of an EMT (Nakayama et al., 2020; Lu et al., 2021). The dissemination patterns are generally divided into three categories: (i) local dissemination, in which disseminated mCherry-positive cells exist in close proximity to the liver; (ii) abdominal dissemination, in which the cells spread throughout the abdomen; and (iii) distant dissemination, in which the cells are observed over a broad region from the trunk to the tail (Figure 1A).
This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the discovery of anti-metastasis drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test chemical through comparing the frequencies of the fish showing the abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Previous studies confirmed that ki16425 (a LPA1 inhibitor) or Y27632 (an inhibitor of Rho-associated coiled-coil-containing protein kinase), which have been reported to suppress metastasis in mice models of metastasis (Itoh et al., 1999; Boucharaba et al., 2006), could suppress cell dissemination in the fish model. In vivo drug screen using this model identified adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), as having a potential to suppress metastatic dissemination of cancer cells (Figure 1B and 1C). Furthermore, pharmacologic and genetic inhibition of HSD11β1 were validated to suppress metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model (Nakayama et al., 2020 and 2021a). Taken together, our model offers an in vivo drug screening platform for the identification of anti-metastatic drugs.
Figure 1. Twist1a-ERT2/xmrk double transgenic zebrafish offers an in vivo drug screening platform for the identification of anti-metastatic drugs. A. Representative images of the dissemination of mCherry-labeled hepatic cells from the liver in Twist1a-ERT2/xmrk double transgenic zebrafish at 16 days post-fertilization (dpf). The fish were treated with doxycycline and 4-hydroxytamoxifen (4-OHT). Some of the disseminated mCherry-positive cells are indicated by arrowheads. The images are shown as Z-stack images using 100× magnification. Scale bar, 200 μm. B. Representative images of the effect of adrenosterone on the dissemination of mCherry-positive cells in the fish at 16 dpf. Fish were treated with either vehicle (left) or adrenosterone (right). The images are shown as Z-stack images using 100× magnification. Scale bar, 100 μm. C. Mean frequencies of the fish showing the dissemination patterns of mCherry-positive cells in the vehicle- or adrenosterone-treated groups. Each value is presented as mean ± SEM from three independent experiments. Images are reprinted from Nakayama et al. (2020).
Materials and Reagents
150 mm dish (Corning, catalog number: 430599)
100 mm dish (Corning, catalog number: 430167)
6-well flat-bottom plastic plates (Corning, catalog number: CLS3335)
Micron powder food (food for zebrafish larvae) (Sera)
27 G needle tip (Terumo, catalog number: NN-2719S)
Plastic tea strainer (purchased from a local supermarket)
Transgenic zebrafish line Tg (fabp10a:mCherry-T2A-Twist1a-ERT2)
Transgenic zebrafish line Tg (fabp10a:TA; TRE:xmrk; krt4:GFP) known as xmrk
Note: Tg (fabp10a:mCherry-T2A-Twist1a-ERT2) and Tg (fabp10a:TA; TRE:xmrk; krt4:GFP) are available upon request from Prof. Zhiyuan Gong, Department of Biological Sciences, National University of Singapore, Singapore.
Doxycycline (Sigma-Aldrich, catalog number: D9891)
4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich, catalog number: H6278)
Methylcellulose (Sigma-Aldrich, catalog number: M7027)
Phenoxyethanol (Sigma-Aldrich, catalog number: 77699)
NaCl (Sigma-Aldrich, catalog number: S3014)
KCl (Sigma-Aldrich, catalog number: P9541)
MgSO4·7H2O (Sigma-Aldrich, catalog number: M2773)
CaCl2 (Sigma-Aldrich, catalog number: C4901)
Doxycycline stock solution (see Recipes)
4-hydroxytamoxifen stock solution (see Recipes)
E3 medium (see Recipes)
30% methylcellulose (w/v %) (see Recipes)
Phenoxyethanol (v/v %) (see Recipes)
Equipment
Recirculating aquaculture system (Aquatic Habitat)
Fluorescence microscope (Olympus, catalog number: MVX10)
Leica TCS SP5X confocal microscope system (Leica)
Incubator for zebrafish embryos (AQUALYTIC, catalog number: 2418210)
External tank, a component of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTE)
Perforated internal tank, a component of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTI)
Polycarbonate divider, a component of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTD)
Polycarbonate lid (Tecniplast, catalog number: ZB10BTL)
Software
Image analysis software, Imaris 8 (Bitplane)
Procedure
Zebrafish spawning (Day 0)
On the night before collecting the embryos, arrange pairs of male xmrk fish and female Twist1a-ERT2 fish in a zebrafish breeding tank with a divider.
Early in the morning, remove the divider to allow the fish to spawn.
Scoop out the embryos with a plastic tea strainer.
Transfer the embryos into a 150 mm plastic dish containing E3 medium.
Remove dead embryos with a pipette.
Maintain the embryos for six days in an incubator set at 27 °C.
Change the E3 medium and remove dead embryos with a pipette daily.
Notes:
Adult Twist1a-ERT2 and xmrk transgenic zebrafish lines were maintained in a recirculating aquaculture system with an ambient water temperature of approximately 28 °C in Singapore. 6–8 pairs of female heterozygous Twist1a-ERT2 transgenic fish and male heterozygous xmrk transgenic fish were crossed in the breeding tank (one pair per tank). Young adult zebrafish (3–9 months of age) were preferentially used for the crossing. Electric lighting in the facility was switched on from 07:00 to 19:00. Brine shrimps were fed to both zebrafish lines five times a day, at 08:30, 10:30, 12:30, 14:30, and 16:30.
The Fish facility at the National University of Singapore connects to the outdoors. Therefore, the facility does not have air conditioning and the room temperature in the facility fluctuates between 25 °C and 30 °C. Embryos and larvae are maintained in an incubator set at 27 °C. Therefore, all larvae zebrafish were maintained at 27 °C to ensure consistency of the zebrafish development.
If a specific developmental stage is required, the embryos should be grown at the optimal condition of 28.5 °C, in order to allow a standard development of the embryos and larvae (Westerfield, 2007).
Feeding of the zebrafish larvae began at 5 dpf. A pinch of micron powder was diluted in 1 mL of E3 medium and added to the zebrafish larvae every morning. The zebrafish larvae were transferred into each well of a 6-well plate at a time point between 8 and 11 dpf, and a few drops of the E3 medium were added to each well every morning.
Collect Twist1a-ERT2/xmrk double transgenic zebrafish (Day 6)
Transfer the zebrafish larvae into a 50 mL tube.
To anesthetize the fish, add 2% (v/v) phenoxyethanol to the E3 medium with the micron powder that contains the fish. Thus, the final concentration of phenoxyethanol is 0.02%.
Array the fish on a lid of a 150 mm plastic dish.
Visualize under a fluorescence microscope and use a pipette to collect the fish expressing green fluorescent protein (GFP) in the skin (Figure 2).
Figure 2. A Twist1a-ERT2/xmrk double transgenic zebrafish expresses green fluorescent protein (GFP) in a skin-specific manner (A) and mCherry in a liver-specific manner (B). Representative images of GFP and mCherry signals in Twist1a-ERT2/xmrk double transgenic zebrafish at 6 days post-fertilization (dpf). Scale bar, 200 μm.
Among the GFP-positive fish, visualize under a fluorescence microscope and use a pipette to collect those expressing mCherry in the liver (Figure 2).
Transfer the collected fish into a 150 mm plastic dish containing E3 medium.
Maintain the fish for two days in an incubator set at 27 °C.
Notes:
At 6 dpf, Tg (fabp10a:mCherry-T2A-Twist1a-ERT2) begins to express the gene coding mCherry-T2A-Twist1a-ERT2 in a liver-specific manner. Tg (fabp10a:TA; TRE:xmrk; krt4:GFP) expresses GFP in a skin-specific manner. Therefore, Twist1a-ERT2/xmrk double transgenic zebrafish is indicated as mCherry- and GFP-positive in the liver and skin, respectively.
The screening process is divided into two steps. Firstly, zebrafish possessing the xmrk transgene are screened at 3–5 dpf and then the xmrk transgenic zebrafish possessing the Twist1a-ERT2 transgene are screened at 6 dpf.
The zebrafish larvae should not be overcrowded in the 150 mm plate, since overcrowding negatively affects their viability. By 6 dpf, 100–300 zebrafish larvae can be maintained in the 150 mm plate. After 6 dpf, a maximum of 100 zebrafish larvae should be maintained in the 150 mm plate.
Primary tumor induction (Day 8)
Aliquot a maximum of 100 zebrafish larvae into a 150 mm plate along with 100 mL of E3 medium.
At 8 dpf, treat the fish with 30 μg/mL of doxycycline in E3 medium for three days.
Change the E3 medium containing doxycycline every two days.
Note: 100 μL of doxycycline stock solution (30 mg/mL) is added to 100 mL of E3 medium.
Chemical treatment (Day 11)
Aliquot approximately 20 zebrafish larvae into each well of a 6-well plate with 8 mL of E3 medium containing doxycycline (30 μg/mL) at a time point between 8 and 11 dpf.
Add each test chemical to each well of the plate at a final concentration of 5 μmol/L.
Notes:
All test chemicals are dissolved in DMSO at a concentration of 10 mmol/L (stock solution).
4 μL of each stock solution (10 mmol/L) is diluted with 100 μL of E3 medium and added into each well of the plate containing 8 mL of E3 medium.
Metastatic dissemination induction in the presence of a chemical (Day 11.5)
Beginning 12 h after the addition of the test chemical, treat the fish with 0.1 μmol/L of 4-OHT in E3 medium for five days.
Change the E3 medium containing doxycycline, 4-OHT, and the test chemical every two days.
Notes:
The quality of the E3 medium is critical for the survival of the zebrafish larvae. The fish excrete waste materials including urine. Waste materials and leftover foods are harmful for the viability of the fish. Thus, the E3 medium should be changed before a high concentration of waste materials is reached.
We recommend changing the E3 medium every two days after 8 dpf.
Data analysis
Timing: Day 16
At five days after the first 4-OHT addition, transfer the zebrafish larvae into a 50 mL tube.
To anesthetize the fish, add 160 μL of 2% phenoxyethanol (v/v) into E3 medium containing the fish. The final concentration of phenoxyethanol is 0.02% (v/v) in a total volume of 16 mL.
Array the fish on the lid of a 100 mm plastic dish.
Embed the fish with a drop of 30% methylcellulose.
Manually orient the fish into a lateral view using a 27 G needle tip.
Determine the pattern of cell dissemination in the fish under a fluorescence microscope.
Count the number of fish showing each dissemination pattern of mCherry-labeled cells from the liver.
For taking images, capture serial sections of the fish in 8 μm Z-step intervals using a Leica TCS SP5X confocal microscope system. Process Z-stack images using the image analysis software Imaris (Bitplane).
Notes:
The dissemination patterns are generally divided into three categories: (i) local dissemination, in which disseminated mCherry-positive cells exist in close proximity to the liver; (ii) abdominal dissemination, in which the cells spread throughout the abdomen; and (iii) distant dissemination, in which the cells are observed over a broad region from the trunk to the tail (Figure 1).
The suppressor effect of a test drug is evaluated by comparing the frequencies of the fish showing the abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group.
The dissemination patterns are classified by viewing the fish under a fluorescence microscope. To avoid bias, the experimenter should be blinded to the treatments.
Limitations
There is a limitation on the number of Twist1a-ERT2/xmrk double transgenic zebrafish that can be prepared (a few hundred). This limitation determines how many chemicals can be tested in one screening session. To test one chemical, approximately 20 double transgenic fish are needed. Following Mendel's laws, the rate of the double transgenic fish production is 25% when a heterozygous Twist1a-ERT2 transgenic zebrafish is crossed with a heterozygous xmrk transgenic zebrafish. If 20 chemicals are to be tested, at least 400 double transgenic fish would be required. To prepare 400 double transgenic fish, pairs of Twist1a-ERT2 and xmrk fish would need to generate approximately 2,000 embryos. Thus, when following the protocol described above, a maximum of 20 test chemicals can be evaluated.
Recipes
Doxycycline stock solution
Reagent Final concentration Amount
Doxycycline 50 mg/mL 500 mg
ddH2O n/a 10 mL
Total n/a 10 mL
4-hydroxytamoxifen stock solution
Reagent Final concentration Amount
4-OHT 100 mM 387 mg
Ethanol n/a 1,000 mL
Total n/a 1,000 mL
E3 medium
Reagent Final concentration Amount
NaCl 5.0 mM 0.292 g
KCl 0.17 mM 0.013 g
MgSO4·7H2O
CaCl2
ddH2O
Total
0.33 mM
0.33 mM
n/a
0.081 g
0.048 g
1,000 mL
1,000 mL
30% methylcellulose (w/v %)
Reagent Final concentration Amount
Methylcellulose 30% 30 g
ddH2O n/a 100 mL
Total n/a 100 mL
Phenoxyethanol (v/v %)
Reagent Final concentration Amount
Phenoxyethanol 0.02% 20 μL
ddH2O n/a 100 mL
Total n/a 100 mL
Acknowledgments
Graphical abstract was drawn by Ami Inoue (Kyoto University of the Arts). This study was funded by Ministry of Education of Singapore (MOE2019-T2-2-018) to Z. Gong. This protocol originates from Nakayama et al. (2020).
Competing interests
J.N., H.M., and Z.G. declare no conflict of interest.
Ethics
The study protocol was approved by the Institutional Animal Care and Use Committee of the National University of Singapore (protocol number: 096/12).
References
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Chaffer, C. L. and Weinberg, R. A. (2011). A perspective on cancer cell metastasis. Science 331(6024): 1559-1564.
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Letrado, P., de Miguel, I., Lamberto, I., Díez-Martínez, R. and Oyarzabal, J. (2018). Zebrafish: Speeding Up the Cancer Drug Discovery Process. Cancer Res 78(21): 6048-6058.
Lu, J.-W., Sun, Y., Lin, L.-I., Liu, D. and Gong, Z. (2021). Exacerbation of Liver Tumor Metastasis in twist1a+/ xmrk+ Double Transgenic Zebrafish following Lipopolysaccharide or Dextran Sulphate Sodium Exposure. Pharmaceuticals (Basel) 14(9): 867.
Lu, W. and Kang, Y. (2019). Epithelial-Mesenchymal Plasticit in Cancer Progression and Metastasis. Dev Cell 49(3): 361-374.
Westerfield, M. (2007). The Zebrafish Book, 5th Edition. A guide for the laboratory use of zebrafish (Danio rerio), Eugene, University of Oregon Press. Kimmel, C. B. et al. (1995). Stages of embryonic development of the zebrafish. Dev Dyn 203(3): 253-310.
Nakayama, J. and Gong, Z. (2020). Transgenic zebrafish for modeling hepatocellular carcinoma. MedComm 1(2): 140-156.
Nakayama, J., Konno, Y., Maruyama, A., Tomita, M. and Makinoshima, H. (2022a). Cinnamon bark extract suppresses metastatic dissemination of cancer cells through inhibition of glycolytic metabolism. J Nat Med 76(3): 686-692.
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Nakayama, J., Makinoshima, H. and Gong, Z. (2022b). Gastrulation Screening to Identify Anti-metastasis Drugs in Zebrafish Embryos. Bio Protoc 12(19): e4525.
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Nakayama, J., Tan, L., Li, Y., Goh, B. C., Wang, S., Makinoshima, H. and Gong, Z. (2021b). A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis. Elife 10: e70151.
Nakayama., J., Lu., J.-W., Makinoshima., H. and Gong., Z. (2020). A Novel Zebrafish Model of Metastasis Identifies the HSD11β1 Inhibitor Adrenosterone as a Suppressor of Epithelial-Mesenchymal Transition and Metastatic Dissemination. Mol Cancer Res 18(3): 477-487.
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Drug Discovery > Drug Screening
Cancer Biology > Invasion & metastasis > Drug discovery and analysis
Cell Biology > Cell movement > Cell motility
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Direct Adeno-associated Viruses Injection of Murine Adipose Tissue
SW Shao-Chin Wu
CL Chi-Hung Lin
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4674 Views: 844
Reviewed by: Mohammed Mostafizur RahmanRachael E. Hokenson Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Communications Jul 2022
Abstract
The adipose tissue is a central metabolic organ that regulates whole-body energy homeostasis. The abnormal expansion of adipose tissue leads to the progression of obesity. The adipose tissue microenvironment is affected by pathological hypertrophy of adipocytes, highly correlated with systemic metabolic disorders. In vivo genetic modification is a great tool for understanding the role of genes involved in such processes. However, obtaining new conventional engineered mice is time consuming and costly. Here, we provide a simple and speedy method to efficiently transduce genes into adipose tissue by injecting the adeno-associated virus vector serotypes 8 (AAV8) into the fat pads of adult mice.
Keywords: Adipose tissue AAV Gene transfer Mice Virus
Background
Obesity is a severe global health problem characterized by excess adipose tissue expansion and strongly associated with metabolic diseases such as diabetes, cardiovascular and hepatic lipid diseases, and some types of cancers (Haslam and James, 2005; Swinburn et al., 2011; Wabitsch et al., 2015). The growth of the adipose tissue can be attributed to the enlargement of the existing adipocytes (hypertrophy) or the formation of new adipocytes (hyperplasia) (Salans et al., 1973; Spalding et al., 2008; Jo et al., 2009). Unlike the protected role of adipocyte hyperplasia (Vishvanath and Gupta, 2019), hypertrophic adipocytes are more responsible for lipid homeostasis disorders and pathological consequences (Haczeyni et al., 2018). Moreover, besides its usage as a container for lipid storage, the adipose tissue also plays crucial roles in regulating metabolic and endocrine functions. Therefore, there is an urgent need to clarify the obesity pathogenic mechanism, develop safe anti-obesity therapeutic strategies, and further avoid the pandemic dimensions of obesity.
Genetic engineering of mice is a commonly used approach to clarify gene function in vivo. However, the production of new transgenic mice is time consuming and costly (Jimenez et al., 2013; Bates et al., 2020). Some undesired side effects can be observed in conventional genetically modified mice: the manipulated gene that works on the whole body may interfere with its primary function on a specific tissue. In vivo gene transfer to the target organ has become a faster, lower-cost, and more specific strategy. Using this strategy, the time points of gene delivery can be arranged based on experimental requirements. It can also eliminate the undesired effects on embryo development.
Adeno-associated virus (AAV) vectors, discovered in the 1960s, are considered one of the safest and most promising tools for in vivo delivery of gene therapies (Atchison et al., 1965; Wang et al., 2019). They are small (25–26 nm in diameter), non-enveloped viruses composed of an icosahedral capsid that contains a linear single-stranded DNA genome (approximately 4.7–4.9 Kb). In contrast to the adenovirus, retrovirus, and lentivirus, AAVs show a relatively safe profile with apathogenicity and low immunogenicity (Cao et al., 2011). They can transduce genes into dividing and non-dividing cells (Flotte et al., 1994), allowing long-term transgene expression in such tissues. Among the 13 AAV serotypes that have been identified (Pipe et al., 2019), at least three serotypes (AAV2/8/9) exhibit efficient gene transfer to adipose tissue of adult mice (O'Neill et al., 2014; Uhrig-Schmidt et al., 2014; Bates et al., 2020). Recently, we successfully applied this protocol on direct AAV8 injection into the subcutaneous adipose tissue. In this protocol, we demonstrate a detailed procedure for efficient gene delivery into adipocytes of adult mice.
Materials and Reagents
Animals: 8–12-week-old C57BL/6JNarl mice (National Laboratory Animal Center)
Rodent MD’sTM Rimadyl (Carprofen, 2 mg/tablet) (Bio-Serv, catalog number: SMD150-2)
AAV8 vectors carrying enhanced green fluorescent protein (GFP) as reporter gene driven by CMV promoter (obtained from National RNAi Core Facility at Academia Sinica, Taiwan)
PBS (Gibco, catalog number: 10010023)
75% ethanol
FORANE® isoflurane (Abbott, catalog number: B506)
Bacitracin-neomycin ointment (Shiteh, catalog number: 022990)
Equipment
Matrx® anesthesia machine (Midmark, catalog number: VIP3000) (Figure 1, ①)
Anesthetic gas recovery machine (Step, catalog number: R-600) (Figure 1, ②)
Anesthetized mouse facemask (RWD, catalog number: 68635) (Figure 1, ③)
Surgery tweezers (Shineteh, catalog number: ST-TW011) (Figure 1, ④)
Surgery scissor (Shineteh, catalog number: ST-S011PK) (Figure 1, ⑤)
50 µL syringe (Hamilton, catalog number: 80901); needles sold separately (model: 1705 LT) (Figure 1, ⑥)
Needle (30 G × ½) (BD Precision GlideTM, catalog number: 305106) (Figure 1, ⑦)
Needle holder (Shineteh, catalog number: ST-H212) (Figure 1, ⑧)
Sterilized Suture, 4-0, 12 mm 3/8 circle (UNIK, catalog number: SC124) (Figure 1, ⑨)
Biosafety Cabinet (ClassII)
Hair clipper (Orbaner, catalog number: MB-022)
Figure 1. Setup of experiment. ① Anesthesia machine, ② Anesthetic gas recovery machine, ③ Anesthetized mouse facemask, ④ Surgery tweezers, ⑤ Surgery scissor, ⑥ Syringe, ⑦ 30 G needle, ⑧ Needle holder, ⑨ Sterilized suture.
Procedure
Replace the diet with rodent MD’sTM Rimadyl 16 h before surgery to prevent postoperative pain.
Anesthetize mice in an anesthesia chamber filled with isoflurane at a flow rate of 5% in 100% oxygen.
Assess the level of anesthesia by pinching the mice’s hind toes or tail end (no withdrawal should be observed).
Transfer mice to a biosafety cabinet and put on an anesthetized mouse facemask. Adjust the rate of isoflurane to 1.5%–2.5% in 100% oxygen.
Shave a small area in the flanks and proximal hip joints with a hair clipper (Figure 2A).
Video 1 shows the procedures in steps 5–10.
Video 1. Procedures of direct adeno-associated virus (AAV) injection of murine adipose tissue. The video was produced at National Yang Ming Chiao Tung University (NYCU), where all procedures followed guidelines from the NYCU and were approved by the Institutional Animal Care and Use Committee (IACUC) of NYCU (IACUC approved number: 1090706, 1090706r, 1100332).
Clean the shaved region with 75% ethanol.
Prepare an AAV8 solution in PBS. AAV8 will be administered at a total of 1 × 1011–2 × 1011 viral genomes (VG) AAV8 in a 30 μL volume for one fat pad per mouse. Fit a 30G needle securely with Hamilton syringe. Preload syringe with the AAV8 solution. Avoid air in the syringe.
Tent the skin with the tweezers and make a 0.5–1 cm incision in the skin using surgical scissors.
Expose the fat pad and carefully insert a 30G needle until the needle bevel enters the tissue. Inject the AAV8 slowly into 4–5 distinct spots (6–8 μL per spot) of the fat pad. Hold the needle in place for a little while after each injection to prevent backflow into the syringe (Figure 2B).
Hold the needle of the 4-0 braided silk with a needle holder and suture the incision (Figure 2C). (Alternative: close the incision with Michel's suture clips.)
Apply ointment to the incision region until it heals to prevent post-operative infection.
Transfer the mice to a clean cage on a heating pad. Monitor the mice closely until they regain consciousness.
Each experimental mouse must be single housed in a cage after surgery until the incision heals. Provide mice with Rimadyl within 48 h after the surgery to alleviate pain.
After one week from injection, the GFP signal is detectable.
Figure 2. Direct adipose adeno-associated virus (AAV) injection procedure. A. Shave and clean the surgery area at the flank and proximal hip joints. The surgical incision region is shown as a double-headed arrow. B. Make a 0.5 cm incision and carefully inject the AAV into multiple spots in the fat pad. C. Suture the incision using 4-0 braided silk.
Notes
All materials used in this experiment must be sterilized or autoclaved to prevent contamination.
Observe the mice daily to ensure there are no surgical complications such as infection, bleeding, or poor wound healing. Apply the ointment to the incision region or prolong the Rimadyl feeding if particular surgical complications arise.
Acknowledgments
We thank the National RNAi Core Facility at Academia Sinica in Taiwan for providing AAV8 reagents and related services. This work was financially supported by the “Cancer Progression Research Center, National Yang Ming Chiao Tung University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. This work was supported by the MOST 111-2740-B-A49-001 and NSTC 111-2634-F-A49-014. We have successfully applied this protocol to our current publication (Wu et al., 2022).
Competing interests
The authors declare that no competing financial interests exist.
Ethics
All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University. (IACUC #1090706, 1090706r, 1100332) and housed on a 12:12 h light/dark cycle at 22 °C.
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4,675 | https://bio-protocol.org/en/bpdetail?id=4675&type=0 | # Bio-Protocol Content
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Quantifying Single and Dual Channel Live Imaging Data: Kymograph Analysis of Organelle Motility in Neurons
LD Laura Digilio
LM Lloyd P. McMahon
AD Alois Duston
CY Chan Choo Yap
BW Bettina Winckler
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4675 Views: 1009
Reviewed by: Xi FengJose Martinez Hernandez Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Neuroscience Jun 2022
Abstract
Live imaging is commonly used to study dynamic processes in cells. Many labs carrying out live imaging in neurons use kymographs as a tool. Kymographs display time-dependent microscope data (time-lapsed images) in two-dimensional representations showing position vs. time. Extraction of quantitative data from kymographs, often done manually, is time-consuming and not standardized across labs. We describe here our recent methodology for quantitatively analyzing single color kymographs. We discuss the challenges and solutions of reliably extracting quantifiable data from single-channel kymographs. When acquiring in two fluorescent channels, the challenge becomes analyzing two objects that may co-traffic together. One must carefully examine the kymographs from both channels and decide which tracks are the same or try to identify the coincident tracks from an overlay of the two channels. This process is laborious and time consuming. The difficulty in finding an available tool for such analysis has led us to create a program to do so, called KymoMerge. KymoMerge semi-automates the process of identifying co-located tracks in multi-channel kymographs and produces a co-localized output kymograph that can be analyzed further. We describe our analysis, caveats, and challenges of two-color imaging using KymoMerge.
Keywords: Kymograph KymoMerge Time-lapse imaging Co-localization FIJI Dual-channel imaging Neurons Dendrites
Background
Time-lapse imaging using fluorescence microscopy is a useful tool for studying vesicle trafficking in neurons. Information about vesicle behaviors (such as speed, directionality, pause times, etc.) needs to be quantified in order to understand and compare how different vesicle populations behave under different conditions. Extracting quantitative information from live imaging data is time consuming and carried out differently by different labs. Kymographs are often used to easily display vesicle behavior over time in a figure but can also be used to quantify these behaviors. A kymograph shows the directionality on the x-axis and time along the y-axis. This technique has applications across a wide variety of studies: it is used in studying microtubule growth (Zwetsloot et al., 2018), kinetochore movement (Hertzler et al., 2020), lamellipodial advance or collapse (Menon et al., 2014), and, probably most commonly, vesicle movement in neurons (Maday and Holzbaur, 2016; Farías et al., 2017; Farfel-Becker et al., 2019). Neuronal processes are particularly amenable to the use of kymograph analysis because of their inherent linear morphology. The highly polarized structure of axons and dendrites provides built-in tracks along one axis where movement of a variety of biological structures can be followed. With fluorescent labeling, either from adding fluorescent tracers or by transfection with plasmids encoding fluorescent proteins, various processes and structures can be analyzed, such as trafficking of organelles (Wang et al., 2009; Yap et al., 2018) or cytoskeletal elements (Liang et al., 2020; Ganguly and Roy, 2022).
Kymographs contain a great deal of information about trafficking dynamics. Parameters available for analysis include the number of anterograde, retrograde, and stationary events, event speed, pause time, and event distance. Quantifying these parameters can be done manually or by several available software programs suitable for one-channel images. If these parameters are to be measured under the condition where two proteins are trafficking together, events must be identified that coincide in both channels. A review of the literature shows that current kymograph analysis is largely limited to tracking one channel at a time (Lasiecka et al., 2010; Chien et al., 2017; Boecker et al., 2020). The common method of creating kymographs from time-lapse fluorescent microscope data is done on one channel, producing one independent kymograph per marker with no direct connection between them, even though multiple channels can easily be acquired. To analyze two objects that co-traffic together, one has to carefully examine the kymographs from both channels and decide which tracks are the same, or one could try to identify the coincident tracks from an overlay of the two channels (see Lasiecka et al., 2014 as an example). Tracks can merge and diverge, and such events are not easy to identify when looking at two separate images, so marking them can be a challenge. The whole process is laborious and time consuming. Detailed information from two kymographs would only be useful if all the data of interest were in one image. Our lab has been studying the dynamics of a variety of endosomal compartments and their inter-relationship in neurons using more than one endosomal marker (Yap et al., 2008, 2017 and 2018). The difficulty in finding an available application for such analysis has led us to create a program to do so, called KymoMerge (McMahon et al., 2021). KymoMerge addresses the issues discussed by automating the process of identifying co-located tracks in multi-channel kymographs and producing an output that can be analyzed directly.
Software
FIJI open-source image analysis software (https://imagej.net/software/fiji/downloads)
Procedure
Live imaging
Live imaging was done as previously described (Yap et al., 2018). Brief procedure description:
Neuronal cultures are prepared from E18 rat hippocampi combining all embryos from one litter (as described in Lasiecka and Winckler, 2016). Other cultured neurons can also be used.
Cells are plated on a 35 mm glass-bottomed microwell dish coated with poly-L-lysine. After 4 h, the plating medium is removed and replaced with serum-free medium supplemented with B27, and neurons are cultured for 7–10 days in vitro (DIV) for experimental use.
Transfections are performed with Lipofectamine 2000 according to manufacturer’s instructions.
All live imaging is performed on a 37 °C heated stage in a chamber with 5% CO2 on an inverted LSM880 confocal microscope using a 40× water objective (LD-C Apochromat 1.2W). Spinning disk confocal microscopes are also well suited for imaging cultured neurons live. Higher magnification objectives can also be used, as per the research question.
Images are collected from single or dual channels using the bidirectional scan-frame mode with the lowest laser power that allows visualization of object of interest. Spinning disk confocal microscopy is also very well suited for imaging live organelle movements in cultured neurons. Images are captured at 1–2 frames per second, but faster moving organelles may require more frequent image acquisition. We routinely capture 400–500 frames (approximately 8 min) before photobleaching becomes substantial. If longer imaging times are desired, frame capture rates can be less frequent (one per 3 s, one per 10 s) with the caveat that very fast–moving events will be missed. A single focal place is chosen for imaging such that the most organelles are in focus. The speed of most motile organelles precludes z-stack acquisition during live imaging for microscope in common use.
Images are saved as TIFF files and opened in FIJI.
Note: We always carry out all control and experimental conditions for any experiment on the same cultures on the same day and image on the same day. This ensures that any differences in motility are not due to culture-to-culture variation, but due to the experimental manipulation. We have found this to be an important aspect of experimental design and rigor.
Single-channel kymograph production
Open the live imaging image file in FIJI and display the first frame of the live imaging file. Since the position of the dendrites do not change over the course of the live imaging, the first frame can be used for tracing.
Open the ROI manager (Analyze > Tools > ROI manager).
Using the segmented line tool in FIJI, trace the dendrite or axon starting at the soma and extend out to desired end point, and add this line to the ROI manager. Continue drawing, tracing the rest of the dendrites, and add them to the ROI manager (Figure 1). Dendrites with a lot of crisscrossing or bundling are omitted. Click Show All and Labels boxes to view all traces.
Highlight the trace in ROI manager and run the Multiple Kymograph Plugin in FIJI, which is included in its updated version. When prompted for the line width, enter the pixel width of the dendrite you wish to make a kymograph from. With our imaging conditions, typical values of line width are 3 pixels for axons and 5 pixels for dendrites. Once you enter line width, the kymograph will generate.
Save the kymograph of this dendrite as a TIFF file. Continue making kymographs (as in steps B3 and B4) from the remaining dendrites of the same cell. In our imaging conditions (40× objective), all dendrites of one neuron are captured simultaneously in the same live imaging (see example in Figure 1) and multiple kymographs can be obtained from one live imaging session.
Figure 1. Tracing dendrites and adding to the ROI manager. Single plane image of the first time-lapse frame of a neuron transfected with GFP-RILP (Rab-interacting lysosomal protein) is shown. Dendrites are traced using the segmented line tool and added to the ROI manager. One trace is omitted because of crisscrossing dendrites.
Once you are finished making the kymographs from the cell, save the image with the traced ROI on top of it. To do this, click Image > Overlay > From ROI Manager and then save as TIFF file for reference. This will allow you to go back to each cell at any point in the future and see which dendrite each kymograph came from.
Note: Potential problem—if there is bleaching during the imaging, use the bleach correcting plugin for FIJI (Miura, 2020; https://imagej.net/plugins/bleach-correction). If one experiences microscope stage drift during the imaging, a drift correction plugin exists in FIJI (https://imagej.net/plugins/manual-drift-correction).
Kymograph analysis
Multiple software packages exist. We have used manual analysis (Lasiecka et al., 2010 and 2014) as well as Kymograph Clear/Kymograph Direct software (Barford et al., 2018). These are briefly described in the Note below. Other programs exist and will also work.
Most recently, we have used KymoButler (Yap et al., 2022), a web-based software (Jakobs et al., 2019) available to customize with machine learning for a fee. A free version is available, which outputs velocities, track duration, and distances. https://www.wolframcloud.com/objects/deepmirror/Projects/KymoButler/KymoButlerForm. KymoButler is also available through a plugin for FIJI https://github.com/fabricecordelieres/IJ-Plugin_KymoButler_for_ImageJ.
Analysis: KymoButler output can be customized to fit desired criteria for the questions of interest. For our purposes, KymoButler was customized to identify individual events and calculate data for each event, including speed, duration of movement or pausing, distance moved, or direction of movement. KymoButler also creates vesicle trajectories, which are comprised of multiple individual events. Data can be compiled for trajectories in addition to individual events. Creating trajectories from individual events can often not be accomplished for all vesicles for the entirety of the time-lapse imaging, but shorter unambiguous trajectories can usually be identified. In our experience, the trajectories created manually by a human user are almost identical to those created by KymoButler, since the same limitations exist for trajectory assignment by both (Figure 2).
Figure 2. Software output for kymograph analysis. Original kymographs (A) are assigned events per user preference (for instance, defining motile events as ≥2 µm movements) (B) or trajectories (C). All events or trajectories identified by the software can be quantified in multiple ways, including speed, directionality, run lengths, or pause times.
We have found it useful to use trajectory data to determine the net motility of individual vesicles over a longer time of the whole live imaging experiment instead of individual events. Net retrograde/anterograde/stationary data can be obtained from these measurements. For our purposes, we distinguished stationary trajectories (zero net movement) from short (<2 µm) and long (>2 µm) net movements. Theses cutoffs can be chosen by the user for their own questions. The raw data includes a full readout of distances traveled and can be binned into stationary vs. motile as per the user’s wishes. Since endosomes in dendrites pause and reverse direction a lot, we were more interested in the net behavior of vesicles and less interested in individual event measurements. For other questions, the user might be interested in individual event data. We create kymographs from all dendrites on any imaged cell for analysis (see Figure 1) and do not exclude dendrites unless they are not in focus or crisscrossing or bundling. We usually combine data from all the dendrites from one cell into one data point of motility data. So, the number of samples (n) for statistical purposes is usually one cell and not one dendrite or one vesicle. We find that imaging of ~20 cells (80–100 dendrites) gives good statistical power for endosome motility in dendrites. We recommend imaging at least 4–5 cells from 3–4 independent cultures and making kymographs from all dendrites for statistical analysis.
Note: We repeatedly trained the machine learning function of KymoButler on 20 kymographs for which ground truths had been established manually. Once the output from KymoButler closely matched the manual ground truth established by a human user, the same settings were used for all analyses. This initial training might need to be repeated for different types of fluorescent markers, because background haze might differ, and the same settings might not give satisfactory results. The event and trajectory assignments for each kymograph can be visually verified after KymoButler has finished to ensure accuracy.
Notes:
1. Manual analysis: Using the line tool in FIJI, manually trace all tracks on the kymograph and save them to the ROI manager as above. Using the known pixel size as determined from the objective and camera used, one can determine average velocities, retrograde and anterograde events, and pause events/times. This is laborious and time consuming, but the user has maximal control over the output. We usually validate software automation by comparing their output to manually established ground truth measurements for 10–20 kymographs.
2. Kymograph Clear/Kymograph Direct: open-source programs (Mangeol et al., 2016) available online for download and use with FIJI. These are sequential kymograph production and analysis programs that give user information regarding particle velocities, intensities, directionality, and run lengths. http://sites.google.com/site/kymographanalysis.
Double-channel kymograph: preparation and analysis
For generating double-channel kymographs, we created a plugin for ImageJ FIJI, called KymoMerge (currently only functional when using a Mac; McMahon et al., 2021).
Install KymoMerge: download from https://github.com/alduston/kymomerge. To begin using the merge tool, open FIJI and open the KymoMerge.py script in the FIJI console. The program can be installed as a plugin or run from the console.
Input the directory folders for the kymographs from each channel (Figure 3A).
Figure 3. KymoMerge workflow. (A) Input directory window. This window will be used to choose the folders containing the kymographs from each channel. (B) Image thresholding window. This window will be used to input threshold values determined by the user for all images individually from each channel. Thresholding depends on the level of background fluorescence and the brightness of the signal. Each user needs to visually determine the optimal threshold value for their particular image.
The files from each input directory (individual kymograph files) should have identical naming. Input directory should be a directory containing a series of named .tif files, using the naming convention ‘filename’-‘i’.tif, where i is the given .tif files ‘index’ in the folder. For instance, given three files in ‘groupA’ directory, using ‘kymo’ as file name, the .tif files would be given “kymo-1.tif, kymo-2.tif, kymo-3.tif.”
Input directory 2 should be a directory containing a series of named .tif files, using the naming convention ‘filename’-‘i’.tif, where i is the given .tif files ‘index’ in the folder. This index and file name should correspond to the index of input directory 1. The algorithm will compare files with corresponding indexes. For instance, given three files in ‘groupB’ directory, using ‘kymo’ as the file name as before, the .tif files would be given “kymo-1.tif, kymo-2.tif, kymo-3.tif.”
Kymograph image thresholding
The dialog box and image on the left comes up and shows the original kymograph along with an input to set the threshold (Figure 3B). The contrast for the original image is automatically increased so the data are clearly visible. When a value is chosen for the threshold by the user (typically 10–100 depending on background fluorescence and brightness of signal), the binarized image appears and can be compared to the original, as shown with the dialog box and images on the right. At this point, the user can accept the value by typing in Yes or choosing OK or try a new value by typing in No or choosing Cancel. Once a value is accepted, the next image opens. Once all images from the input folders are thresholded, they will be processed, and the output (individual binary images and merged image) will be available for further analysis as described above. Should the user want to quit the analysis at any point, simply type Quit into the dialog box.
Once the threshold is set, it is applied, and the image is converted to eight bits from the bit depth of the original image. This is then converted to a binary file and saved. Each pixel in the two channels is now set to a value of either 0 or 254.
Note: 8-bit images are used at this point because 16–32 bit formats interpolate locally to achieve greater color depth and make direct manipulation of individual pixel values more difficult.
The program then goes pixel by pixel between the two kymographs comparing values and creating a new image. If the values are both zero, or one is zero and the other is 254, the new image is set at zero at that pixel. If they are both 254 (positive signal in both channels), then the value is set at 254. The result is an image consisting only of those pixels where a positive signal is in both channels. This produces a kymograph containing tracks where the two proteins of interest are co-located, and the dynamics of the co-localized tracks can now be analyzed. The folder of co-located kymographs is saved in a new “output folder” in the folder containing the folder of original kymographs. The output folder contains the binarized kymographs from the input folders and the new, co-located kymograph. The program will open a file from the Group A directory of kymograph files (channel 1) for the user to threshold, followed by the corresponding file from the Group B directory (channel 2). This will continue until all the images are thresholded.
Notes
KymoMerge vs. manual kymograph analysis
Two sample data sets are used to test and validate the reliability and efficiency of the program. One consists of the neuronal membrane proteins NSG1-cherry and NSG2-GFP. NSG1 and NSG2 are members of a neuron-specific gene family of proteins. Both proteins are highly expressed in neurons and localized to a variety of endosomal compartments in dendrites (Yap et al., 2017). Our previous live imaging data showed that both proteins co-trafficked in neurons, and thus serve as a good example for analyzing the dynamics of two highly co-localized membrane cargos with low background noise signals (Figure 4A). The second data set contains NSG1-cherry with GFP-RAB7, a late endosome marker. RAB7 is a small GTPase involved in regulating transport to late endocytic compartments. We have previously shown that RAB7 co-trafficked and regulated the endocytic transport of NSG1 in neurons (Yap et al., 2017). Like other Rabs, RAB7 is constantly cycling between the activated form (GTP-bound) when it is recruited to membranes and inactivated form (GDP-bound) after hydrolysis and when it is cytosolic. This feature allows us to test the ability of our program in extracting membrane-bound signals from cytosolic high background signals (Figure 4C).
Visualizing co-localized tracks
In Figure 4, we give examples of typical output from KymoMerge. Figure 4A shows kymographs from the NSG1 and NSG2 data set with NSG1 in red and NSG2 in green, while Figure 4C shows example kymographs from the GFP-RAB7 and NSG1-mcherry data set. The top row shows the original 32-bit single-channel images followed by an overlay of the two channels. Below are the binary outputs from KymoMerge. Careful comparison of the original images in Figure 4A, C with the binary output of KymoMerge (Figure 4B and 4D) shows very similar tracks between the two. Due to the binary nature of the KymoMerge output, it does not show intensity differences as in the original files, but this is not a parameter that is usually of interest in kymograph analysis. Intensity differences are only used initially in determining background levels and true signal during the thresholding stage before the binary output is created. Comparison of the overlayed and merged files also shows similar characteristics.
Figure 4. Comparison of original images vs. KymoMerge-generated images. Dual live imaging was carried out for two transmembrane proteins, NSG1-mCh and NSG2-GFP (A and B), and for NSG1-mCh with a cytosolic regulator GFP-RAB7 (C and D). The original kymographs for each channel and a merged kymograph are shown in (A) and (C). The binarized single-channel kymographs created by KymoMerge are shown in (B) and (D), together with the co-localized output kymograph. This final output of co-localized pixels can be used for further quantitative analysis.
One of the principal strengths of our approach is that any track seen in the merged binary data is a co-located track. In contrast, it can be difficult to discern coincident tracks in the original data.
Testing the robustness of KymoMerge
Our approach is useful only if the program produces results that are robust and reliable. We find that KymoMerge produces results comparable to those from a careful count done by hand, which is often how kymograph data is analyzed. For this purpose, we took the two data sets referenced in Figure 4A, B and 4C, D and independently counted anterograde and retrograde events (≥ 2 μm) manually, referencing either the original and overlayed data (as shown in Figure 4A and 4C) or using the merged binary output from KymoMerge (as shown in Figure 4B and 4D). The results in Figure 5A and 5B show that there was no statistical difference between the two methods (Mann-Whitney test).
Figure 5. Comparison between manual counts of anterograde and retrograde events (≥ 2 μm) using original 32-bit images and KymoMerge created kymographs. (A) n = 26 dendrites from seven neurons in the NSG1-mCherry/NSG2-GFP data set. (B) n = 36 dendrites from seven neurons in the NSG1-mCherry/GFP-RAB7 data set. Statistical results from Mann-Whitney test between anterograde or retrograde pairs.
Caveats on binary thresholding
Thresholding of images is often necessary in image analysis, but it can also be somewhat subjective and, therefore, potentially problematic. With KymoMerge, thresholding requires attention particular to the process of creating binary images. There is a difficulty inherent to the process of creating the binary images: if one region is noisier than another, the noise can be of the same magnitude as the signal elsewhere. The result is that the set threshold will either eliminate actual signal if it is at a level suitable for the noisier regions, or potentially background will be left, creating false co-localized tracks. Careful thresholding with KymoMerge can produce an accurate and interpretable merged kymograph. For kymographs with areas of high background, it may be necessary to crop excessively noisy areas before analysis. Since these areas would generally be uninterpretable even with manual analysis, analyzable data are not being lost and the rest of the image will produce accurate results. It is important to understand that any data set is only a sampling of the system being studied. It is better to err on the side of excluding false positive co-localized tracks and analyze only the resulting tracks that are truly co-localized. Because KymoMerge allows for individual screening and thresholding of the individual channels before the merged images are created, any problematic kymographs can be identified by the user and properly processed. More examples are shown in McMahon et al. (2021), including direct comparisons of two different thresholds used on the same data sets.
Acknowledgments
This research was supported by NIH R01NS083378. The initial publication where this method is published: Yap et al. (2022).
Competing interests
No competing financial interests for this study.
Ethics
Neuronal cultures used for live imaging were prepared from E18 rat embryos as approved by the University of Virginia Animal Care and Use Committee. All experiments were performed in accordance with relevant guidelines and regulations (ACUC #3422).
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4,676 | https://bio-protocol.org/en/bpdetail?id=4676&type=0 | # Bio-Protocol Content
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Measuring in vitro ATPase Activity with High Sensitivity Using Radiolabeled ATP
SV Sarina Veit
TP Thomas Günther Pomorski
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4676 Views: 879
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Original Research Article:
The authors used this protocol in Journal of Fungi Jul 2021
Abstract
ATPase assays are a common tool for the characterization of purified ATPases. Here, we describe a radioactive [γ-32P]-ATP-based approach, utilizing complex formation with molybdate for phase separation of the free phosphate from non-hydrolyzed, intact ATP. The high sensitivity of this assay, compared to common assays such as the Malachite green or NADH-coupled assay, enables the examination of proteins with low ATPase activity or low purification yields. This assay can be used on purified proteins for several applications including the identification of substrates, determination of the effect of mutations on ATPase activity, and testing specific ATPase inhibitors. Furthermore, the protocol outlined here can be adapted to measure the activity of reconstituted ATPases.
Graphical overview
Keywords: ABC transporter ATPase ATP hydrolysis Radioactive assay 32P-ATP P-type ATPase
Background
Through all the kingdoms of life, ATPases are the common enzyme class to catalyze reactions that would otherwise not take place due to high energy barriers. All ATPases use the dephosphorylation of ATP to ADP, resulting in the release of inorganic phosphate. Additionally, the decomposition of ATP provides energy that ATPases use for conformational changes in a variety of ways. Since most ATPases are transmembrane proteins, conformational changes enable the transport of substrates along the membrane and even against the concentration gradient.
Despite intensive research, many details on the mechanism(s) of these ubiquitous enzymes remain to be elucidated. The cellular environment is too complex to investigate one specific protein without the influence of other proteins or molecules. Therefore, most research uses a purification approach to investigate the protein of interest exclusively in the (detergent)-solubilized state or after reconstitution in model membranes, in the case of transmembrane proteins. Since substrate transport is coupled to ATP hydrolysis, one major aspect of the protein characterization is the ATPase activity. Furthermore, it is stimulated by the substrate, pH, ionic strength of the buffer, or in the case of transmembrane proteins, the lipid surroundings.
Many techniques have been used to quantify the in vitro ATPase activity of purified proteins. The most common approach to determine the ATP hydrolysis activity of ATPases is the Malachite green assay, developed in 1966 by Itaya and Ui (Itaya and Ui, 1966). This assay has been improved several times over the last few decades by different groups (Carter and Karl, 1982; Baykov et al., 1988; Geladopoulos et al., 1991; Feng et al., 2011). The method relies on the formation of a green complex when malachite green/molybdate reacts with inorganic phosphate under acidic conditions. The amount of green molybdophosphoric acid complexes can be measured with a spectrophotometer and is directly correlated with the concentration of free inorganic phosphate in the reaction. Another widely used ATP hydrolysis assay is the NADH-coupled assay (Warren et al., 1974). Here, ATP is regenerated by the pyruvate kinase, which simultaneously converts phosphoenolpyruvate to pyruvate. The latter is subsequently converted to lactate by the lactate dehydrogenase and coupled to the oxidation of NADH to NAD+. The absorption spectra of both substances at 340 nm vary significantly, allowing live absorbance- or fluorescence-based tracking of NADH consumption, and therefore, indirectly, the coupled ATP hydrolysis (Radnai et al., 2019). Based on the same principle, other fluorescence-based assays use a coupled enzyme system that catalyzes a reaction of a fluorescent substrate with a free phosphate to a product with low fluorescence (Banik and Roy, 1990). Other assays are based on a fluorescent substrate to detect free phosphate in the ATPase reaction that loses its fluorescence by binding to phosphate (Brune et al., 1994). In general, assays utilizing coupled enzyme systems have the disadvantage of being sensitive to the assay conditions, e.g., pH and/or presence of lipids. Furthermore, some ATPases cannot be purified in reasonable quantities, or their ATPase activity is low. Thus, large amounts of purified protein would be needed in the experiment, since neither the Malachite green, the NADH-coupled assay, nor the fluorescence-based assays are sensitive enough with detection limits in the low micromolar or maximum nanomolar range.
Here, we describe an in vitro ATPase assay utilizing radiolabeled [γ-32P]-ATP based on previous protocols (Gorbulev et al., 2001), with the ability to detect free phosphate in the femtomolar range. In the assay presented, the active ATPase will liberate radiolabeled gamma-phosphate from [γ-32P]-ATP. Excess radiolabeled ATP is subsequently separated from liberated radiolabeled gamma-phosphate by molybdate-phosphate extraction, in which the molybdate-phosphate complex and non-hydrolyzed ATP partition into the organic and aqueous phase, respectively. The radioactive assay provides a direct and sensitive quantification of ATPase activity. In contrast to colorimetric and fluorescent assays, this assay is not disturbed by turbidity, which can be caused, e.g., by detergents and/or lipids present in the samples. The major limitations of radioactive ATPase assays are the hazards of handling radiolabeled isotopes and the unsuitability of this assay format for large-scale high-throughput screening.
Materials and Reagents
Biological materials
The exemplary detergent-solubilized ATPase was a C-terminally GFP-tagged version of the Cryptococcus neoformans P4-ATPase Apt1p, in complex with its β-subunit Cdc50p, C-terminally FLAG-tagged (Cdc50p-Flag). The membrane transporter complex was heterologously expressed from a pESC-URA plasmid in the Saccharomyces cerevisiae strain dnf1Δdnf2Δdrs2Δ (ZHY709, Hua et al., 2002) under the control of a galactose inducible bidirectional promoter. Apt1p-GFP/Cdc50p-Flag was purified via anti-FLAG affinity chromatography, resulting in 1.026 mg/L protein in assay buffer [20 mM HEPES-NaOH, pH 7.5, 20% (w/v) glycerol, 150 mM NaCl] supplemented with 0.04% (w/v) n-Dodecyl-β-D-maltoside (DDM), stored at -80 °C (Stanchev et al., 2021).
Note: The protocol can also be applied to other ATPases, either purified or reconstituted (Gorbulev et al., 2001; van der Does et al., 2006; Marek et al., 2011; Laub et al., 2017; Theorin et al., 2019). Whether the ATPase activity of the protein remains during storage needs to be tested.
Materials
Polypropylene reaction tubes: 1.5, 2, and 15 mL (e.g., Sarstedt, catalog numbers: 72.690.001, 72.691, 62.554.502)
15 mL glass tubes with screwable lid (e.g., Oehmen, catalog numbers: 7920120, 6702588)
Note: To avoid phosphate contamination, glassware should be washed in phosphate-free detergent prior to use.
Filter tips: 10, 200, and 1,000 μL (e.g., Starlab, catalog numbers: S1120-3810, S1120-8810, S1126-7810)
5 mL pipette tips (e.g., Gilson, catalog number: F161571)
4 mL scintillation vials (PerkinElmer, catalog number: 1200-421)
Glass beakers (100 mL)
Glass flasks (500 mL)
Measuring flasks (100 mL)
Magnetic stir bars
Safety goggles
Gloves
Chemicals
N-Dodecyl β-maltoside (DDM) (Glycon, catalog number: D97002)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Roth, catalog number: 6763.3)
Sodium chloride (NaCl) (Fisher Scientific, catalog number: 10284640)
Glycerol, >99% (VWR, catalog number: 24388.295)
Sodium hydroxide (NaOH) (Signa-Aldrich, catalog number: 30620)
Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: M2670)
Isobutanol, 99% (Alfa Aesar, catalog number: B23091)
Cyclohexane, ≥99.5% (VWR, catalog number: 23224.293)
Acetone, ≥99.5% (Sigma-Aldrich, catalog number: 904082)
Adenosine-5’-triphosphate disodium salt hydrate (non-radioactive ATP) (Sigma, catalog number: A3377)
Ammonium heptamolybdate tetrahydrate, ≥99% (Roth, catalog number: 3666.1)
Scintillation fluid for hydrophilic and lipophilic samples (Roth, catalog number: 0016.3)
Hydrochloric acid (HCl) (VWR, catalog number: 20252.290)
85% (w/w) orthophosphoric acid (H3PO4) (VWR, catalog number: 20624.295)
Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P564060)
[γ-32P]-ATP 3,000 Ci/mmol, 5 mCi/mL, 250 μCi (PerkinElmer, catalog number: BLU502H). Store at 4 °C, half-life is 14.29 days
Note: We recommend using this 'easy Tide' version, which includes a green dye in the liquid to aid pipetting. Furthermore, this ATP can be stored at 4 °C to avoid freeze-thaw cycles. [γ-33P]-ATP can also be used.
Sodium orthovanadate (Sigma, catalog number: 450243)
Assay buffer (100 mL) (see Recipes)
20% (w/v) DDM stock (5 mL) (see Recipes)
1 M HCl (100 mL) (see Recipes)
Reagent A (15 mL) (see Recipes)
Reagent B (33.3 mL) (see Recipes)
20 mM H3PO4 (5 mL) (see Recipes)
100 mM ATP (10 mL) (see Recipes)
500 mM MgCl2 (10 mL) (see Recipes)
700 mM orthovanadate (35 mL) (see Recipes)
Equipment
Analytical balance (Sartorius Entris-i II, 220 g/0.1 mg, Buch Holm, catalog number: 4669128)
Eppendorf Research® plus pipettes P20, P200, P1000, P5000 (e.g., Eppendorf, catalog numbers: 3123000039, 3123000055, 3123000063, 3123000071)
Thermomixer (e.g., Eppendorf, catalog number: 5382000015)
β-radiation protection shield (e.g., Thermo Fisher Scientific, catalog number: 6700-1812)
Beta Counter (e.g., Berthold, catalog number: LB 1210B)
Vortex mixer (e.g., Vortex Genie 2, Scientific Industries Inc., catalog number: SI-0236)
Scintillation counter (e.g., MicroBeta2, PerkinElmer, catalog number: 2450-0020) with sample cassette for 4 mL scintillation vials (PerkinElmer, catalog number: 1450-117)
pH meter (e.g., SevenCompact S220, Mettler Toledo, catalog number: 30019028)
Magnetic stir platform with heating option (e.g., RCT basic, IKA, catalog number: 0025005927)
Fume hood
Software
MicroBeta2 Windows Workstation Version 2.3.0.12
Microsoft Excel
Procedure
This assay is performed by mixing the purified or reconstituted ATPase, 5 μCi [γ-32P]-ATP, and 1 mM non-radioactive ATP in a reaction buffer that contains Mg2+. Control reactions include a sample without protein addition and an incubation of the ATPase in the presence of 1 mM orthovanadate, a classical inhibitor of P-type ATPases. In addition, a catalytically inactive mutant of the protein can serve as a negative control for the reaction to evaluate the presence of potentially contaminating ATPases present in protein preparations. The amount of non-radioactive ATP used in each reaction has to be in excess, so that it is not rate-limiting. Typically, approximately 1 mM of non-radioactive ATP is used per reaction. However, the quantity of protein, the ATP concentration, and the incubation time for the ATP hydrolysis reaction should initially be tested. For an overview, the workflow is visualized in Figure 1. The ATPase reaction is terminated by placing the samples on ice and acid addition. Subsequently, reaction components are separated by molybdate-phosphate extraction and the amount of liberated inorganic phosphate is determined via scintillation counting. Comparison to a buffer sample without protein is used for background correction. A calibration reference sample with a known volume from the [γ-32P]-ATP stock allows calibration of the measured scintillation signal to [γ-32P] in the sample, and thus a quantification of the hydrolyzed ATP.
Figure 1. Experimental design and workflow for measuring ATPase activity.
After preparing the samples, the ATPase mix is added, and the reaction is started by transferring the samples to the heating block. The reaction is terminated by placing the samples on ice and adding reagent A, which contains molybdate that forms a complex with free inorganic phosphate. The solution is transferred to a glass tube containing orthophosphoric acid, and reagent B is added for phosphate extraction by phase separation. After separation, 500 μL of the upper phase is added to the scintillation fluid in the scintillation vial and taken for measurement.
Sample preparation
We recommend preparing each sample as duplicates or triplicates.
Prepare test samples with a final volume of 45 μL in separate 2 mL reaction tubes on ice. An exemplary pipetting scheme is shown in Table 1. Typically, 40–250 ng of purified ATPase is used per reaction. If the specific ATPase activity range of the protein is unknown, several dilutions should be tested. Use the assay buffer to make a total of 45 μL volume.
Note: It is recommended to store purified proteins at -80 °C in small aliquots to avoid multiple freeze-thaws. Thawing has to be done slowly on ice before an experiment is performed.
Table 1. Exemplary pipetting scheme for samples and buffer in duplicates and calibration reference in triplicates. 10× ATPase reaction mix is added at a later step (see Section C).
Sample
Repl.
Protein (μL)
Inhibitor (μL)
Buffer (μL)
10× ATPase reaction mix (μL)
Buffer
I
-
-
45
5
II
-
-
45
5
Sample
I
39
-
6
5
II
39
-
6
5
Sample + inhibitor
I
39
1
5
5
II
39
1
5
5
Calibration reference
I
-
-
-
0.2 (2 μL of 1:10 dilution)
II
-
-
-
0.2 (2 μL of 1:10 dilution)
III
-
-
-
0.2 (2 μL of 1:10 dilution)
Prepare test samples with inhibitor using the same quantity of purified ATPase as before with the addition of inhibitor. Use the assay buffer to make a total of 45 μL volume. Incubate the samples for 20 min at room temperature to allow proper inhibition.
Note: If sodium orthovanadate is used as an inhibitor, it has to be boiled for 15 min at 95 °C and subsequently stored on ice until use to destroy polymers.
Prepare a blank by adding 45 μL of assay buffer in a separate 2 mL reaction tube.
Store samples (after incubation if inhibitors are used) on ice until the assay is started.
Preparation of the 10× ATPase reaction mix
To take the rate of decay of [γ-32P]-ATP into consideration, calculate its activity for the day the assay is conducted. Manufacturers specify a calibration or reference date that corresponds to the indicated activity of a radiolabeled reagent. This date can be used to determine the residual activity of the radioactive isotope on the day the assay is conducted using the following equation:
A(t) = activity at time point t
A0 = initial activity (at t = 0)
t = time point (days after t = 0)
Furthermore, the reference number of the [γ-32P]-ATP (BLU502H) can be used to obtain both the concentration in mCi/mL and the specific activity in Ci/mmol of the stock solution via the PerkinElmer Homepage:
https://www.perkinelmer.com/tools/calculatorrad#/product/BLU502H
Note: The recommended [γ-32P]-ATP (BLU502H) contains 50 μL only. The earlier the experiment is performed after the calibration date, the more samples can be tested.
Calculate the volumes needed for each component for the preparation of the 10× ATPase reaction mix (1 mM ATP, 5 mM MgCl2, 0.4 μCi/μL [γ-32P]-ATP in assay buffer) as displayed in Table 2. For each sample, 5 μL of mix is needed and an excess of one sample should be prepared for the preparation of calibration reference samples. Based on the calculation, first prepare the 10× ATPase reaction mix without [γ-32P]-ATP.
Table 2. Exemplary pipette scheme for the 10× ATPase reaction mix for one and six samples.
For every sample, 5 μL of the mix with a total of 2 μCi [γ-32P]-ATP is needed. The amount of [γ-32P]-ATP has to be calculated on the basis of the half-life and calibration date (exemplified here with a concentration on the date of the experiment of 5 μCi/μL). The total volume of the reaction mix should always be adjusted to 5 μL per sample using assay buffer.
10× ATPase reaction mix per sample (μL) For 6+1 samples (μL) Final conc.
ATP (100 mM) 0.5 3.5 1 mM
MgCl2 (500 mM) 0.5 3.5 5 mM
Assay buffer 3.6 25.2 -
[γ-32P]-ATP 0.4 2.8 0.4 μCi/μL
Total volume 5 35 -
From this step onwards, radioactive material handling precautions have to be taken. These include the use of appropriate shielding materials such as Perspex shielding (3/8 inches thick), behind which all work should be done, and Perspex Eppendorf tube holders. Surfaces should be routinely monitored by Geiger counters, and ring dosimeters can be used to monitor personal exposure. Radioactive isotopes should be used only by authorized personnel in designated places following the institution’s regulations. Requisition and storage of radioactive material, solid and liquid radioactive waste disposal, and spill decontamination should be carried out according to the institution’s regulations.
Finalize the 10× ATPase reaction mix by addition of the appropriate volume of [γ-32P]-ATP from the solution stock, as calculated in B.2.
ATPase assay
Add 5 μL of 10× ATPase reaction mix supplemented with [γ-32P]-ATP to each sample on ice.
Note: The excess 10× ATPase reaction mix is later used for the preparation of reference samples in section D.
Start the ATPase reaction by transferring the samples to a thermomixer at 28 °C under gentle shaking (do not exceed 700 rpm).
Note: The optimal temperature will depend on the ATPase and should initially be tested.
Incubate for 60 min.
Note: Since the assay is based on end-point determination, it is important that the reaction is not limited by the amount of ATP available. To prevent a complete ATP consumption and thereby underestimation of sample activity, several ATPase dilutions should be tested.
Stop the reaction by placing the samples on ice and subsequently adding 1.5 mL of Reagent A (Recipe 4).
Note: All ATPases should be inactivated and the reaction stopped completely. However, to prevent autohydrolysis of [γ-32P]-ATP, the samples should be processed directly by extraction of phosphate.
Molybdate-phosphate extraction
Note: Molybdate-phosphate extraction is needed to separate inorganic 32P-phosphate from non-hydrolyzed 32P-ATP, as illustrated in Figure 2.
Figure 2. Illustration of the molybdate-phosphate extraction procedure.
After the ATPase reaction, free 32P-phosphate and non-hydrolyzed γ-32P-ATP are present in the same solution. Reagent A, containing ammonium molybdate, is added and forms a complex with the inorganic phosphate. Addition of reagent B results in phase separation in the lower aqueous phase and the upper organic phase. The molybdate-phosphate complexes localize to the upper organic phase, while the non-hydrolyzed ATP remains in the aqueous phase. A sample of the organic phase is taken for scintillation counting, to determine the amount of inorganic phosphate formed.
Transfer each sample to separate 15 mL glass tubes containing 15 μL of 20 mM H3PO4 (Recipe 6).
Note: The phosphate of H3PO4 serves as a carrier for the inorganic phosphate of the ATPase reaction in the next step.
Add 3 mL of Reagent B to all samples, fasten the lid, and vortex for 30 s.
Note: Reagent B is volatile. Work under the fume hood.
Incubate on ice for 10 min to allow phase separation.
Add 2 mL of scintillation fluid to 4 mL scintillation vials; separate tubes are needed for every sample and three calibration references.
Note: The scintillation counter used here measures through the lid. Therefore, the tubes should be labeled on the sides of the vial, not on the lid. Clarify how your device is measuring and change the labeling position accordingly.
After phase separation, take 500 μL of yellow, upper, organic phase containing the radioactive orthophosphate-molybdate complex and add it on top of the scintillation fluid in scintillation vials.
Note: The organic phase has low surface tension and tends to drop out of the pipette.
Note: Do not pipette up and down in the vial, as scintillation fluid gets stuck in the tip.
To allow quantification of the scintillation measurement, prepare a 1:10 dilution of 10× ATP reaction mix (2 μL + 18 μL H2O) and add 2 μL of 1:10 dilution to the scintillation tube with scintillation fluid. Make triplicates of this calibration reference.
Scintillation counting using MicroBeta2
Tighten the lids of the scintillation vials properly.
Place vials in the measuring cassette, invert it several times for mixing, and place it in the scintillation counter with the stop cassette below.
Start the MicroBeta2 Windows Workstation program.
Create a protocol for 32P measurement, as shown in Figure 3.
Set Label 1 to 32P and Label 2 to None.
Set Plate/Filter to 4 mL plate 6 × 4.
Set Assay Type to Normal.
Set counting time to 1 min.
Set Detector Normalization and Background correction to None.
Set counting precision to 0.2 2 sigma%.
Set cycling parameter to 1 time per plate.
Figure 3. User interface of MicroBeta2 Windows Workstation program showing the protocol settings. A: General settings; B: Correction settings; C: Counting Control settings; D: Other settings.
Change the plate setup to only measure the slots that include a vial.
Start the measurement.
Extract the cpm values per sample as .txt files.
Data analysis
Extract the signal intensities from the .txt file and load it into Microsoft Excel for analysis.
Data analysis:
First, the amount of hot ATP in the reference sample must be calculated based on its specific activity on the day of the experiment and the volume taken from the 10× ATPase reaction mix. Correlating the amount of hot ATP in the calibration reference sample with its measured cpm value allows calibration of measured cpm values. Exemplary sample values for calculation are listed in Table 3.
Next, the sample signal can be background corrected by the buffer sample signal, and the amount of hydrolyzed hot ATP can be quantified.
After correction for the total upper phase volume and extrapolation for hydrolyzed non-radiolabeled ATP, the total amount of hydrolyzed ATP can be specified per minute and per milligram of protein to yield the specific ATPase activity.
Table 3. Exemplary data from scintillation measurement. Calibration reference, buffer and sample are prepared as triplicates and duplicates, respectively. Each sample included 40 ng of protein.
Sample Sample signal [cpm]
Calibration reference I 221,331
Calibration reference II 218,048
Calibration reference III 221,200
Buffer I 8,874
Buffer II 8,444
Sample I 50,037
Sample II 53,292
Reference calculation for quantification:
For the reference calculation, 0.2 μL was taken from the 10× ATPase reaction mix with a concentration of 0.4 μCi/μL [γ-32P]-ATP, resulting in 0.08 μCi [γ-32P]-ATP.
In the exemplary experiment, these 2.667 × 10-11 mmol of the reference sample referred to the calibration reference average of 220,193 cpm.
Sample analysis:
i. The counts for duplicates of each sample are averaged, and the background correction is applied for the averaged background signal.
Note: The buffer serves not only as a control for the spurious extraction of intact [γ-32P]-ATP in the upper organic phase, but also for the amount of [γ-32P]-ATP that is already hydrolyzed in the stock or in the time course of the experiment, as well as phosphate contaminations in the used solutions.
ii. The buffer corrected counts for each sample are normalized to millimole of hydrolyzed hot ATP via the reference calculation.
iii. The calculated amount of hydrolyzed hot ATP refers to 500 μL taken from the upper phase for analysis only. Therefore, for the complete upper organic phase of 3 mL, the calculated value has to be multiplied by six, the volume correction factor (CFVol).
iv. Since the ratio of [γ-32P]-ATP to non-radiolabeled ATP was 1:75,000 (mol:mol), the calculated value has to be further multiplied by 75,000, the ratio correction factor (CFRatio), to account for all ATP present.
v. In the last step, the calculated amount of hydrolyzed ATP has to be normalized for the amount of protein (mprotein) in the sample and for the reaction time of 60 min, resulting in a final specific ATPase activity in millimole hydrolyzed ATP per milligram of protein per minute.
vi. In summary, the specific activity can be calculated based on the background corrected cpm values as follows:
ref. = reference sample
CFVol = correction factor for total upper phase volume
CFRatio = correction factor for ratio hot to cold ATP
mprotein = amount of protein
vii. The final specific ATPase activity can be compared between samples with and without an inhibitor. Here, the sample without inhibitor is set to 100% to allow quantification of specific inhibition in percentage (Figure 4).
Figure 4. Exemplary result of ATPase assay with detergent-solubilized Apt1p/Cdc50p. ATPase activity of purified Apt1p/Cdc50p assayed in the absence (-) and presence (+) of 1 mM orthovanadate, an inhibitor of P-type ATPases. Adapted from Stanchev et al. (2021).
Recipes
Note: Since low quantities of free phosphate are quantified, a phosphate contamination of the used reagents and material should be minimized. A control sample only containing buffer should be used for detection of the background signal and data correction.
Assay buffer (100 mL)
20 mM HEPES (4.766 g), 150 mM NaCl (8.766 g), 20% (w/v) glycerol (20 g), 0.05% (w/v) DDM (250 μL of 20% stock; see Recipe 2), pH 7.4.
Weigh NaCl, glycerol, and HEPES and fill up to 90 mL with ddH2O. Adjust the pH with 1 M NaOH to 7.4. Add DDM and fill up to 100 mL with ddH2O.
Note: For other ATPases, this buffer may be adapted.
20% (w/v) DDM stock (5 mL)
Dissolve 1 g of DDM in 3 mL of ddH2O in a 15 mL polypropylene screw-cap tube and incubate with head-over-head rotation until completely dissolved. Let the foam set and fill up to a total of 5 mL with ddH2O. Store at 4 °C.
1 M HCl (100 mL)
Dilute 8.3 mL of concentrated (37%) HCl to a total volume of 100 mL with ddH2O.
Reagent A (15 mL)
Dissolve 0.185 g of ammonium heptamolybdate tetrahydrate in a total volume of 15 mL in 1 M HCl.
Note: Prepare fresh on the day of the experiment. Per sample, 1.5 mL of Reagent A is needed. We recommend preparing a sufficient volume for one extra sample.
Reagent B (33.3 mL)
Mix isobutanol, cyclohexane, acetone, and Reagent A in a glass flask in a ratio of 5:5:1:0.1 (v:v) by circular movements, resulting in 45% (v/v) isobutanol (15 mL), 45% (v/v) cyclohexane (15 mL), 9% (v/v) acetone (3 mL), and 0.1% Reagent A (0.3 mL) for 33.3 mL.
Note: Substances used are volatile and toxic. Work under the fume hood. Prepare fresh on the day of the experiment. Per sample, 3 mL of Reagent B is needed. We recommend preparing a sufficient volume for one extra sample.
20 mM H3PO4 (5 mL)
Dilute 6.5 μL of 85% (w/w) orthophosphoric acid to a total of 5 mL in ddH2O.
100 mM ATP (10 mL)
Dissolve 551 mg of ATP in a total volume of 8 mL in ddH2O. Adjust pH to 7.0 with 1 M KOH. Fill up to 10 mL with ddH2O. Freeze aliquots of e.g., 200 μL in 1.5 mL polypropylene reaction tubes at -20 °C. Avoid repeated freeze-thawing.
500 mM MgCl2 (10 mL)
Dissolve 1.017 g of MgCl2 hexahydrate in a total volume of 10 mL in ddH2O. Store at RT.
700 mM orthovanadate (35 mL)
Add 4.5 g of sodium orthovanadate to 30 mL of ddH2O in a glass beaker and stir until completely dissolved.
Adjust the pH to 10 using 1 M NaOH or 1 M HCl.
Note: Acidification leads to a color change to yellow.
Boil the solution on a heating plate with continuous stirring until the solution loses its color.
Let cool to room temperature and measure pH.
Titrate again to pH 10 using 1 M NaOH or 1 M HCl and repeat boiling and titration until pH = 10 is stable at room temperature.
Fill up with ddH2O to 35 mL.
Store aliquots of e.g., 200 μL in 1.5 mL polypropylene reaction tubes at -20 °C.
Directly before use, heat the solution for 15 min at 95 °C to break polymeric species (Goodno, 1982) and place it on ice until use.
Note: Orthovanadate is toxic. Work under the fume hood and wear protective gear.
Acknowledgments
This protocol was adapted from our previous work (Marek et al., 2011; Laub et al., 2017; Theorin et al., 2019; Stanchev et al., 2021). This work was funded by a grant from the Novo Nordisk Fonden (NNF18OC0034784) to T.G.P.; S.V. is a scholar of the Studienstiftung des Deutschen Volkes. Figures were created in BioRender.
Competing interests
The authors declare that there are no competing interests.
References
Banik, U. and Roy, S. (1990). A continuous fluorimetric assay for ATPase activity. Biochem J 266(2): 611-614.
Baykov, A. A., Evtushenko, O. A. and Avaeva, S. M. (1988). A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171(2): 266-270.
Brune, M., Hunter, J. L., Corrie, J. E. and Webb, M. R. (1994). Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33(27): 8262-8271.
Carter, S. G. and Karl, D. W. (1982). Inorganic phosphate assay with malachite green: an improvement and evaluation. J Biochem Biophys Methods 7(1): 7-13.
Feng, J., Chen, Y., Pu, J., Yang, X., Zhang, C., Zhu, S., Zhao, Y., Yuan, Y., Yuan, H. and Liao, F. (2011). An improved malachite green assay of phosphate: mechanism and application. Anal Biochem 409(1): 144-149.
Geladopoulos, T. P., Sotiroudis, T. G. and Evangelopoulos, A. E. (1991). A malachite green colorimetric assay for protein phosphatase activity. Anal Biochem 192(1): 112-116.
Goodno, C. C., (1982). [12] Myosin active-site trapping with vanadate ion. In: Methods in Enzymology, Structural and Contractile Proteins Part B: The Contractile Apparatus and the Cytoskeleton (pp. 116-123). Academic Press.
Gorbulev, S., Abele, R. and Tampé, R., (2001). Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP. Proc Natl Acad Sci U S A 98(7): 3732-3737.
Hua, Z., Fatheddin, P. and Graham, T.R., (2002). An Essential Subfamily of Drs2p-related P-Type ATPases Is Required for Protein Trafficking between Golgi Complex and Endosomal/Vacuolar System. MBoC 13: 3162-3177.
Itaya, K. and Ui, M. (1966). A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta 14(3): 361-366.
Laub, K. R., Marek, M., Stanchev, L. D., Herrera, S. A., Kanashova, T., Bourmaud, A., Dittmar, G. and Günther Pomorski, T. (2017). Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p. PLoS One 12(9): e0184236.
Marek, M., Milles, S., Schreiber, G., Daleke, D.L., Dittmar, G., Herrmann, A., Müller, P. and Pomorski, T.G., (2011). The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: Purification, characterization, and the effect of lipids on its activity. J Biol Chem 286: 21835-21843.
Radnai, L., Stremel, R. F., Sellers, J. R., Rumbaugh, G. and Miller, C. A. (2019). A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors. J Vis Exp (150).
Stanchev, L. D., Rizzo, J., Peschel, R., Pazurek, L. A., Bredegaard, L., Veit, S., Laerbusch, S., Rodrigues, M. L., Lopez-Marques, R. L. and Günther Pomorski, T. (2021). P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity. J Fungi (Basel) 7(10).
Theorin, L., Faxén, K., Sørensen, D.M., Migotti, R., Dittmar, G., Schiller, J., Daleke, D.L., Palmgren, M., López-Marqués, R.L. and Günther Pomorski, T., (2019). The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase. Biochem J 476: 783-794.
van der Does, C., Presenti, C., Schulze, K., Dinkelaker, S. and Tampé, R., (2006). Kinetics of the ATP Hydrolysis Cycle of the Nucleotide-binding Domain of Mdl1 Studied by a Novel Site-specific Labeling Technique. J Biol Chem 281, 5694-5701.
Warren, G. B., Toon, P. A., Birdsall, N. J., Lee, A. G. and Metcalfe, J. C. (1974). Reconstitution of a Calcium Pump Using Defined Membrane Components. Proc Natl Acad Sci U S A 71(3): 622-626.
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Agrobacterium-mediated Genetic Transformation of Cotton and Regeneration via Somatic Embryogenesis
Alka Srivastava
AS Anoop K. Shukla
Subhi Srivastava
RD Rama S. Dubey
PS Pradyumna K. Singh
PV Praveen C. Verma
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4677 Views: 1859
Reviewed by: Amey Redkar Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Biotechnology Oct 2016
Abstract
Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide.
Keywords: Cotton Agrobacterium Somatic embryogenesis Callus induction Stable genetic transformation Transgenic cotton
Background
Genetic engineering has opened the doors to enhanced yield, disease resistance, and improved nutritional quality and quantity. Genetically modified (GM) crops ensure that the needs of the growing population are met worldwide. The altered attributes in GM crops are due to introduced transgenes, which is achieved by different tools as particle bombardment, chloroplast transformation, pollen tube pathway transformation, protoplast electroporation, Agrobacterium-mediated genetic transformation, and many more. Amongst these, Agrobacterium tumefaciens–mediated transformation is the preferred one due to its exceptional trait of transferring a DNA segment into the host cell via a specialized plasmid (Ziemienowicz, 2014). This technique has become favorable for scientists and industries because of its simple and easy approach, its ability to transfer a low copy number of the gene of interest, and its efficiency of transfer of a large DNA fragment into the host cell—all of this at a very low cost. Agrobacterium-mediated genetic transformation has been employed to transform many economically important crops using plant tissue culture, which helps to regenerate a large number of transgenic plants at a single time point (Somleva et al., 2002; Manickavasagam et al., 2004; Jones et al., 2005; Sun et al., 2006).
Transgenic cotton was one of the first genetically modified organisms to be globally accepted and commercially developed. Now and again, researchers have been trying to improve the quality of cotton crops, either by enhancing its fiber yield or by imparting pests resistance. Developing a transgenic cotton plant is tedious and time consuming because of its recalcitrant nature and high production of phenolics, which hinders the regeneration of the plant. Although Agrobacterium-mediated genetic transformation has enabled the development of stable transgenic cotton, various efforts have been made to improve the transformation process (Qandeel-E-Arsh et al., 2021). A successful transformation does not depend on a single parameter; instead, it heavily relies on multiple factors such as Agrobacterium strain, type and age of explants, duration of co-cultivation between Agrobacterium and the plant tissue, bacterial cell density during transformation, regeneration method for the plant, selection marker, and type of vector to use (Firoozabady et al., 1987; Satyavathi et al., 2002; Jadhav and Katageri, 2017). To develop transgenic cotton plants, scientists have used the hypocotyls, cotyledons, and shoot tips, as well as the embryogenic callus, pollen tube, embryo, and meristems as explants (Jin et al., 2005; Pathi and Tuteja, 2013; Wang et al., 2013). The use of meristems is very cumbersome and labor intensive and requires particle bombardment (with the drawback of high copy number). The use of the embryonic callus is a relatively good option, but these take several months to be obtained, and the subsequent process does not guarantee proper plantlet germination (McCabe and Martinell, 1993). Transformation of the shoot tip, the embryo, and the pollen tube is easier than others, but the potential for stable transgenic plants is low. Most of these techniques fall into the category of transient transformation, skipping the inserted gene in subsequent generations. Direct organogenesis has also been tried in order to facilitate regeneration, but when the transformation is involved, it becomes sluggish and dreary, and chimeric plants are often obtained. Many of these issues can be resolved by using hypocotyls or cotyledons as the explant, as the process involves somatic embryogenesis that produces somatic embryos, which are single cell–derived. Furthermore, this promotes high-end vegetative output, ensuring more transgenic plants.
Various regeneration protocols have been proposed by several researchers. They have fiddled with different concentrations and types of plant hormones to induce callus, manipulated growth media to induce embryogenesis in proliferating calli, and changed carbohydrate sources as well (Shoemaker et al., 1986; Finer, 1988; Hemphill et al., 1998; Aydin et al., 2004; Ikram-ul-Haq and Zafar, 2004; Kumar and Tuli, 2004). All these attempts have successfully regenerated cotton genotypes, but the development of stable transgenic lines requires an appropriate combination of cotton regeneration and transformation. Currently, the most commonly used approaches are the pollen tube pathway, embryo transformation, and meristem-mediated methods (Bajwa et al., 2015), but these do not guarantee stable gene transfer. Using these methods, elucidation of the applied hypothesis is possible, but only a few have been able to be applied in practical use for stable transgenic development.
Here, we propose a protocol for Agrobacterium-mediated genetic transformation and regeneration in cotton, resulting in transgenic production (Figure 1). This protocol is based on the approach described by Kumar and Tuli (2004), with various modifications that accelerate the achievement of the transgenic development. We have optimized the media for the Agrobacterium to be ready to infect the explants, the co-cultivation period, the appropriate quantity and type of phytohormones to be used, and the environment required for callus induction, its proliferation, and further embryogenesis, and we have reduced the abnormalities in the embryos produced. To summarize, this protocol is an approach towards obtaining a stable transgenic line, via transforming the explants with a gene of interest and regenerating them with early callus induction, improved embryogenesis, and culture requisites. We have used a 14 kb construct harboring a CamV35S promoter, the pectin methylesterase (PME) gene, the neomycin phosphotransferase II (nptII), and the nopaline synthase (Nos) terminator. The protocol has been used to develop several distinct transgenic lines and is currently being used to alter numerous unique genes in cotton for genome editing.
Figure 1. Schematic representation of Agrobacterium-mediated genetic transformation of cotton hypocotyl explants and regeneration (created with BioRender.com)
Materials and Reagents
Glassware
Glass rod
Conical flask, 250 mL (Borosil, catalog number: 4984021)
Glass bottle, 1,000 mL (Borosil, catalog number: 1501029)
Culture vials, 30 mL (Borosil, catalog number: 9910010)
Test tubes
Wide-mouth bottles, 100 mL
Plasticwares
Petri plates, 90 × 20 mm (Tarsons, catalog number: 960096)
Petri plates, 90 × 15 mm (Tarsons, catalog number: 960091)
Petri plates, 100 × 20 mm (Genetix, catalog number: 310100)
Parafilm tape (Tarsons, catalog number: 380020)
Stainless steel sieve
Filter paper (Whatman, catalog number: 1003-917)
Computer paper
Planton plant tissue culture container, 7.5 × 7.5 × 10 cm (Tarsons, catalog number: 020080)
Frames for planton boxes (Genetix, catalog number: 310074)
Spirit lamp
Plastic bags
Falcon tubes, 50 mL
Sterile syringe
Test tube caps (Tarsons, catalog number: 020070)
Plant earthen pots, 14”
Plant plastic pots, 5”
Sterile syringe filters, 0.22 μm
Electroporation cuvettes (gene pulser 0.4 cm gap) (Bio-Rad, catalog number: 1652086)
Toothpick
Scalpel
Forceps
Surgical blade
Biological materials
Matured, defuzzed, and delinted cotton seeds. Variety: Coker-312
Agrobacterium tumefaciens strain LBA4404
Binary vector incorporating gene of interest. Here, PME (pectin methylesterase gene) is cloned in pBI121 vector under the promoter CaMV35S, neomycin transferase II (nptII) as selectable marker, and Nos terminator.
Chemicals and reagents
Sodium bicarbonate (Himedia, catalog number: MB045), store at room temperature (RT)
Sulfuric acid (Himedia, catalog number: RM6224), store at RT
Mercuric chloride (HgCl2) (Sigma-Aldrich, catalog number: M1136), store at RT
Ethanol (Merck, catalog number: 1.00983), store at RT
Acetosyringone (Himedia, catalog number: PCT1301), store at 4 °C
Glucose (Sigma-Aldrich, catalog number: G7021), store at RT
Sucrose (Sigma-Aldrich, catalog number: S0389), store at RT
Phytagel (Sigma-Aldrich, catalog number: P8169), store at RT
Agar powder (Himedia, catalog number: MB053), store at RT
Myo-inositol (Sigma-Aldrich, catalog number: I7508), store at RT
Magnesium chloride (Sigma-Aldrich, catalog number: M4880), store at RT
Potassium chloride (Sigma-Aldrich, catalog number: P5405), store at RT
Calcium chloride (Sigma-Aldrich, catalog number: C5670), store at RT
MES buffer (Sigma-Aldrich, catalog number: M3671), store at RT
Murashige & Skoog basal salt (Sigma-Aldrich, catalog number: M5524), store at 4 °C
Potassium nitrate (Sigma-Aldrich, catalog number: P8291), store at RT
Magnesium sulfate (Sigma-Aldrich, catalog number: M2773), store at RT
Potassium phosphate monobasic (Sigma-Aldrich, catalog number: P5655), store at RT
Calcium nitrate tetrahydrate (Sigma-Aldrich, catalog number: C2786), store at RT
Ammonium phosphate monobasic (Sigma-Aldrich, catalog number: 216003), store at RT
Calcium chloride dehydrate (Sigma-Aldrich, catalog number: C7102), store at RT
Sodium molybdate dehydrate (Sigma-Aldrich, catalog number: M1651), store at RT
Boric acid (Sigma-Aldrich, catalog number: B7901), store at RT
Potassium iodide (Sigma-Aldrich, catalog number: 221945), store at RT
Manganese sulfate tetrahydrate (Sigma-Aldrich, catalog number: 1.02786), store at RT
Zinc sulfate (Sigma-Aldrich, catalog number: Z0251), store at RT
Copper sulfate pentahydrate (Sigma-Aldrich, catalog number: C8027), store at RT
Cobalt chloride hexahydrate (Sigma-Aldrich, catalog number: C8661), store at RT
Sodium EDTA (Sigma-Aldrich, catalog number: 03650), store at RT
Ferrous sulfate heptahydrate (Sigma-Aldrich, catalog number: F8633), store at RT
Nicotinic acid (Sigma-Aldrich, catalog number: N0761), store at RT
Pyridoxine HCl (Sigma-Aldrich, catalog number: P6280), store at RT
Thiamine HCl (Sigma-Aldrich, catalog number: T1270), store at RT
Kinetin (Sigma-Aldrich, catalog number: K3375), store at 4 °C
2,4-Dichlorophenoxyacetic acid (2,4-D) (Sigma-Aldrich, catalog number: D76724), store at 4 °C
6-Benzylaminopurine (BAP) (Sigma-Aldrich, catalog number: B3408), store at 4 °C
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418), store at RT
Methanol (Sigma-Aldrich, catalog number: 34860), store at RT
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465), store at RT
Yeast extract (Sigma-Aldrich, catalog number: Y1625), store at RT
Beef extract (Himedia, catalog number: RM002V), store at RT
Peptone (Sigma-Aldrich, catalog number: P5905), store at RT
Murashige & Skoog basal salt mixture (4.3 g/L) (Sigma-Aldrich; catalog number: M2909)
Antibiotics (see Recipes)
Kanamycin 50 mg/L (Himedia, catalog number: MD026), store at 4 °C
Streptomycin 250 mg/L (Himedia, catalog number: MD048), store at 4 °C
Rifampicin 50 mg/L (Himedia, catalog number: MD045), store at 4 °C
Augmentin 250 mg/L (Himedia, catalog number: PCT1115), store at 4 °C
Cefotaxime 250 mg/L (Himedia, catalog number: MD015), store at 4 °C
Phytohormones (see Recipes)
2,4-D (stock: 10 mg/mL; working: 0.5 mg/mL)
Kinetin (stock: 10 mg/mL; working: 0.2 mg/mL)
BAP (stock: 10 mg/mL; working: 0.2 mg/mL)
Acetosyringone (see Recipes)
YEB media (see Recipes)
Induction media (see Recipes)
Hoagland media (see Recipes)
Murashige & Skoog stock (see Recipes)
Culture media (see Recipes)
Equipment
Incubator/shaker (Kuhner, model: ISF-1-W)
Laminar flow hood
Autoclave (TOMY, model: ES-315)
Ultracentrifuge (Eppendorf, model: 5430R)
pH meter (Eutech, pH tutor)
Magnetic stirrer (Abdos, Swirl top)
Thermal cycler (Bio-Rad, C1000 thermal cycler)
-80 °C ultra-low temperature freezer (Thermo Scientific, Forma 89000 Series)
-20 °C low temperature freezer (Celfrost)
Weighing balance (Sartorius, SQP-F)
Electroporation machine (MicroPluserTM Bio-Rad)
Greenhouse
Culture room
Soil mix for plants
Procedure
Transformation of Agrobacterium and seed germination for transformation
Fresh, uniform culture of transformed Agrobacterium is required for efficient plant transformation.
LBA4404 competent cells’ transformation with pBI121 (14.7 kb) construct (Figure 2):
Figure 2. Construct map of the vector used for Agrobacterium-mediated genetic transformation
Place the competent cells (LBA4404) on ice and let them thaw (do not rub the Eppendorf to force thawing). Once the cells thaw, add 1 μL of the desired plasmid to the competent cells. (Plasmid concentration: 10–100 ng/μL.)
Mix the plasmid and cells by gentle pipetting and let it sit for 5 min.
Transfer the cell mixture to an electroporation cuvette and mix by gentle pipetting.
Place the cuvette into the cuvette chamber and start the electroporation machine. Press the pulse button until a beep sound is heard, indicating that the pulse has been given.
Remove the cuvette, transfer the entire cell mixture into a 1.5 mL Eppendorf, and add 950 μL of YEB media (see Recipes).
Gently mix the cell mixture by pipetting and then place it in the incubator shaking at 200 rpm for 2 h in the dark at 28 °C. Dark conditions help the bacteria to repair and prepare itself for growth and multiplication.
After 2 h, remove the Eppendorf and centrifuge at 3,500× g for 2 min at RT.
Discard the supernatant (used YEB media) and add 300 μL of YEB media again. Mix the pelleted cell mixture by pipetting.
Once mixed properly, spread 50–100 μL of cells on YEB agar plates containing 50 mg/L kanamycin, 250 mg/L streptomycin, and 50 mg/L rifampicin. Spreading should result in a moistureless plate (this leads to appropriate single colony formation).
Wrap the plates with foil and place them in an incubator at 28 °C for 48 h in the dark.
After 48 h, single colonies start to appear. Pick 10–12 single colonies and check by PCR if the cells are transformed.
Create a master plate by streaking the positive colonies on YEB agar plates containing 50 mg/L kanamycin, 250 mg/L streptomycin, and 50 mg/L rifampicin. These Agrobacterium colonies consist of our gene of interest.
Incubate the master plate for 48 h in an incubator at 28 °C.
Store the plate in the dark at 4 °C.
Cotton seed sterilization and germination (Figure 3):
Figure 3. Cotton seed sterilization and germination (created with BioRender.com)
Defuzz the cotton bolls. After defuzzing, remove the remaining lint by delinting the seeds.
To delint 100 seeds, prepare a liter of saturated sodium bicarbonate solution by mixing sodium bicarbonate in 1 L of double-distilled water until it stops dissolving.
Add 25 seeds to a 100 mL wide-mouth bottle and pour 7–8 drops of concentrated sulfuric acid on it. Stir the seeds continuously until the whole lint is removed.
Once the lint is removed, add saturated sodium bicarbonate solution to the bottle and simultaneously keep stirring the seeds. Stir the seeds until the effervescence ceases.
Discard the solution and rinse the seeds with tap water by vigorous shaking. Repeat this 4–5 times to remove traces of sodium bicarbonate.
Once washed properly, place the seeds on a blotting sheet and let them dry.
For sterilization, prepare a 0.1% (w/v) solution of HgCl2 by mixing 100 mg of HgCl2 with 100 mL of autoclaved double-distilled water in a 250 mL conical flask.
In a laminar flow hood, add 25–30 dried seeds to 0.1% HgCl2 solution and stir vigorously for 5 min. Try not to exceed 5 min. Excess sterilization will degrade the seed quality. Discard the solution and rinse the seeds 5–6 times for 30 s each by adding 100 mL of double-distilled water to the flask after every rinse.
After sterilizing the seeds, place half in a stainless-steel strainer and scorch them with ethanol. Do not burn the seeds. Scorching loosens the seed coat and aids in better germination (Video 1).
Video 1. Seed scorching
Let the seeds cool down for 5 min.
Cut two pieces of filter paper in the shape of a Petri plate (90 × 15 mm). Pour autoclaved double-distilled water, just enough to dampen the filter paper. Next, place the seeds on it and cover them with cut pieces of filter paper. Once again, pour enough autoclaved double-distilled water to dampen it.
Seal the Petri plate with parafilm tape and cover it with foil. Place the plate in an incubator at 28 °C for 48–72 h.
Radicals start appearing within two days. Once proper radicals are seen, place them on paper boats (made of computer paper) in test tubes containing Hoagland media (see Recipes, Table 1), with the radical in a downward position. Seeds can also be grown on ½ MS media, but Hoagland media is preferred as its carbohydrate absence leads to fewer chances of contamination.
Place the test tubes in the test tube stand and keep the stands in a culture room set at 28 °C.
Plantlets appear in 10 days, ready to be used for transformation (Figure 4).
Figure 4. Seed germination. a) Test tube with a paper boat immersed in Hoagland media, on which a cotton plantlet has been grown. b) Fully grown 10-day-old plantlet with a slender and proper hypocotyl. c) 10-day-old plantlets grown in ½ Murashige & Skoog media with phytagel, ready for transformation (shown in red box).
Plant transformation and transgenic development
Agrobacterium culture preparation and induction for plant transformation (Figure 5):
Figure 5. Agrobacterium culture preparation and induction for plant transformation (created with BioRender.com)
Using a toothpick, pick a single colony from the master plate and add to it 5 mL of YEB media (with 50 mg/L kanamycin, 250 mg/L streptomycin, and 50 mg/L rifampicin) in a 30 mL culture vial. Incubate the culture for 2–3 days in an incubator with shaking at 200 rpm at 28 °C. This primary will be used for culturing a secondary culture.
After the culture grows in 5 mL of YEB media, take 100 μL of culture and inoculate it in 50 mL of YEB media containing 50 mg/L kanamycin, 250 mg/L streptomycin, and 50 mg/L rifampicin in a 250 mL conical flask. Incubate it for 16 h with shaking at 200 rpm at 28 °C.
Check the OD of the secondary culture after 16 h. A culture OD600 = 0.8–1.6 is used for transformation.
Once the desired OD is attained, transfer the cultures to 50 mL falcon tubes. Use two autoclaved 50 mL falcons for 50 mL of culture, equally dividing 25 mL into each falcon. Pellet down the cells at 3,500× g for 10 min at 4 °C.
Discard the supernatant and dissolve the pellets, each in 50 mL of induction media, and transfer the total 100 mL of media to a 250 mL conical flask. Incubate for 4 h with shaking at 175 rpm at 27 °C.
After 4 h, centrifuge the culture at 3,500× g for 10 min at 4 °C. Dissolve the pellet in 100 mL of Murashige & Skoog-0 (MSO; see Recipes, Table 3) media and incubate for 2 h with shaking at 150 rpm at 26 °C. Later on, this culture will be used for transformation.
Plant transformation:
In a laminar flow hood, take the hypocotyls from the 10-day-old plantlet and cut them into 1 cm long pieces. After cutting, place them on a plain MS media plate, so as not to dry them out (Video 2).
Video 2. Explant preparation
Meanwhile, the Agrobacterium culture should be ready in MSO for further transformation processes. Add the explants to 100 mL of MSO and incubate the mixture of explants and culture at 25 °C with shaking at 125 rpm for 30 min.
Remove the explants from the culture after 30 min and blot them dry with an autoclaved filter paper sheet.
Arrange the explants on MSP-1 culture media (without any antibiotics; see Recipes, Table 3) for co-cultivation. Wrap the plates with foil and keep in the dark for 48 h.
After 48 h, wash the explants with 100 mL of autoclaved double-distilled water in a conical flask containing 250 mg/L cefotaxime. Shake the flask for 5 min and then rinse 4–5 times again with autoclaved water.
Once the explants are properly washed, dry them on an autoclaved filter paper.
Callus induction and somatic embryogenesis (Figure 6):
Figure 6. Regeneration from transformed explants and transgenic development. a) Callus induction. b) Callus initiation. c) Callus proliferation. d) Embryogenic callus. e) Germinated embryo in ½ Murashige & Skoog with phytagel media. f) Developed plantlet. g) Hardening of plantlets. h and i) Fully grown transgenic plants.
After drying the explants, place 8–9 explants on each selection media (MSP-1; see Recipes, Table 3) plate. Place the culture plates in a culture room set at 28 °C with 16 h light and 8 h dark conditions.
Subculture the explants after 21 days on selection media. At this stage, callus starts appearing from the ends of the explants.
After two cycles of MSP-1, place the explants on MSP-2 (see Recipes, Table 3) media for a third and fourth cycle. A total of four cycles (each consisting of 21 days) of antibiotics ensure proper selection of positive callus.
At this point, the calli starts proliferating, and lots of friable, yellow-colored beady calli can be seen. Remove the callus from the explants with caution and place it on MSA-1 (see Recipes, Table 3) for further proliferation. Subculture the callus every 21 days for an extra 2–3 times. Lots of yellowish-colored, friable calli emerge when proper subculturing is done. Embryogenic callus will have a tinge of pinkish color, which is due to presence of the anthocyanin pigment. Presence of pink color shows onset of embryogenesis (Figure 7).
Figure 7. Different types of regenerated callus. a–c) Non-viable calli can be green, light brown, or dark brown in color. These types of calli are either too hard (a) or soggy and wet (b and c). d–f) Viable calli, which are friable, beady, and have a slight pinkish color.
Transfer the friable callus to MSA-2 (see Recipes, Table 3) media for embryogenesis. Normal friable callus shows embryogenesis on plain MS media too, but the absence of NH4NO3 results in more anthocyanin production, which is an indication of stress for plants. As such, embryogenesis starts early in callus. Also, placing the proliferating callus at lower temperature (2–3 °C) accelerates the embryogenesis process.
Once the globular embryos can be seen converting to bipolar (Figure 8), subculture them on ½ MSP (see Recipes, Table 3) plates. When placed on ½ MSP, the root develops appropriately, giving the required anchorage for growth (Video 4).
Figure 8. Distinguishing stages of embryos. a) Globular embryo development after a few subcultures. b) Development of bipolar embryos. Once developed, bipolar embryos leave the connection with callus and have their own root and shoot apex with vascular connections.
Remove the bipolar embryos from the plates after 10 days and place them in planton boxes containing approximately 100 mL of ½ MSP media. This ensures the required space for plantlet development.
Let the plantlets grow a decent and stable root system. During this time interval, the shoot system starts touching the planton box cap, so replace the cap with another autoclaved planton box, attaching both the boxes with a coupler frame.
Transfer the developed transgenic plant into a 5” plastic pot containing autoclaved soil mixture, and water it with Hoagland media (see Recipes). Cover the pot with a polythene bag. Sprinkle a few drops of water inside the bag to maintain moisture.
Place the pots in a plastic tray and pour Hoagland media in the tray to maintain the water level in the pots.
Transfer these pots to the culture room. After 10 days, shift them to a greenhouse for hardening. One week later, make a cut at the top of polythene bag. After another week, remove the polythene bag from the pots. This ensures appropriate acclimatization of transgenic plants.
After 10–15 days, when the plants are 15–20 cm long, transfer them into 14” earthen pots containing cotton field soil. Place them in a greenhouse at 28 °C until the harvest.
Extract DNA from the plants and conduct PCR to confirm the insertion of the gene of interest in the cotton plants (Figure 9).
Figure 9. PCR check for the presence of the AnPME gene in putative transgenic lines. 1–9: transgenic lines; N: negative control; P: positive control; NTC: non-template control.
Notes
A few drops of sulfuric acid are sufficient for delinting. Adding excess sulfuric acid will damage the seeds. Dry the seeds properly. Keep the seeds in a 30 °C incubator for several hours before use.
Adding antibiotics to hot media will break down the antibiotics. Therefore, check the temperature of the media before adding antibiotics; it should be bearable when touched with a bare hand.
Bacterial cell suspension culture density must be between 0.8 and 1.6. OD at 600 nm. A lower cell density results in no transformation at all; higher cell density leads to increased Agrobacterium contamination after/during co-cultivation. This causes rotting of the explants. OD600 = 1.2 is best suited for transformation.
Using fresh secondary culture is recommended for transformation. The cell culture density must be attained overnight. Cultures growing for 24–48 h to get the desired OD, or cultures exceeding the recommended point that are further diluted to use for transformation purposes do not ensure efficient transformation.
Augmentin (250 mg/L) ceases the Agrobacterium proliferation after transformation. Therefore, adding it to the selection media ensures efficient transformation.
Callus subculturing is the crucial step in cotton somatic embryogenesis. Yellowish, beady, and friable calli are best suited for regeneration (Video 3). Friable calli can also appear alongside non-viable callus. Therefore, the viable calli should be separated from the non-viable ones and subcultured on a different MSA-2 plate (see Video 3). Some calli appear to be friable, but are hard from the inside when handled with forceps (Figure 7); this type of callus should be discarded. Sometimes, calli become soggy or can get contaminated; these should be removed before subculturing onto the next media.
Video 3. Viable callus subculturing
Embryogenic callus should be subcultured with care and must be separated from the normal callus. Once the embryogenic callus is segregated, embryogenesis is accelerated.
Different stages of embryos should be taken care of. Globular embryos are very small but visible via the naked eye when they appear (Figure 8); take care not to injure them during subculturing. When the embryos convert to the bipolar stage, they leave the connection with the callus and develop their own vascular system (Figure 8). At this stage, embryos can also be removed manually and placed in ½ MSP for further growth (Video 4).
Video 4. Embryo subculturing
It is recommended to transfer plantlets that have developed suitable bipolar growth to the planton boxes. Embryos transferred to ½ MSP growth medium plates may proliferate rapidly but may not have yet developed roots. We recommend using a 100 × 20 mm Petri plate to allow room for growing plantlets. Appropriate space and additional media create the right environment for plant development.
Plantlets are grown in culture media where phytagel is used as a hardening base. Phytagel ensures smooth development of the roots. It also plays an important role while transferring the plantlets to the pots for hardening, as roots can be extracted easily from the phytagel media as it is comparatively less hard than agar.
There can be abnormal embryo formation too. Gibberellic acid (GA3; 1 mg/L) can be used in the ½ MSP culture media to salvage those embryos. Adding GA3 to the media reduces the abnormality.
Precise and cautious removal of media from the roots is extremely necessary, as leftover media decays the root.
During the hardening process, sudden exposure to environmental changes should be avoided. Gradual shifting from culture room to glass house is recommended.
Recipes
Antibiotics
Kanamycin (stock: 100 mg/mL; working: 50 mg/L)
Dissolve 1 g of kanamycin powder in 9.8 mL of double-distilled water. Filter-sterilize with a 0.22 μm syringe filter and store at 4 °C in 1 mL aliquots. Use 500 μL/L in media.
Streptomycin (stock: 250 mg/mL; working: 250 mg/L)
Dissolve 1 g of streptomycin powder in 3.8 mL of double-distilled water. Sterilize with a 0.22 μm syringe filter and store in 1 mL aliquots at 4 °C. Use 1 mL/L in media.
Rifampicin (working: 25 mg/L; always prepared fresh)
Dissolve 25 mg of rifampicin powder in 1 mL of methanol and sterilize with a 0.22 μm syringe filter. Use 1 mL/L in media. Do not store.
Augmentin (working: 250 mg/L; prepared fresh)
Dissolve 1 g of augmentin powder in 3.8 mL of double-distilled water. Sterilize with a 0.22 μm syringe filter and store in 1 mL aliquots at 4 °C. Use 1 mL/L in media. Avoid storing this antibiotic.
Cefotaxime (stock: 250 mg/mL; working: 250 mg/L)
Dissolve 1 g of cefotaxime powder in 3.8 mL of double-distilled water. Sterilize with a 0.22 μm syringe filter and store in 1 mL aliquots at 4 °C. Use 1 mL/L in media.
Phytohormones
2,4-D (stock: 10 mg/mL; working: 0.5 mg/mL): Dissolve 100 mg of 2,4-Dichlorophenoxyacetic acid powder in 1 mL of ethanol and adjust to 10 mL with double-distilled water. Store at 4 °C.
Kinetin (stock: 10 mg/mL; working: 0.2 mg/mL): Dissolve 100 mg of kinetin powder in 1 mL of 1 N NaOH and adjust to 10 mL with double-distilled water. Store at 4 °C.
BAP (stock: 10 mg/mL; working: 0.2 mg/mL): Dissolve 100 mg of BAP powder in 1 mL of 1 N NaOH and adjust to 10 mL with double-distilled water. Store at 4 °C.
Acetosyringone
Dissolve 19.3 mg of acetosyringone powder in 1 mL of DMSO. Filter-sterilize it with a 0.22 μm syringe filter. Store at 4 °C and in the dark. If the solution is prepared correctly, it freezes at 4 °C. Use 100 μL in 100 mL media.
YEB media (1 L), pH = 7.2
Yeast extract 1 g
Beef extract 5 g
Peptone 5 g
Magnesium sulphate heptahydrate 500 mg
Sucrose 5 g
Agar powder 1.5%
Autoclave at 121 °C for 20 min and 15 psi
Induction media (1 L), pH = 6.0
Ammonium chloride 1 g
Magnesium sulphate heptahydrate 0.3 g
Potassium chloride 0.15 g
Calcium chloride 0.01 g
Ferrous sulphate heptahydrate 0.0025 g
Potassium phosphate monobasic 0.272 g
MES buffer 0.390 g
Acetosyringone 100 μm (added after autoclaving)
Glucose 5 g
Autoclave at 121 °C for 20 min and 15 psi
Hoagland media (1 L)
Major salts 50 mL
Minor salts 1 mL
Table 1. Media composition for Hoagland’s media
S. No. Components g/L
Major salts (1 L), stock 50×
1 Potassium nitrate 121.32
2 Calcium nitrate tetrahydrate 18.9
3 Ammonium phosphate monobasic 4.6
4 Magnesium sulphate heptahydrate 4.9
Minor salts (500 mL) 1,000×
5 Potassium chloride 1.86
6 Boric acid 0.773
7 Manganese sulphate hydrate 0.169
8 Zinc sulphate heptahydrate 0.2875
9 Copper (II) sulphate 0.0625
10 Sodium molybdate 0.505
Murashige & Skoog stock preparation
All the components are mixed accordingly and stored in reagent bottles. Stocks can be stored up to one month at 4 °C. MS-III requires an amber bottle for storing.
If ammonium nitrate is not available, Murashige & Skoog basal salt mixture (4.3 g/L) can be used instead of preparing stocks. While using MS basal salt mixture, GAM vitamins need to be added additionally, equal to the quantity given in the table below. However, it is recommended to use stocks for media preparation.
Table 2. Murashige & Skoog’s media composition
S. No. Components g/L g/L (final standard)
MS-I (major salts), stock 50×
1 Ammonium nitrate 82.5 1.650
2 Potassium nitrate 95.0 1.900
3 Magnesium sulphate heptahydrate 18.5 0.370
4 Potassium phosphate monobasic 8.5 0.170
MS-II (CaCl2), stock 100×
5 Calcium chloride dihydrate 44.0 0.440
MS-III (minor salts), stock 1,000×
6 Sodium molybdate 0.250 0.250
7 Boric acid 6.200 6.200
8 Potassium iodide 0.830 0.830
9 Manganese sulphate tetrahydrate 22.300 22.30
10 Zinc sulphate heptahydrate 8.6 8.6
11 Copper sulphate trihydrate 0.025 0.025
12 Cobalt chloride hexahydrate 0.025 0.025
MS-IV (Fe-EDTA), stock 200×
13 Sodium EDTA 7.460 37.3
14 Ferrous sulphate heptahydrate 5.560 27.8
MS-V (GAM Vitamin), stock 1,000×
15 Nicotinic acid 1.00 1
16 Pyridoxine HCl 1.00 1
17 Thiamine HCl 1.00 1
Culture media preparation
Prepare culture media components either in stock form or use fresh components. Even when stocks (Table 2) are used for media preparation, mix all the components accordingly and autoclave at 121 °C for 20 min and 15 psi. Once cooled, add antibiotics, give a good stir, and pour in 90 × 20 mm Petri dishes in a laminar flow hood.
Table 3. List of different media used at different stages of transformation and regeneration of cotton
Culture media (per liter)
S. No. Component MSO ½ MSP MSP-1 MSP-2 MSA-1 MSA-2
1 MS major 20 mL 10 mL 20 mL 20 mL 20 mL 20 mL (without NH4NO3 and double KNO3)
2 MS minor 1 mL 0.5 mL 1 mL 1 mL 1 mL 1 mL
3 GAM vitamin 1 mL 0.5 mL 1 mL 1 mL 1 mL 1 mL
4 Calcium chloride 10 mL 5 mL 10 mL 10 mL 10 mL 10 mL
5 Fe-EDTA 5 mL 2.5 mL 5 mL 5 mL 5 mL 5 mL
6 Myo-inositol 100 mg 100 mg 100 mg 100 mg 100 mg
7 Glucose 10 g 30 g 30 g 30 g 30 g 30 g
8 Magnesium chloride 750 mg 750 mg 750 mg - -
9 BAP - 0.2 mg - - -
10 Kinetin - 0.2 mg - - -
11 2,4-D - 0.5 mg - - -
12 Phytagel 2.2 g 2.2 g 2.2 g - -
13 Agar - - - 8 g 8 g
14 Kanamycin - 50 mg 50 mg - -
15 Augmentin - 250 mg 250 mg - -
16 MES buffer 1.950 g - - - - -
17 pH 6.5 5.8 5.8 5.8 5.8 5.8
MSO: Murashige & Skoog-0 media
MSP: Murashige & Skoog with phytagel
MSA: Murashige & Skoog with agar
Acknowledgments
We are grateful to Scientific and Engineering Board (SERB), India for providing us research grant (CRG/2021/005998) to support our research work. We are also thankful to Council of Scientific and Industrial Research (CSIR), India for providing the research facilities. This protocol has been derived from our previous research paper by Shukla et al. (2016). Institutional Manuscript ID No. CSIR-NBRI_MS/2023/02/02.
Competing interests
The authors declare that there are no existing conflicts of interest as to their knowledge.
References
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Plant Science > Plant transformation > Agrobacterium
Molecular Biology > DNA > Transformation
Biological Sciences > Biological techniques > Microbiology techniques
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THRIFTY—A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection
SE Sebastian Edman *
OH Oscar Horwath *
WA William Apró
(*contributed equally to this work)
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4678 Views: 569
Reviewed by: Alessandro DidonnaMarco Pagliusi Jr.Xin Xu
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Original Research Article:
The authors used this protocol in The Journal of Physiology Oct 2022
Abstract
Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type–dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type–isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology.
Graphical Overview
Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).
Keywords: Muscle fiber type Myosin heavy chain Fiber type identification Muscle
Background
Skeletal muscle consists of a mixture of muscle fiber types, unique in their contractile and metabolic properties, as well as in their proteomic profile (Murgia et al., 2021). At a whole-muscle level, the relative abundance of the fiber types determines whether the muscle is primarily adapted to low-intensity repetitive activities or to short bursts of high-intensity contractions. The relative abundance of fiber types has also been linked to a number of physiological outcomes at a whole-body level, such as obesity and weight loss (Tanner et al., 2002), glucose-stimulated insulin secretion (Blackwood et al., 2022), and risk of cardiovascular disease (Karjalainen et al., 2006). In addition, many reports have demonstrated that skeletal muscle responds to acute exercise stimuli in a fiber type–dependent manner (Koopman et al., 2006; Tannerstedt et al., 2009; Kristensen et al., 2015). Similar observations have been reported for muscle adaptations in response to long-term training regimens, such as muscle fiber hypertrophy, improved glucose transport, and increased mitochondrial content (Daugaard et al., 2000; Verdijk et al., 2009; Skelly et al., 2021). Understanding the molecular intricacies of the different fiber types of skeletal muscle is therefore important for both disease prevention and physical performance.
Fiber type–specific analyses of isolated muscle fibers are typically associated with a tedious and time-consuming workflow, including isolating and fiber typing of hundreds to thousands of individual fibers prior to the experimental analysis. Even though the process of fiber type identification was recently improved by the introduction of the dot blot protocol (Christiansen et al., 2019), this method is still limited by the time required to fiber type large quantities of muscle fibers. The dot blot protocol also requires the entire muscle fiber to be denatured, thus limiting the number of analytical applications that can be performed on the same sample lysate. Therefore, we set out to develop the THRIFTY (high-THRoughput Immunofluorescence Fiber TYping) method to further facilitate the workflow associated with fiber type identification of single muscle fibers. The THRIFTY protocol presented herein will drastically reduce the time consumption of fiber type identification, thus allowing researchers to more easily capture a representative portion of the total muscle fiber pool. Our method also allows for experimental techniques in which a large sample mass is required to obtain proper measurement resolution, e.g., analyses of muscle protein synthesis using stable isotope tracers.
Materials and Reagents
Microscope slides (VWR, catalog number: 631-1554)
Cover slips 24 × 50 mm (VWR, catalog number: 631-1574)
Test tubes, soda glass 40 × 8 mm (VWR, catalog number: 212-0011)
Versilic peroxide-cured silicone stoppers (Lab Pure, catalog number: 263006-50)
CryoPure tubes 1.2 mL (Sarstedt, catalog number: 72.377)
Silica gel, SiO2 (VWR, catalog number: 83001.260)
Milli-Q ultrapure water
Acetone (C3H6O) (Fisher Scientific, catalog number: 1000220500)
Phosphate buffered saline (PBS) tablets (Fisher Scientific, catalog number: 10388739)
TritonTM X-100 (Sigma-Aldrich, catalog number: T8787)
Normal goat serum (NGS) 10% (Thermo Fisher Scientific, catalog number: 50062Z), store at 4 °C
Mouse anti-myosin heavy chain isoform I (DSHB, catalog number: BA-F8-c), store at 4 °C
Mouse anti-myosin heavy chain isoform II (DSHB, catalog number: SC-71-c), store at 4 °C
Alexa Fluor goat anti-mouse IgG2b 488 (Thermo Fisher Scientific, catalog number: A-21141), store at 4 °C in the dark
Alexa Fluor goat anti-mouse IgG1 647 (Thermo Fisher Scientific, catalog number: A-21240), store at 4 °C and in the dark
ProlongTM Gold antifade mountant (Thermo Scientific, catalog number: P36934)
Triton 10% stock solution (see Recipes)
Phosphate buffered saline (PBS) solution (see Recipes)
Primary antibody solution (see Recipes)
Secondary antibody solution (see Recipes)
Equipment
Freeze-dryer system (Heto FD 1.0 cooling unit, Edwards VacuumTM nXDS6i Vacuum Pump and Vacuubrand VAP5 Vacuum Gauge)
Climate controlled room (<40% humidity)
VisiScope® Stereo microscope (VWR, catalog number: 630-3073)
Dumont forceps (FST, catalog number: 11252-00)
Needles (sharpened sewing needles glued to a wooden handle)
Scalpel (Swamm-Morton, carbon steel scalpel, catalog number: 11)
HamiltonTM syringe 10 μL (Fisher Scientific, catalog number: 203560)
Black A4 paper (Common office supply)
Set of micropipettes (Tacta®, mechanical pipettes 0.5–1,000 μL, Sartorius)
Pipette tips (Safetyspace®, filtered pipette tips 0.5–1,000 μL, Sartorius)
Vortex-Genie® (VWR, catalog number: 444-5900)
Magnetic stirrer (Heidolph Instruments, catalog number: 503-02000-00)
Polypropylene slide mailer (Histolab, catalog number: 05309)
Celena S fluorescence microscope (Logos Biosystems)
Celena S LED filter cube (EYFP Ex500/20, Em535/30; Logos Biosystems, catalog number: I10106)
Celena S LED filter cube (Cy5 Long Pass Ex620/60, Em665 lp; Logos Biosystems, catalog number: I10112)
Pen and paper
Software
Celena S Digital Imaging Software (Logos Biosystem)
Procedure
Prepare microscope slides and grid system on black and white A4 paper
Print grid on a microscope slide with solvent-resistant ink using any commercially available printing service with glassware-printing capabilities (Figure 1A).
Using a regular pencil and ruler, draw a grid system corresponding to the printed microscope slide on black A4 paper (gridded paper reusable; Figure 1B).
Using a word processing software (e.g., Microsoft Word), create a similar grid system as in the previous step and print on white A4 office paper for later use during step F6 (Figure 1C).
Figure 1. The coordinate grid system used throughout the THRIFTY protocol.
A. Microscope slide with a custom coordinate grid system printed with solvent-resistant ink. Each square measures 1.05 × 1.05 mm, with a line thickness of 0.5 mm. The entire microscope slide measures 76 × 26 mm. B. A regular A4-sized black paper with a corresponding grid system for keeping track of the remainder of each muscle fiber. C. A matching grid system created in Microsoft Word and printed on regular A4 office paper. This paper is used to note the fiber type identity of each fiber segment when the microscope slide is visualized in the microscope. Type I and type II fiber segments are marked in red (I) and blue (II), respectively.
Isolate individual muscle fibers
Freeze-dry a muscle biopsy sample overnight and store at -80 °C. Before freeze-drying, start the cooling system connected to the vacuum pump and place the muscle sample in a cryotube with small holes in the lid to allow for evaporation. Place the sample in a plastic container (specific to the freeze-dryer) and put it in the -80 °C freezer. Once the plastic container has reached -80 °C, quickly remove it from the freezer and attach it to the freeze-dryer. The sample is then left to dry at a pressure below 0.03 mbar overnight.
Prior to dissection, thaw the biopsy sample to room temperature on silica gel for 30 min.
In a climate-controlled room (<40% humidity), manually isolate individual muscle fibers using needles and forceps under a stereomicroscope (Figure 2). Do this by first locating a fiber bundle of appropriate length, and then splitting the fiber bundle into smaller segments containing 2–10 fibers. Thereafter, fixate the bundle with the forceps and, using the needle, carefully remove individual fibers without breaking them.
Store dissected fibers in glass tubes sealed with silicone stoppers at -80 °C (it is important not to store fibers in plastic tubes, as it may be challenging to retrieve them from the test tubes prior to fiber type identification due to static electricity).
Figure 2. Manual isolation of individual freeze-dried muscle fibers. Isolation is performed under a stereomicroscope using Dumont forceps and custom needles created from sharpened sewing needles attached to a handle.
Mount fiber segment onto the microscope slide
Place the glass tubes with the fibers on silica gel for 30 min to allow thawing to room temperature.
Under a stereomicroscope, and using a needle and scalpel, cut a short segment (<1 mm) of an individual fiber (Figure 3A and 3B).
Place the fiber segment in the first square of the microscope slide (Figure 3C).
Add a droplet of distilled water (dH2O) onto the fiber segment using a Hamilton syringe (attachment occurs as the droplet slowly dries; Figure 3D and 3E).
Place the remaining (large) piece of the muscle fiber directly into the corresponding square of the gridded black A4 paper (Figure 3F).
Repeat steps C2–C5 until all squares of the microscope slide are filled with fiber segments.
Mount one to five microscope slides with fiber segments prior to staining.
Figure 3. Mounting fiber segments and utilizing the coordinated grid system for fiber tracking. A. Isolate a fiber under the microscope. B. Cut the fiber into a large and a small fiber segment using a scalpel. C. Place the smaller fiber segment onto a square of the gridded microscope slide. D and E. Mount the small fiber end onto the microscope slide by adding a small droplet of distilled water onto it using a Hamilton syringe and letting it dry. F. Place the larger fiber end on the corresponding square of the gridded black A4 paper, to facilitate tracking which fiber end represents each of the remaining fiber pieces. All steps that require moving the fibers are done using needles, as muscle fibers generally stick to the needle due to static electricity.
Prepare stock solutions and the primary antibody solution
Prepare a Triton 10% stock solution (see Recipes).
Prepare PBS solution (see Recipes).
Prepare a primary antibody solution (see Recipes).
Staining protocol
Carefully submerge the mounted microscope slide into a polypropylene slide mailer filled with room temperature acetone and incubate for 3 min.
Let the microscope slides dry for approximately 3 min.
Carefully submerge the microscope slides in the primary antibody solution and incubate for 45 min at room temperature.
During incubation with primary antibodies, prepare secondary antibody solution (see Recipes; not recommended to reuse).
Wash the microscope slides three times for 5 min in cold PBS (4 °C) solution by carefully submerging them into mail sliders filled with PBS solution. The slides should be washed without any shaking movements to avoid possible risk of detachment.
Incubate the microscope slides in the secondary antibody solution at room temperature in the dark.
Wash the microscope slides again three times for 5 min in cold PBS (4 °C) solution in the dark.
Carefully dry excess PBS solution from the microscope slide and apply 2–3 droplets of the antifade mounting agent onto the fibers, followed by mounting onto a cover glass.
Let the antifade mounting agent dry for 15 min in the dark.
Fiber type identification in the Celena S microscope
Locate the first fiber segment using the light microscope setting (4× magnification).
Adjust the focus to clearly visualize the fiber segment.
Set the microscope to the following image settings: Light 100%, Gain (18 dB), and 40- and 10 ms, respectively, for the EYFP and Cy5 Long pass channels. Use the function pseudo-color to more easily detect the different fiber types. Here, we used green as a pseudo-color for the EYFP channel and red as a pseudo-color for the Cy5 Long pass channel. For simplicity, the channels will hereafter be referred to as green (EYFP) and red (Cy5 Long pass).
Control the fiber end in both the green and red channels.
Identify the fiber type using the following scheme: type I fibers are stained green, type II fibers are stained red, type I/II hybrid fibers are stained yellow, and more than one fiber segment of different colors in a square indicates a contamination, either because two adjacent fibers were not separated properly or because a residual fiber segment attached to the main fiber during the dissection process. See Figure 4 for an example.
Make a note of the identified fiber type for each fiber mounted on the microscope slide on the gridded white A4 paper.
Figure 4. Examples of fiber type classification using the THRIFTY protocol. Fiber segments can either be classified as type I, type II, hybrid, two fiber segments, or a contamination. Image modified from Horwath et al. (2022).
Locate the next fiber on the gridded microscope slide.
Repeat steps F4–F7 for the remainder of the fiber segments mounted onto the microscope slide.
Collect fibers individually or pool fibers according to their fiber type
The fiber type identity of each fiber placed on the black gridded A4 paper is now provided using the notes from the previous step.
This information can now be used to collect the fibers, either individually or pooled in line with their fiber type identify.
Proceed according to the requirement of the downstream analysis.
Notes
Printing service:
To create the customized glass slide containing a white grid system, we contacted a commercial printing company (Creative Reklam i Sverige AB, Ludvika, Sweden), which printed a 10 × 20 white grid system using solvent-resistant ink with a line thickness of 0.5 mm. Each square in the grid measured 1.05 × 1.05 mm. However, if microscope systems other than the Celena S are to be used, these dimensions can be adjusted accordingly to optimize usability.
To minimize the risk of fiber segments detaching from the microscope slide during the staining procedure, antibody solutions and PBS solution should not be poured over the microscope slide when placed in the slide mailer. Instead, the microscope slide should be carefully submerged into a pre-filled mailer. This includes all incubation and washing steps.
For the same reason as described above, plastic slide mailers are preferred over ordinary Coplin jars to minimize the potential for micro-vibrations, which could cause the fiber segments to detach during staining.
The authors strongly advise users to determine fiber type identity on the basis of the signal obtained in both the green and red channels (i.e., strong signal in the green channel and no signal in the red channel), rather than classifying a fiber segment on the basis of only a single channel (i.e., strong signal in the green channel). This is due to the large risk of misclassifying hybrid fibers, dual fiber segments, or contaminated fibers using this approach.
If speed of the staining procedure is crucial, primary and secondary antibody incubations can be reduced to 10 min each (incubation in 37 °C) and PBS solution washes can be performed once for 5 min instead of three times for 5 min. This allows for a staining protocol of approximately 30 min with a staining quality close to the original protocol.
The protocol described here is for freeze dried muscle; however, the THRIFTY method also works on fresh, non-freeze-dried muscle tissue.
Recipes
Triton 10% stock solution
Add 25 mL of Triton X-100 into 225 mL of dH2O.
Stir gently using a magnetic stirrer as Triton X-100 otherwise tends to create a layer of foam.
Store at room temperature or 4 °C.
Phosphate buffered saline (PBS) solution
Using a magnetic stirrer, dissolve two PBS tablets in 1 L of dH2O.
Store at 4 °C.
Primary antibody solution
Mix 6 mL of NGS with 4.8 mL of PBS solution.
Add 1.2 mL of Triton 10% stock into the solution; do not vortex yet.
As the antibody concentration varies between different batches, calculate the amount of mouse anti-myosin heavy chain isoform I and mouse anti-myosin heavy chain isoform II antibodies required for 30 and 50 μg of the solution, respectively.
Add antibodies to the solution based on the calculations.
Vortex.
Store at 4 °C.
Primary antibody solution can be reused multiple times if kept at 4 °C between uses (over 20 uses over a period of several months).
Secondary antibody solution
Prepare this solution fresh every use, preferably during primary antibody incubation.
Add 1 mL of Triton 10% stock solution and 1 mL of NGS into 8 mL of PBS solution.
Add 10 μL of Alexa Fluor goat anti-mouse IgG2b 488 and Alexa Fluor goat anti-mouse IgG1 647, respectively.
Vortex.
Keep the secondary antibody solution in the dark. The solution can be stored at room temperature if prepared during primary antibody incubation prior to use.
Acknowledgments
This protocol is based on a previous article published in The Journal of Physiology (Horwath et al., 2022). The project was funded by project grants (P2020-0058, P2021-0173) and an Early Research Fellowship (No. D2019-0050), both from the Swedish National Council for Sport Science awarded to W.A.
Competing interests
The authors have no competing interests.
Ethics
All experiments in the development of this method were conducted on human skeletal muscle samples. The muscle samples used to develop this protocol were obtained from three ongoing research projects approved by the Swedish Ethical Review Authority (DNR 2017/2107–31/2, DNR 2017/2034–31/2, and DNR 2019-0038 1). All projects were in agreement with the declaration of Helsinki, and all human volunteers gave both their oral and written consent prior to enrollment in experiments.
References
Blackwood, S. J., Horwath, O., Moberg, M., Pontén, M., Apró, W., Ekblom, M. M., Larsen, F. J. and Katz, A. (2022). Extreme Variations in Muscle Fiber Composition Enable Detection of Insulin Resistance and Excessive Insulin Secretion. J Clin Endocrinol Metab 107(7): e2729-e2737.
Christiansen, D., MacInnis, M. J., Zacharewicz, E., Xu, H., Frankish, B. P. and Murphy, R. M. (2019). A fast, reliable and sample-sparing method to identify fibre types of single muscle fibres. Sci Rep 9(1): 6473.
Daugaard, J. R., Nielsen, J. N., Kristiansen, S., Andersen, J. L., Hargreaves, M. and Richter, E. A. (2000). Fiber type-specific expression of GLUT4 in human skeletal muscle: influence of exercise training. Diabetes 49(7): 1092-1095.
Horwath, O., Edman, S., Andersson, A., Larsen, F. J. and Apró, W. (2022). THRIFTY: a novel high-throughput method for rapid fibre type identification of isolated skeletal muscle fibres. J Physiol 600(20): 4421-4438.
Karjalainen, J., Tikkanen, H., Hernelahti, M. and Kujala, U. M. (2006). Muscle fiber-type distribution predicts weight gain and unfavorable left ventricular geometry: a 19 year follow-up study. BMC Cardiovasc Disord 6: 2.
Koopman, R., Zorenc, A. H., Gransier, R. J., Cameron-Smith, D. and van Loon, L. J. (2006). Increase in S6K1 phosphorylation in human skeletal muscle following resistance exercise occurs mainly in type II muscle fibers. Am J Physiol Endocrinol Metab 290(6): E1245-1252.
Kristensen, D. E., Albers, P. H., Prats, C., Baba, O., Birk, J. B. and Wojtaszewski, J. F. (2015). Human muscle fibre type-specific regulation of AMPK and downstream targets by exercise. J Physiol 593(8): 2053-2069.
Murgia, M., Nogara, L., Baraldo, M., Reggiani, C., Mann, M. and Schiaffino, S. (2021). Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study. Skelet Muscle 11(1): 24.
Skelly, L. E., Gillen, J. B., Frankish, B. P., MacInnis, M. J., Godkin, F. E., Tarnopolsky, M. A., Murphy, R. M. and Gibala, M. J. (2021). Human skeletal muscle fiber type-specific responses to sprint interval and moderate-intensity continuous exercise: acute and training-induced changes. J Appl Physiol (1985) 130(4): 1001-1014.
Tanner, C. J., Barakat, H. A., Dohm, G. L., Pories, W. J., MacDonald, K. G., Cunningham, P. R., Swanson, M. S. and Houmard, J. A. (2002). Muscle fiber type is associated with obesity and weight loss. Am J Physiol Endocrinol Metab 282(6): E1191-1196.
Tannerstedt, J., Apro, W. and Blomstrand, E. (2009). Maximal lengthening contractions induce different signaling responses in the type I and type II fibers of human skeletal muscle. J Appl Physiol (1985) 106(4): 1412-1418.
Verdijk, L. B., Gleeson, B. G., Jonkers, R. A., Meijer, K., Savelberg, H. H., Dendale, P. and van Loon, L. J. (2009). Skeletal muscle hypertrophy following resistance training is accompanied by a fiber type-specific increase in satellite cell content in elderly men. J Gerontol A Biol Sci Med Sci 64(3): 332-339.
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© 2023 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 > Tissue analysis > Tissue imaging
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Determination of Interleukin-17A and Interferon-γ Production in γδ, CD4+, and CD8+ T Cells Isolated from Murine Lymphoid Organs, Perivascular Adipose Tissue, Kidney, and Lung
KC Kevin Comeau
AC Antoine Caillon
Pierre Paradis
ES Ernesto L. Schiffrin
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4679 Views: 1252
Reviewed by: Meenal SinhaJulie WeidnerClara Lubeseder-Martellato
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Original Research Article:
The authors used this protocol in Hypertension Research Jan 2023
Abstract
T cells localized to the kidneys and vasculature/perivascular adipose tissue (PVAT) play an important role in hypertension and vascular injury. CD4+, CD8+, and γδ T-cell subtypes are programmed to produce interleukin (IL)-17 or interferon-γ (IFNγ), and naïve T cells can be induced to produce IL-17 via the IL-23 receptor. Importantly, both IL-17 and IFNγ have been demonstrated to contribute to hypertension. Therefore, profiling cytokine-producing T-cell subtypes in tissues relevant to hypertension provides useful information regarding immune activation. Here, we describe a protocol to obtain single-cell suspensions from the spleen, mesenteric lymph nodes, mesenteric vessels and PVAT, lungs, and kidneys, and profile IL-17A- and IFNγ-producing T cells using flow cytometry. This protocol is different from cytokine assays such as ELISA or ELISpot in that no prior cell sorting is required, and various T-cell subsets can be identified and individually assessed for cytokine production simultaneously within an individual sample. This is advantageous as sample processing is kept to a minimum, yet many tissues and T-cell subsets can be screened for cytokine production in a single experiment. In brief, single-cell suspensions are activated in vitro with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Golgi cytokine export is inhibited with monensin. Cells are then stained for viability and extracellular marker expression. They are then fixed and permeabilized with paraformaldehyde and saponin. Finally, antibodies against IL-17 and IFNγ are incubated with the cell suspensions to report cytokine production. T-cell cytokine production and marker expression is then determined by running samples on a flow cytometer. While other groups have published methods to perform T-cell intracellular cytokine staining for flow cytometry, this protocol is the first to describe a highly reproducible method to activate, phenotype, and determine cytokine production by CD4, CD8, and γδ T cells isolated from PVAT. Additionally, this protocol can be easily modified to investigate other intracellular and extracellular markers of interest, allowing for efficient T-cell phenotyping.
Keywords: T-cell activation Intracellular staining Flow cytometry Cytokines Phenotyping Hypertension
Background
Adaptive immune cells, including CD4+, CD8+, and γδ T cells, play an important role in hypertension and vascular injury (Caillon et al., 2019; Higaki et al., 2019; Higaki et al., 2021). T-cell subtypes are programmed to produce interleukin-17 (IL-17) or interferon-γ (IFNγ). Naïve T cells can also be induced to produce IL-17 through stimulation of the IL-23 receptor. Hypertension is associated with increased infiltration of T cells in perivascular adipose tissue (Caillon et al., 2017), with both IL-17 and IFNγ having been demonstrated to contribute to hypertension (Madhur et al., 2010; Itani et al., 2016). We have therefore developed a protocol to profile CD4+, CD8+, and γδ T-cell subtypes producing IL-17A and/or IFNγ in tissues relevant to hypertension. This is accomplished by performing intracellular staining with fluorescent-labeled monoclonal antibodies and flow cytometry. This protocol was used to investigate the role of the IL-23 receptor in the regulation and homeostasis of IL-17A-producing γδ T cells, specifically those localized to mesenteric vessels and perivascular adipose tissue (MV/PVAT) in an angiotensin II–induced model of hypertension (Shokoples et al., 2022). We showed that IL-23 receptor deficiency caused a reduction in IL-17A-producing γδ T cells and expansion of IFNγ-producing CD4+, CD8+, and γδ T cells. Angiotensin II treatment led to expansion of IFNγ-producing CD4+, CD8+, and γδ T cells in IL-23 receptor knockout mice. However, only IFNγ-producing γδ T cells expanded in angiotensin II–treated wild-type mice. Accordingly, profiling cytokine-producing T-cell subtypes in tissues relevant to hypertension provides useful information regarding immune activation.
Here, we describe methods used to obtain single-cell suspension from spleens, mesenteric lymph nodes (mLNs), MV/PVAT, lungs, and kidneys from four wild-type C57BL/6 mice. This is followed by an in vitro T-cell activation in the presence of the Golgi blocker monensin, which allows for signal amplification by trapping cytokines within activated cells. Next, cells are stained for viability and surface marker expression, which is then followed by IL-17A and IFNγ intracellular staining. Finally, IL-17A- and IFNγ-producing CD4+, CD8+, and γδ T cells are profiled by flow cytometry. This protocol has been extensively tested and optimized for use with samples derived from male C57BL/6 mice, but it should work equally well with other mouse strains as well as female mice. However, some optimization may be required. It is important to note that this technique cannot be used to quantify the cytokine produced, which may represent a limitation. However, this protocol can precisely identify cytokine-producing cells within less numerous T-cell subsets, as there is no requirement for cell sorting before the activation step, so sample loss is minimized. The methods presented in this protocol are advantageous as they provide the ability to screen several tissues for cytokine-producing T cells simultaneously, which can then be followed up with quantitative methods in subsequent experiments.
The methods described below are designed for processing four mice by one person. Someone not experienced in these techniques should start with two animals per day. It is also possible to scale up this protocol by getting help from other colleagues to collect tissues and prepare single-cell suspensions. The remaining part of the protocol can easily be handled by one experienced person. The fluorochrome-conjugated monoclonal antibodies used to determine surface marker expression can be tailored to the individual experiment, as this protocol only contains the minimum number of markers required to phenotype IL-17A and IFNγ production in T cells. This protocol also works when using primary and secondary antibodies to investigate surface marker expression. The protocol is optimized to look at the production of IL-17A and IFNγ in T cells and may need to be modified to investigate the production of other cytokines and/or the expression of other intracellular markers.
Figure 1. A flow diagram outlining the whole protocol. mLNs: mesenteric lymph nodes; MV/PVAT: mesenteric vessels and perivascular adipose tissue; PMA: phorbol 12-myristate 13-acetate; FMO: Fluorescence Minus One.
Materials and Reagents
Ethyl alcohol anhydrous, USP (Commercial alcohols by Greenfield Global, catalog number: P016EAAN)
Double-distilled water
Centrifuge tubes, 50 mL (VWR, catalog number: 89039-656)
Sterile 100 mm tissue culture dishes, standard (Sarstedt, catalog number: 83.3902)
Cell strainers, 70 μm nylon mesh (Sarstedt, catalog number: 83.3945.070)
Tuberculin syringes, 1 mL with luer slip tip (BD Biosciences, catalog number: BD309659)
Luer lock syringes, 3 mL (BD Biosciences, catalog number: BD309657)
Falcon serological pipette, 10 mL (VWR, catalog number: CA53300-523)
Pipette tips: 10 μL for Gilson PIPETMAN Classic P2 (Sarstedt, catalog numbers: 70.3010 for bag and 70.3010.200 for racked tips), 200 μL for Gilson PIPETMAN Classic P20 and P200 (Gilson, catalog number 70.3030 for bag and 70.3050.200 for racked tips), and 1,000 μL for Gilson PIPETMAN Classic P1000 (Gilson, catalog number 70.3050 for bag and 70.3050.200 for racked tips); 250 μL pipette tips RC LTS used for Rainin multichannel pipette (METTLER TOLEDO, catalog number: 17001118 for bag and 30389243 for racked tips)
Falcon 5 mL polystyrene round-bottom tubes (VWR, catalog number: 352058)
Racked mini tubes, 1.1 mL (VWR, catalog number: 89005-572)
Microtubes, 1.5 mL with conical base and attached flat cap (Sarstedt, catalog number: 72.690.300)
Microtubes, 5 mL with conical base and attached flat cap (Sarstedt, catalog number: 72.701.400)
Sterile Falcon 24-well tissue culture plates, flat bottom with lid (VWR, catalog number: 353047)
Nunc 96-well polystyrene conical bottom microwell plates (Thermo Fisher Scientific, catalog number: 249570)
Isoflurane (CDMV, catalog number: 55868225)
Oxygen gas (O2)
Surgical sutures for small animal surgery, non-absorbable silk 7-0 (Braintree Scientific, catalog number: SUT-S 103)
Phosphate buffered saline (PBS) pH 7.4, Ca2+ and Mg2+ free, 500 mL (Thermo Fisher Scientific, catalog number: 10010023)
Roswell Park Memorial Institute 1640 (RPMI), with L-glutamine and phenol red, HEPES-free (Thermo Fisher Scientific, catalog number: 11875093)
Collagenase A from Clostridium histolyticum, lyophilized, ≥0.15 U/mg protein (Roche, catalog number: 10103586001)
Collagenase type 2, lyophilized, >125 U/mg protein (Worthington Biochemical, catalog number: LS004176)
Elastase, lyophilized, ≥3 U/mg protein (Worthington Biochemical, catalog number: LS002292)
Hyaluronidase, lyophilized, ≥300 U/mg protein (Sigma-Aldrich, catalog number: H2126-500MG)
Trypsin inhibitor, soybean, purified (Worthington Biochemical, catalog number: LS003570)
Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P1585-1MG)
Ionomycin (Sigma-Aldrich, catalog number: I0634-1MG)
Monensin 1,000× solution (BioLegend, catalog number: 420701). Monensin solution is supplied by BioLegend as a 1,000× working solution in 70% ethanol. The concentration is 2 mM. Store the monensin 1,000× solution at 4 °C until used
LIVE/DEAD Fixable Aqua Dead Cell Stain kit (405 nm excitation, 200 assays) (Thermo Fisher Scientific, catalog number: L34957). The kit contains five vials of stain (component A) and one vial of anhydrous dimethyl sulfoxide (DMSO, component B)
Fetal bovine serum (FBS), qualified, Canada origin, 500 mL (Thermo Fisher Scientific, catalog number: 12483020)
Mouse Fc blocking reagent: purified rat anti-mouse CD16/CD32 Fc-block (BD Biosciences, catalog number: 553142, clone 2.4G2, 0.5 mg, 0.5 mg/mL)
Antibodies for extracellular and intracellular staining are presented in Table 1
Table 1. Antibodies for extracellular and intracellular staining
Antigen Antibodies Clone, company (catalog #)
CD45 BV786-conjugated rat anti-mouse CD45 antibody 30-F11, BD Biosciences (564225)
CD3 BUV395-conjugated rat anti-mouse CD3 antibody 17A2, BD Biosciences (740268)
CD4 PerCP-eFluor710-conjugated rat anti-mouse CD4 antibody RM4-5, eBioscience (46-0042-82)
CD8a AF700-conjugated rat anti-mouse CD8a antibody 53-6.7, BioLegend (100730)
TCR γδ PE-CF594-conjugated hamster anti-mouse TCR δ antibody GL3, BD Biosciences (563532)
IL-17A APC-conjugated rat anti-mouse IL-17A antibody eBio17B7, eBioscience (17-7177-81)
IFNγ AF488-conjugated rat anti-mouse IFNγ antibody XMG1.2, eBioscience (53-7311-82)
AF488 and AF700: Alexa-Fluor 488 and 700; APC: allophycocyanin; BUV395: Brilliant Ultraviolet 395; BV786: Brilliant Violet 786; CD: cluster of differentiation; CF594: cyanine‐based fluorescent dye 594; IL-17A: interleukin-17A; IFNγ: interferon gamma; PE: phycoerythrin; PerCP: peridinin-chlorophyll-protein.
Paraformaldehyde 10% aqueous solution (PFA), 10 × 10 mL (Electron Microscopy Sciences, catalog number: 15712)
Probumin® bovine serum albumin (BSA), 100 g, life science grade (Sigma-Aldrich, catalog number: 810683)
Saponin, 100 g (Calbiochem, catalog number: 558255)
InvitrogenTM UltraComp eBeadsTM (Thermo Fisher Scientific, catalog number: 01-2222-42)
ArcTM Amine-Reactive Compensation Bead kit (Thermo Fisher Scientific, catalog number: A10346). The kit contains ArC-reactive beads (Component A) that bind any of the amine-reactive dyes and provide a positive signal, and ArC-negative beads (Component B), which have no reactivity and provide a negative compensation control)
The following reagents and equipment need to be prepared prior to beginning the protocol
Detailed descriptions can be found under Recipes.
Reagents and equipment to prepare in advance (see Recipes)
70% ethanol
Sterile pipette tips
Sterile dissecting tools
Digestion stock solution (8×)
Hyaluronidase stock solution (8×)
PMA 1 mg/mL stock solution
Ionomycin stock solution (1 mg/mL)
LIVE/DEAD stain stock solution
FBS aliquots
Permeabilization buffer
Reagents to prepare within three days of the experiment (see Recipes)
Pre-diluted antibody mixtures
Reagents to prepare on the day of the experiment (see Recipes)
24-well tissue culture plate for the collection of tissues
Labeled 50 mL centrifuge tube for collecting the intestines and mesenteric bed
T-cell activation cocktail (1 mL/sample)
PFA 1% solution
LIVE/DEAD staining solution
Equipment
Small animal isoflurane anesthesia machine with adequate gas scavenging system or filter, an induction chamber constructed of a see-through material (glass, polycarbonate, etc.), and a mouse anesthesia nosecone or mask (Dispomed)
Necropsy tools
1× surgical scissors, sharp-blunt, 14.5 cm (Fine Science Tools, catalog number: 14001-14)
1× fine scissors, sharp, 9 cm (Fine Science Tools, catalog number: 14061-09)
1× delicate suture tying forceps, 9 cm, tip: angled 45° and smooth (Fine Science Tools, catalog number: 11063-07)
2× Graefe forceps, 10 cm, tip: curved and serrated (Fine Science Tools, catalog number: 11051-10)
Dissecting tools
2× splinter forceps (tweezers), number 5 long points, 11 cm (Almedic, catalog number: A10-704)
1× student Vannas spring scissors, tips: sharp, cutting edge: 5 mm, tip diameter: 0.35 mm, length: 9 cm (Fine Science Tools, catalog number: 91500-09)
Biological safety cabinet, class II (Thermo Fischer Scientific)
Drummond Portable Pipet Aid XP (Cole-Parmer)
Single-edged surgical carbon-steel blades (Thermo Fisher Scientific, catalog number: S17302)
Ice packs stored in a -20 °C freezer
Binocular dissecting microscope Leica MS 5 with variable magnification (Leica Microsystems)
VWR Boekel hybridization incubator oven at 37 °C equipped with a rocker platform (VWR)
Cell culture incubator (5% CO2, 37 °C, Thermo Fischer Scientific)
Refrigerator at 4 °C (Thermo Fisher Scientific)
Dissection Petri dish: 100 × 10 mm Pyrex Petri dish (Cole-Parmer, catalog number: UZ-34551-02) with wax bottom lining (Thermo Fisher Scientific, catalog number: S80769) for pinning down samples
BD PrecisionGlide needle 26 G × 5/8 Sub-1 (BD, catalog number: 305115) for pinning down the mesenteric bed in the dissection Petri dish
Centrifuge 5810 R with swinging bucket rotor A-4-81 and adaptors for 96-well plates and 5 and 50 mL Eppendorf tubes (Eppendorf)
P2, P20, P200, and P1000 Gilson PIPETMAN Classic single-channel pipettors (Thermo Fisher)
Rainin Pipet-Lite L200 8-Channel Multichannel 20–200 μL LTS Pipette (METTLER TOLEDO, see Note 1)
Multicolour flow cytometer: BD LSR Fortessa (BD Biosciences), equipped with 355, 405, 488, 561, and 640 nm excitation lasers and appropriate bandpass filters
Software
FlowJo V10 (FlowJo LLC, BD, https://www.flowjo.com)
Procedure
Tissue collection (see Note 2)
Anesthetize a mouse with 3% isoflurane mixed with O2 at 1 L/min following the standard operating procedure of your institution and verify depth of anesthesia by the absence of the pedal withdrawal reflex to rear foot squeezing.
Perform a laparotomy to expose the intestines.
Dissect and transfer the spleen to the spleen well of the labeled 24-well tissue culture plate containing cold RPMI. Store on ice (Figure 2A).
Tie off the ends of the intestines using surgical sutures at the beginning of the duodenum (Figure 2B) and the end of the colon (Figure 2C) to prevent excess leakage of intestinal contents. Excise the intestines with the mesenteric bed using scissors, keeping the knots attached to the intestines, and transfer to a 50 mL centrifuge tube containing PBS. Store on ice.
Dissect out the kidneys (Figure 2D) and transfer both kidneys to the kidney well of the labeled 24-well tissue culture plate containing RPMI. Store on ice.
Open the thoracic cage by cutting the ribs laterally to each side of the sternum, and immediately remove the heart to euthanize the animal (Figure 2E). Then, hold the lungs by the bronchi with tweezers and remove both lungs by cutting the trachea with scissors (Figure 2F). Transfer the lungs to the lung well of the labeled 24-well tissue culture plate containing RPMI. Store on ice.
Repeat the above operation with the remaining mice.
Figure 2. Collection of tissues. Perform a laparotomy to expose the intestines and remove the spleen (A). Tie off the beginning of the duodenum (B) and the end of the colon (C) using surgical sutures and excise the intestines. Then, dissect out the kidneys. Open the thoracic cage by cutting the ribs laterally to each side of the sternum and remove the heart (E). Finally, hold the lungs by the bronchi with tweezers and remove both lungs by cutting the trachea with scissors (F).
Preparation of single-cell suspension from the lungs, kidneys, and MV/PVAT
Single-cell suspensions are obtained from the lungs, kidneys, and MV/PVAT by digesting these tissues with a cocktail of digestive enzymes. Begin by digesting the lungs, followed by the kidneys and the MV/PVAT, as the lungs take the longest to digest. Digestions are to be carried out at 37 °C with agitation in a hybridization incubator oven equipped with a rocker platform set at 30 cycles per minute with a displacement amplitude of 3 cm. Digestions are ended by placing the tubes containing digestive mixtures on ice, as the enzymes are non-functional at low temperature.
For four mice, prepare four digestion tubes per tissue as follows. For each lung digestion tube, combine 600 μL of RPMI, 100 μL of 8× digestion stock solution, and 100 μL of 8× hyaluronidase stock solution in a 1.5 mL microtube. For each MV/PVAT digestion tube, add 700 μL of RPMI to a 100 μL aliquot of 8× digestion stock solution. For the kidneys, reserve four undiluted 100 μL aliquots of 8× digestion stock solution. Keep all enzyme digestion tubes on ice until used.
Transfer the lungs from the cold RPMI to a 100 mm tissue culture dish sitting on an ice pack that has been removed immediately before from a -20 °C freezer. Thoroughly mince the lungs with scissors until a paste is formed. Then, transfer the minced tissue using tweezers and/or a razor blade into a lung enzyme digestion tube. Store the tube on ice until all lung samples have been processed as above.
Start the digestion of the lung samples at 37 °C with agitation in a hybridization incubator oven as described above. The digestions should be completed in approximately 2 h. Check the progression of the digestion every 30 min by removing the tubes from the oven and holding them up to a light source. Look at the smoothness of the solution. If chunks of tissue are visible, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, and return the tubes to the oven to continue the digestion for another 30 min. Repeat as necessary until the tissue/digestion solution appears smooth. At the end of the 2 h digestion, if the solution is almost smooth, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, place the tube back into the hybridization oven, and check again in 5 min. When the lungs are fully digested, remove the tubes from the incubator and place on ice to stop the digestion.
While the lungs are being digested, remove the capsule from the kidneys using forceps and surgical scissors and transfer the kidneys to a kidney processing well of the 24-well tissue culture plate containing 500 μL of cold RPMI. Cut out the nylon mesh of a 70 μm cell strainer using a razor blade and place the mesh over the top of the kidneys in the well of the 24-well plate with RPMI using tweezers (Figure 3). Using the back of a sterile 3 mL syringe plunger, gently crush the kidneys under the nylon mesh. Transfer the RPMI and kidney solution using a P1000 pipette along with the nylon mesh using tweezers to an aliquot of undiluted (100 μL) 8× digestion stock solution. Each kidney tube should now contain 100 μL of digestion stock solution, 500 μL of RPMI, and the crushed kidneys. Store the tube on ice until all kidney samples have been processed as above.
Figure 3. Crushing of spleen, kidneys, and mesenteric lymph nodes (mLNs). The tissues are crushed by pressing gently with the back of a syringe plunger on a nylon mesh positioned over the tissues.
Start the digestion of the kidney samples at 37 °C with agitation in a hybridization incubator oven and check the progression of the digestion every 15 min as above. The kidney digestion should be completed in approximately 30 min. If chunks of tissue are visible, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, and return the tubes to the oven to continue the digestion for another 15 min. After 30 min of digestion, if the solution is almost smooth, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, return the tube to the hybridization oven, and check again in 5 min. When the kidneys are fully digested, remove the tubes from the incubator and place on ice to end the digestion.
While the lungs and kidneys are being digested, pour ice-cold PBS into a dissection dish and place the dish over an ice pack that has been removed immediately before from a -20 °C freezer as above. Transfer the mesenteric bed from the 50 mL centrifuge tube into the dissection dish containing cold PBS. Place the dish sitting on the cold ice pack under the direct view of a dissecting microscope on low magnification. Find the cluster of mLNs proximal to the cecum and excise the whole cluster with scissors. In addition, check for small lymph nodes that could reside within the adipose tissue of the mesenteric bed. Lymph nodes are more opaque than adipose tissue and have a white hue and a defined capsule. They can be cut out of the tissue using small scissors. Place the mLNs into the labeled well of a 24-well plate containing 1 mL of cold RPMI and set the plate aside on ice for later.
Set a 100 mm tissue culture dish on another ice pack that has been removed immediately before from the -20 °C freezer.
Stretch out the mesenteric vascular bed and pin it down using 26 G needles in the dissection dish to expose the MV/PVAT (Figure 4). Cut out all the MV/PVAT using scissors and transfer to the 100 mm tissue culture dish sitting on an ice pack. Check the tissue again for the presence of any lymph nodes under the microscope and remove any that are found. Thoroughly mince the tissue with scissors and then transfer the minced tissue into a MV/PVAT digestion tube using small forceps. Store the tube on ice until all MV/PVAT samples have been processed as above.
Figure 4. Visualization of mesenteric vessels and perivascular adipose tissue (MV/PVAT) and lymph nodes (mLNs)
Start the digestion of the MV/PVAT samples at 37 °C with agitation in a hybridization incubator oven and check the progression of the digestion after 15 min and at the end of the digestion as above. The digestion should be done in approximately 25 min. If chunks of tissue are visible, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, and return the tubes to the oven to continue the digestion. After 25 min of digestion, if the solution is almost smooth, shake the tube vigorously by hand or vortex the tube at medium-high speed for several short 5 s bursts, return the tube to the hybridization oven, and check again in 5 min. When the MV/PVAT is fully digested, remove the tubes from the incubator and place on ice to stop the digestion.
When all tissues have been digested, filter each suspension through a 70 μm cell strainer into labeled 50 mL Falcon tubes (for the kidneys and lungs) and 1.5 mL microtubes (for the MV/PVAT). Wash the filter with a small amount of RPMI to help free cells from the filter (1 mL of RPMI for the kidneys and lungs and 200 μL of RPMI for the MV/PVAT to account for the smaller tube size). When filtering the suspensions and washing the cell strainer with RPMI, it is recommended to slowly move the pipette tip in small circles just above the nylon mesh while depressing the plunger. This will help to dissociate any remaining clumps of cells and prevent them from clinging to the mesh, thereby minimizing sample loss. The smaller 1.5 mL microtubes are used to minimize loss of cells due to the small number of T cells present in this tissue. Microtubes also allow for the formation of a more robust cell pellet in tissues with fewer immune cells (the small pellet will be more resistant to being lost when pouring out the supernatant after centrifugation in the following steps). Keep filtered suspensions on ice until the in vitro T-cell activation step.
Preparation of single-cell suspensions from spleens and mLNs
Cut out the nylon mesh of a 70 μm cell strainer using a razor blade and place the mesh over the top of a spleen in the well of a 24-well tissue culture plate containing 1 mL of RPMI (Figure 3).
Using the back of a 3 mL syringe plunger, crush the spleen underneath the nylon mesh until dissociated. Repeat for each spleen sample.
Filter each splenocyte suspension through a 70 μm cell strainer placed on top of a 50 mL centrifuge tube. Wash the cell strainer with 1 mL of RPMI to free cells from the filter. Store the 50 mL tube on ice until in vitro T-cell activation step.
Process all remaining spleen samples as above.
Repeat steps C1–C3 with each mLN sample with some modifications. Use the back of a 1 mL syringe plunger to crush mLNs with more precision. Crush mLNs approximately seven times with gentle pressure to avoid damaging immune cells; the mLNs are much more sensitive to this process than the spleen.
Filter mLN samples through 70 μm cell strainers into 1.5 mL microtubes to minimize loss of immune cells and allow for the formation of a more robust cell pellet.
Keep filtered suspensions on ice until in vitro T cell activation step.
Process all remaining mLN samples as steps C5–C7.
In vitro T-cell activation
Prepare one 24-well tissue culture plate for the in vitro activation of T cells from lungs, kidneys, MV/PVAT, spleen, and mLNs as depicted in Figure 5. Label five wells per animal with the tissue names and animal number.
Figure 5. Preparation of a 24-well tissue culture plate for in vitro T-cell activation. The volumes in milliliters of single-cell suspension in T-cell activation cocktail are presented for the lungs, kidneys, mesenteric vessels with perivascular adipose tissue (MV/PVAT), spleen, and mesenteric lymph nodes (mLNs).
Centrifuge all the samples at 410× g for 5 min at 4 °C.
After centrifugation, pour out the supernatant from each tube and resuspend each cell pellet with 1 mL of ice-cold T-cell activation cocktail by pipetting up and down several times. Transfer each cell suspension into the appropriate well of the 24-well tissue culture plate.
Incubate the 24-well tissue culture plate in a cell culture incubator set at 37 °C and 5% CO2 for 4 h.
During the incubation, prepare twenty 1.5 mL microtubes to collect the samples of lung, kidney, MV/PVAT, spleen, and mLN after the in vitro T-cell activation. Label five microtubes per animal with the tissue names and animal number. In addition, label 44 wells of one V-bottom 96-well plate for extracellular and intracellular staining as depicted in Figure 6.
Figure 6. Preparation of the V-bottom 96-well plate for extracellular and intracellular staining. The volumes of cell suspension in microliters to add per unstained control, fluorescence minus one (FMO) control, and sample wells are presented. The unstained and FMO controls are prepared using pooled spleen, lung, and kidney samples. No unstained and FMO control are prepared for mesenteric vessels with perivascular adipose tissue (MV/PVAT) and mesenteric lymph nodes (mLNs), as these tissues do not contain enough T cells. The spleen is used for unstained and FMO controls for these tissues.
Remove the 24-well plate from the incubator and transfer each sample into separate, labeled 1.5 mL microtubes.
Centrifuge all of the samples at 410× g for 5 min at 4 °C.
Distributing activated cell samples to a V-bottom 96-well plate for extracellular and intracellular staining
After centrifugation, pour out the supernatant from each tube and resuspend each cell pellet with the quantity of PBS listed below (specific for each tissue). Transfer aliquots of cell suspensions into unstained-, fluorescence minus one (FMO)-, and sample-labeled wells of the V-bottom 96-well plate according to the volumes listed below and in Figure 6.
Spleen: Resuspend each spleen sample in 500 μL of PBS and transfer 200 μL (40% of the spleen per sample) into the spleen sample wells of the V-bottom 96-well plate. Pool remaining spleen samples in one microtube and transfer 50 μL of pooled splenocytes into each unstained spleen and FMO well (equivalent to 10% of a spleen).
Lungs: Resuspend each lung sample in 500 μL of PBS and transfer 200 μL (40% of the lungs) into the lung sample wells of the V-bottom 96-well plate. Pool remaining lung samples in one microtube and transfer 50 μL of pooled lung cell suspension into each unstained lung and FMO well (equivalent to 10% of the lungs).
Kidneys: Resuspend each kidney sample in 500 μL of PBS and transfer 150 μL (30% of the combined kidneys) into the kidney sample wells of the V-bottom 96-well plate. Pool remaining kidney samples and transfer 50 μL of pooled kidney cell suspension into each unstained kidney and FMO well (equivalent to 10% of two kidneys).
MV/PVAT and mLNs: Resuspend each MV/PVAT and mLN sample in 100 μL of cold PBS and transfer the entire sample into each respective sample well of a V-bottom 96-well plate. These tissues do not have enough T cells present to prepare unstained, FMO, and samples wells. The spleen is used for unstained and FMO controls for these tissues.
Note: The volume of spleen, lung, and kidney cell suspension for unstained and FMO wells could be reduced to 25 μL (5%) if only one animal is being processed.
Centrifuge the V-bottom 96-well plate at 410× g for 5 min at 4 °C.
Live/Dead viability staining
Perform this step out of direct sunlight to prevent bleaching of the fluorochrome.
Pour out the supernatant from the V-bottom 96-well plate into the sink.
Resuspend the cell pellets in the FMO control and sample wells with 100 μL of prepared LIVE/DEAD staining solution. Resuspend the cell pellet in the unstained control wells with 100 μL of PBS.
Cover the V-bottom 96-well plate with aluminum foil (to protect from light) and incubate it at 4 °C in a refrigerator for 20 min.
Centrifuge the V-bottom 96-well plate at 410× g for 5 min at 4 °C.
Extracellular staining
Perform this step out of direct sunlight to prevent bleaching of the fluorochromes.
Note: If using primary and secondary antibodies for determination of marker expression, perform that staining/conjugation protocol at this step.
Pour out the supernatant from the 96-well plate into the sink.
Resuspend the cell pellets in the unstained control, FMO control, and sample wells with 100 μL of the appropriate unstained control, FMO control, or sample pre-diluted antibody mixture for extracellular staining. Refer to Figure 6 for the distribution of unstained control, FMO control, and sample wells.
Cover the plate with aluminum foil and incubate at 4 °C in a refrigerator for 15 min.
Note: This is adequate for binding of most monoclonal antibodies, but incubation time and temperature can be modified to accommodate antibodies with poor binding affinity (i.e., use a higher temperature and/or longer incubation time).
Centrifuge the V-bottom 96-well plate at 410× g for 5 min at 4 °C.
Fixation in 1% PFA
Perform this step out of direct sunlight, as PFA is light-sensitive.
Pour out the supernatant from the V-bottom 96-well plate into the sink.
Gently resuspend the cell pellets with 100 μL of 1% PFA. Do not create air bubbles. Resuspend as gently as possible to avoid shearing the cells by pipetting up and down 7–10 times with a P200 single or multichannel pipette.
Cover the V-bottom 96-well plate with aluminum foil and incubate at 4 °C in a refrigerator for 10 min.
Remove the V-bottom 96-well plate from the refrigerator and gently drip 100 μL of cold PBS into each well to help wash the cells.
Centrifuge the V-bottom 96-well plate at 410× g for 5 min at 4 °C.
Permeabilization/intracellular cytokine staining
Perform this step out of direct sunlight to prevent bleaching of the fluorochromes. The permeabilization and intracellular cytokine staining are performed simultaneously.
Pour out the supernatant from the 96-well plate into the sink.
Resuspend the cell pellets in the unstained control, FMO control, and sample wells with 100 μL of the appropriate unstained control, FMO control, or sample pre-diluted antibody mixtures for intracellular staining. Do not create air bubbles. Resuspend as gently as possible to avoid shearing the cells. Refer to Figure 6 for the distribution of unstained control, FMO control, and sample wells.
Cover the V-bottom 96-well plate with aluminum foil and incubate at 4 °C in a refrigerator for 30 min.
Remove the V-bottom 96-well plate from the refrigerator and gently drip 100 μL of cold PBS into each well to help wash the cells.
Centrifuge the V-bottom 96-well plate at 410× g for 5 min at 4 °C.
Transferring unstained controls, FMO controls, and samples into flow cytometry tubes
Pour out the supernatant from the V-bottom 96-well plate into the sink.
Resuspend the cell pellets with ice-cold PBS as follows. For spleen, kidney, and lung sample wells, resuspend the cell pellets with 150 μL of PBS. For mLNs and MV/PVAT sample wells and all the unstained and FMO control wells, resuspend the cell pellets with 100 μL of cold PBS. Refer to Figure 6 for the distribution of unstained control, FMO control, and sample wells.
Transfer all the cell suspensions into labeled Axygen 1.1 mL polypropylene cluster tubes and store for a maximum of three days at 4 °C in a refrigerator until running the samples on the flow cytometer.
Preparation of compensation beads for flow cytometry compensation (can be prepared in the days leading up to flow cytometry acquisition)
Prepare a V-bottom 96-well plate for staining compensation beads and Axygen 1.1 mL polypropylene cluster tubes for flow cytometry acquisition, by labeling one well and one tube for LIVE/DEAD stain, and one well and one tube per fluorochrome-conjugated monoclonal antibody used for extracellular and intracellular staining.
Add one drop of InvitrogenTM UltraComp eBeadsTM to each fluorochrome-conjugated monoclonal antibody well of the V-bottom 96-well plate.
Add one drop of Arc Amine-reactive compensation beads to the LIVE/DEAD stain well of the V-bottom 96-well plate.
Add 1 μL of each fluorochrome-conjugated monoclonal antibody to the appropriate well of the V-bottom 96-well plate containing UltraComp eBeads. The quantity of stain to use for preparing compensation beads may need to be titrated down if the signal is too high (i.e., if the positive beads are appearing at the edge or off of the axis) when performing flow cytometry acquisition.
Add 0.2 μL of LIVE/DEAD stain stock solution to the LIVE/DEAD stain well of the V-bottom 96-well plate.
Cover the V-bottom 96-well plate with aluminum foil and incubate for 10 min at room temperature; then, add 100 μL of PBS to each well.
Centrifuge the 96-well plate at 410× g for 5 min at 4 °C.
Flick the plate over a waste container or sink to remove the supernatant and resuspend each bead pellet with 200 μL/well of PBS.
Add one drop of Arc-negative compensation beads to the LIVE/DEAD stain well of the V-bottom 96-well plate.
Transfer into Axygen 1.1 mL polypropylene cluster tubes and store the tubes at 4 °C in a refrigerator until running the samples on the flow cytometer.
Flow cytometry sample acquisition using a BD LSR Fortessa flow cytometer running FACSDiva
This section briefly describes the steps to set up the experiment and acquire the samples using a BD LSR Fortessa flow cytometer running FACSDiva. More details and training can be obtained from your flow cytometry core facility. After loading each sample and pressing run, wait several seconds for the event rate to stabilize before acquiring and saving the data.
Verify cytometer optical configuration and ensure cytometer setup and tracking (CST) performance settings are up to date.
Open a new experiment in FACSDiva and select each fluorescent parameter from the parameter dropdown menu.
Load the unstained sample, start acquisition, and adjust the photomultiplier tube (PMT) voltages to bring the lymphocyte population to a fluorescence value of approximately 105 forward scatter area (FSC-A) and 105 side scatter area (SSC-A) in the FSC-A and SSC-A dot plot.
Define the lymphocyte population gate and then adjust PMT voltages for each fluorescent parameter, to have the autofluorescence of the negative (unstained) population at least at three standard deviations above the noise of the instrument for each fluorescent channel. FACSDiva will automatically place a gray box on the plot to guide the adjustment of individual PMT voltages. Voltages should be adjusted until unstained cells appear within this box, but not beyond the right and upper boundary (i.e., unstained cells should not be more positive than the gray box).
Briefly load a fully stained cell sample into the instrument to verify that all populations are visible on the plots and within range. If populations are out of range, increase the scale and/or decrease the PMT voltage.
Save the experiment as a template before calculating compensation for easy setup later if desired.
Run each single-stained compensation bead control to correct for fluorescent spillover and manually gate the positive and negative populations. Acquire 10,000 events for each control and select automatic compensation.
Run each FMO control tube. FMOs are used as a control for gating each cell population. Acquire enough events to clearly see the autofluorescence of the negative population or acquire the whole sample.
Acquire each sample until the event rate begins to drop, indicating that the tube is nearly empty. Quickly end the acquisition and remove the tube to prevent air from entering the flow cell.
Export FCS files for analysis on FlowJo and export EXP files if the experiment will be reopened on FACSDiva later.
Data analysis using FlowJo
This section briefly describes the steps to analyze samples using FlowJo. More details and training can be obtained from your flow cytometry core facility. A representative gating strategy is presented in Figure 7.
Import all FCS files into FlowJo.
Click on the compensation tab and calculate a new compensation matrix using the single-stained bead samples. Look at each plot in the compensation matrix to ensure that there were no errors in the compensation calculation. Manually adjust compensation values, should any fluorochromes be significantly over or under compensated.
Define gates to isolate cells, single cells, live cells, and then each marker of interest.
Use each FMO to determine the location and size of the autofluorescence of the negative population to properly gate positive populations. FMOs are a good starting point for gate placement, especially for fluorochromes that lack clear separation between positive and negative populations. However, manual adjustments from sample to sample are often necessary to account for variability between animals and tissues.
Look at the time parameter for each sample to ensure smooth and uninterrupted flow into the flow cell as a quality control check. Gaps or unevenness across the time plot indicate clotting or air bubbles, which can impact the quality of the data.
Figure 7. Representative gating strategy to determine cytokine production by CD4, CD8, and T cells. A. Cells are first gated in the side scatter area (SSC-A)/forward scatter area (FSC-A) plot. Singlet cells are gated using FSC-A over FSC height (FSC-H). Singlet cells are gated again using SSC-A over SSC height (SSC-H) to further clean up the data. Live cells are gated in the FSC-A/viability dye plot. Then, CD45+ cells are gated in the FSC-A/CD45 plot, followed by CD3+ T cells in the FSC-A/CD3 plot. Then, T-cell receptor (TCR) γδ+ cells are gated from the TCRγδ/CD3 plot, and CD4+ and CD8+ T cells are gated from the CD8a/CD4 plot. B. Determination of IFNγ and IL-17A production within each T-cell subset in each tissue following initial gating above. AF488 and AF700: Alexa-Fluor 488 and 700; APC: allophycocyanin; BUV395: Brilliant Ultraviolet 395; BV786: Brilliant Violet 786; CD: cluster of differentiation; CF594: cyanine‐based fluorescent dye 594; PE: phycoerythrin; PerCP: peridinin-chlorophyll-protein.
Notes
The number of channels should not matter, assuming the multichannel pipette is in good working condition. The choice should be based on how many samples are being prepared to minimize time spent on pipetting steps. In this study, we used an 8-channel multichannel pipette.
The tissues are collected without cardiac perfusion; therefore, cells recovered from tissue represent both tissue-resident immune cells and immune cells within the tissue vasculature. To assess immune cells only in the tissues, cardiac perfusion is required.
The trypsin inhibitor is used to block any contaminating trypsin activity contained within the other digestion enzymes that could cleave flow cytometry surface markers.
It is best to titrate the concentration of antibody used for flow cytometry staining to suit your specific needs. Using too much antibody may result in increased background signal and an overestimation of true positive populations due to nonspecific binding of the antibody. Using too little antibody will result in poor separation and an underestimation of the true positive population, as there will not be enough antibody to fully stain your cell population. In a separate experiment, stain cells from each tissue with a series of eight two-fold dilutions of the antibody of interest. Start with a concentration of antibody above the manufacturers’ recommended quantity (for example, twice as much) to ensure that you do not need more antibody than the company recommends. Your flow cytometry panel may target cells isolated from a different tissue with higher or lower marker expression and different cell number than the manufacturer used in their tests. It is important to titrate using the same fraction of isolated cells that you would use in an experiment. For example, if you plan to use 10% of a spleen for each staining, also titrate using 10% of a spleen for each dilution. Alternatively, you can count cells and use the same number of cells for each dilution that you would use in a real experiment. This will allow for lower noise, more clear separation, more efficient use of reagents, and prevention of over or underestimating the size of your cell population. Be sure to titrate using the same volume of PBS. Optimal staining is found at the lowest dilution that gives distinct separation between positive and negative populations. The more diluted samples than the optimal concentration will begin to show decreased separation between positive and negative populations. A more unbiased metric for determining optimal antibody concentration is the separation index (SI), which finds the optimal separation using the fluorescence intensity of the positive and negative populations (Telford et al., 2009). This index determines the separation of a stained population by dividing the difference between positive and negative signal by the robust standard deviation, which represents the spread of the negative population. The optimal antibody dilution will have the highest SI value. A representative example of a titration of AF700-CD8 is included in Figure 8, along with the formula to calculate SI.
The use of the Golgi blocker monensin allows for signal amplification by trapping cytokines within activated cells. Therefore, individual cells can first be identified using combinations of surface markers, and then assessed for IFNγ and IL-17A production.
Figure 8. An example of Alexa-Fluor 700 (AF700)–CD8a antibody titration. The formula used to calculate separation index (SI) is presented. Splenocytes were stained with LIVE/DEAD Aqua viability dye, BV605-CD3, and eight successive two-fold dilutions of AF700-CD8a to ensure that live lymphocytes were the focus of the titration. The best dilution was determined by the largest SI. Median Positive and Median Negative correspond to the median fluorescence intensity of the positive and negative populations, respectively. The 84% negative refers to the 84th percentile of the fluorescence intensity of the negative population. SI was determined by FlowJo as described previously (UWCCC_Flow_Lab, 2016). The fourth dilution showed maximal separation between positive and negative populations while maintaining minimal background signal, corresponding with the highest SI value. Therefore, the fourth dilution was selected as the appropriate quantity of antibody to use in the experiment.
Recipes
Reagents to prepare in advance
70% ethanol
This solution is made by combining 350 mL of ethyl alcohol anhydrous with 150 mL of double-distilled water.
Sterile pipette tips
Racked tips are sterilized by autoclaving at 121 °C for 30 min followed by 30 min of drying.
Sterile dissecting tools
The tools are sterilized by immersion in 70% ethanol for 5 min.
Digestion stock solution (8×)
The 1× digestion solution is constituted by 0.15 U/mL of collagenase A, 500 U/mL of collagenase type 2, 2 U/mL of elastase, and 0.5 U/mL of trypsin inhibitor (see Note 3). Prepare 8× digestion stock solution in RPMI and store aliquots of 100 μL in 1.5 mL microtubes at -20 °C until used, for up to six months. One hundred aliquots of 8× digestive stock solution can be prepared as described in Table 2.
Table 2. Preparation of 100 aliquots of 100 μL of 8× PVAT/kidney digestion stock solution
Digestion enzyme powders 1× digestion solution 8× digestion solution
Enzyme (U/mg) (U/mL) (mg/mL) (mg/mL) (mg/100 aliquots)
Collagenase A 0.22 0.15 0.67 5.38 53.8
Collagenase type 2 225 500 2.22 17.78 177.8
Elastase 4.67 2.0 0.43 3.43 34.3
Trypsin inhibitor 1.79 0.5 0.28 2.23 22.3
The quantity of each enzyme to prepare 100 aliquots of 100 μL (10 mL) of 8× PVAT/kidney digestion stock solution is calculated as follows. First, the concentration of the enzymes in mg/mL for a 1× solution is obtained by dividing the activity (U)/mL by the activity (U)/mg. Note that the activity (U)/mg is specific for each lot number (#) of the enzyme. Thereafter, the concentration of the enzymes in mg/mL for the 8× solution is determined by multiplying the concentration of the 1× solution by 8. Then, the quantity of enzyme for 100 aliquots is calculated by multiplying the number of aliquots (100) by the volume of each aliquot in milliliters (0.1 mL) and by the concentration of the 8× digestion solution. Finally, the enzymes are weighed, dissolved in a final volume of 10 mL of RPMI, and aliquots of 100 μL are stored in 1.5 mL microtubes at -20 °C until used.
Hyaluronidase stock solution (8×)
The 1× hyaluronidase solution is constituted of 48 U/mL of hyaluronidase. Prepare 8× stock solution in RPMI and store aliquots of 100 μL in 1.5 mL microtubes at -20 °C until used. One hundred aliquots of 8× digestion stock solution can be prepared as described in Table 3.
Table 3. Preparation of 100 aliquots of 100 μL of 8× lung hyaluronidase stock solution
Digestion enzyme powders 1× hyaluronidase solution 8× hyaluronidase solution
Enzyme (U/mg) (U/mL) (mg/mL) (mg/mL) (mg/100 aliquots)
Hyaluronidase 300 48 0.16 1.28 12.8
The quantity of hyaluronidase to prepare 100 aliquots of 100 μL (10 mL) of 8× hyaluronidase stock solution is calculated as follows. First, the concentration of hyaluronidase in mg/mL for a 1× solution is obtained by dividing the activity (U)/mL by the activity (U)/mg. Note that the activity (U)/mg is specific for each lot number (#) of hyaluronidase. Thereafter, the concentration of hyaluronidase in mg/mL for the 8× solution is determined by multiplying by 8 the concentration of the 1× solution. Then, the quantity of hyaluronidase for 100 aliquots is calculated by multiplying the number of aliquots (100) by the volume of each aliquot in milliliters (0.1 mL) and by the concentration of the 8× digestion solution. Finally, hyaluronidase is weighed, dissolved in a final volume of 10 mL of RPMI, and aliquots of 100 μL are stored in 1.5 mL microtubes at -20 °C until used.
PMA 1 mg/mL stock solution
Prepare 1 mg/mL stock solution by dissolving 1 mg of PMA in 1 mL of DMSO and store aliquots of 20 μL at -80 °C until used.
Ionomycin stock solution (1 mg/mL)
Prepare ionomycin stock solution by dissolving 1 mg of ionomycin in 1 mL of DMSO and store aliquots of 20 μL at -80 °C until used.
LIVE/DEAD stain stock solution
Bring a vial of stain and the vial of DMSO to room temperature. Make sure to remove any trace of water from the vials with a paper towel as DMSO/dye solution is unstable when exposed to moisture. Add 50 μL of DMSO to the vial of stain, mix well to dissolve the stain, and store aliquots of 2.5 μL in microtubes at -20 °C until needed or for up to two months.
FBS aliquots
Thaw a bottle of FBS overnight in a refrigerator, equilibrate to room temperature, and heat-inactivate as follows. Heat the bottle of FBS at 56 °C in a water bath for 30 min and swirl the bottle every 10 min or so. For accuracy, use a second bottle of similar size as a control and add an equivalent volume of water to the control bottle. Place a thermometer in the control bottle to check when the water reaches 56 °C. Set your timer for 30 min at this point. Make aliquots of 50 and 1 mL and store them at -20 °C until used.
Permeabilization buffer
This buffer is constituted by sterile PBS supplemented with 0.1% saponin and 0.1% BSA. Make permeabilization buffer by dissolving 100 mg of saponin and 100 mg of BSA in 100 mL of sterile PBS. This solution can be stored at 4 °C for several months.
Reagents to prepare within three days of the experiment
Pre-diluted antibody mixtures
Prepare tubes of unstained controls, FMO controls, and pre-diluted antibody mixtures for extracellular and intracellular staining of samples as described in Tables 4 and 5, respectively. Store them at 4 °C in the dark until used.
Table 4. Pre-diluted antibody mixtures for extracellular staining
Reagents Unstained control FMO controls Full stain
CD45 CD3 CD4 CD8a TCRδ 20 samples and
FMO controls of Table 5
Fc-Block (μL) - 4 4 4 4 4 27
BV786-anti-CD45 antibody (μL) - - 4 4 4 4 27
BUV395-anti-CD3 antibody (μL) - 4 - 4 4 4 27
PerCP-eF710-anti-CD4 antibody (μL) - 4 4 - 4 4 27
AF700-anti-CD8 antibody (μL) - 4 4 4 - 4 27
PE-CF594-anti TCR γδ (μL) - 4 4 4 4 - 27
FBS (μL) 24 24 24 24 24 24 162
PBS (μL) 376 356 356 356 356 356 2,376
The table presents the volume of FBS, PBS, and antibodies to combine per tube of unstained control, fluorescence minus one (FMO) control, and full stain pre-diluted antibody mixtures for 20 samples (four mice with five tissues) and the two FMO controls of Table 5 plus an excess volume of 5% (or at least 100 μL), to ensure proper delivery of 100 μL of pre-diluted antibody mixtures per well of the V-bottom 96-well plate. Note that unstained control and FMO control pre-diluted antibody mixtures are prepared for only three tissues including the spleen, lungs, and kidneys of pooled cells from four mice. The mesenteric vessels with perivascular adipose tissue and mesenteric lymph nodes have too few T cells to prepare unstained and FMO controls. The spleen is used for unstained and FMO controls for these tissues. A dilution of 1/100 is used for all the antibodies in this protocol. Although it works well, the optimal dilution of antibodies should be determined for each tissue type in a previous experiment (see Note 4). Prepare the unstained control and FMO control pre-diluted antibody mixtures in 1.5 mL microtubes and full stain pre-diluted antibody mixtures in a 5 mL microtube, and store them at 4 °C in the dark until used. See Table 1 for details of antibodies.
Table 5. Antibody mixtures for intracellular staining
Reagents Unstained control FMO controls Full stain
IL-17A IFNγ 20 samples and
FMO controls of Table 4
APC-anti-IL-17A (μL) - - 4 37
AF488-anti-IFNγ (μL) - 4 - 37
Permeabilization buffer 400 396 396 3,626
The table presents the volume of permeabilization buffer and antibodies to combine per tube of unstained control, fluorescence minus one (FMO) control, and full stain pre-diluted antibody mixtures for 20 samples (four mice with five tissues) and the five FMO controls of Table 4 plus an excess volume of 5% (or at least 100 μL), to ensure proper delivery of 100 μL of pre-diluted antibody mixtures per well of the V-bottom 96-well plate. Note that unstained control and FMO control pre-diluted antibody mixtures are prepared for only three tissues including the spleen, lungs, and kidneys. The mesenteric vessels with perivascular adipose tissue and mesenteric lymph nodes have too few T cells to prepare unstained and FMO controls. The spleen is used for unstained and FMO controls for these tissues. The permeabilization buffer is constituted of PBS supplemented with 0.1% saponin and 0.1% BSA. A dilution of 1/100 is used for all the antibodies in this protocol. Although it works well, the optimal dilution of antibodies should be determined for each tissue type in a previous experiment (see Note 4). Prepare the unstained control and FMO control pre-diluted antibody mixtures in 1.5 mL microtubes and full stain pre-diluted antibody mixtures in a 5 mL microtube, and store them at 4 °C in the dark until used. See Table 1 for details of antibodies.
Reagents to prepare on the day of the experiment
24-well tissue culture plate for the collection of tissues:
Prepare a 24-well tissue culture plate for the collection of spleen, kidneys, lungs, mLNs, and processing of the kidneys as depicted in Figure 9. Label five wells per animal with the tissue names to collect and animal number. Add 1 mL of cold RPMI per well for tissue collection and 0.5 mL per well for processing kidneys and keep on ice.
Figure 9. Preparation of a 24-well tissue culture plate for collection of spleen, kidneys, lungs, and mesenteric lymph nodes (mLNs), and for processing of the kidneys from four mice. The volumes of RPMI are indicated in milliliter per well.
Label one 50 mL centrifuge tube per animal for collecting the intestines and mesenteric bed and add 30 mL of cold PBS. Keep the tubes on ice until used.
T-cell activation cocktail (1 mL/sample)
This cocktail is constituted by two activators, PMA (0.05 μg/mL) and the calcium ionophore ionomycin (1 μg/mL). The cocktail also contains the protein transport inhibitor monensin (1×, 2 μM), which causes accumulation of protein at the Golgi complex/endoplasmic reticulum, enhancing the intracellular IL-17A and IFNγ staining signal (see Note 5). Prepare enough cocktail to activate T cells in all five tissues from the four mice (1 mL/tissue/mouse). For four mice and five tissues/mouse, prepare 22 mL of T-cell activation cocktail as presented in Table 6 and as follows. First, make 200 μL of 0.1 mg/mL PMA solution by adding 180 μL of PBS to a 20 μL aliquot of 1 mg/mL PMA stock solution. Then, combine 11 μL of PMA 0.1 mg/mL, 22 μL of ionomycin 1 mg/mL stock solution, 22 μL of monensin 2 mM, and 21.945 mL of RPMI in a 50 mL centrifuge tube and keep the tube on ice until used.
Table 6. Preparation of T-cell activation cocktail
Reagents Cocktail
For 1 mL For 22 mL
PMA 0.1 mg/mL (μL) 0.5 11
Ionomycin 1 mg/mL (μL) 1 22
Monensin 2 mM (μL) 1 22
RPMI (μL) 998 21,945
One milliliter of T-cell activation is required per sample. The table shows the volume of reagents to combine to make T-cell activation cocktail for 1 and 20 samples. For the 20 samples, an excess volume of 10% is added to ensure proper delivery of 1 mL per sample. For four mice and five tissues/mouse, prepare 22 mL of T-cell activation cocktail in a 50 mL microtube and keep the tube at 4 °C until used.
PFA 1% solution
Prepare enough PFA 1% to fix all the unstained controls, FMO controls, and samples after extracellular staining (100 μL/well of cells). For four mice, 44 wells of cells need to be fixed (Figure 6). This is done by diluting one 10 mL vial of PFA 10% solution in 90 mL of sterile PBS. Store this solution at 4 °C in the dark until used. Unused solution can be stored for up to one month at 4 °C or for a longer period at -20 °C.
LIVE/DEAD staining solution
Prepare enough LIVE/DEAD Fixable Aqua Dead Cell Stain solution to stain all FMO controls and samples (100 μL/well of cells), except the unstained controls. For four mice, 41 wells of cells need to be stained (Figure 6). To do this, prepare five tubes of LIVE/DEAD stain by adding 1 mL of PBS per 2.5 μL aliquot of LIVE/DEAD stain stock solution, and keep one aliquot of LIVE/DEAD stain stock solution on hand for preparation of compensation beads. Store the tubes at 4 °C in the dark until used.
Prepare all the reagents from section A weeks to months in advance, from section B a few days in advance, and from section C on the day of the experiment.
Acknowledgments
We are grateful to Veronique Michaud for excellent technical support.
Funding: The work of the authors was supported by the Canadian Institutes of Health Research (CIHR) First Pilot Foundation Grant 143348, a Canada Research Chair (CRC) on Hypertension and Vascular Research by the CRC Government of Canada/CIHR Program, a Distinguished James McGill Professorship Award, and by the Canada Fund for Innovation, to ELS, and by the McGill Department of Medicine Gordon Phillips Fellowship to KC.
Competing interests
The authors declare that there are no conflicts of interest, financial or otherwise.
Ethics
The study was approved by the Animal Care Committee of the Lady Davis Institute and McGill University and followed recommendations of the Canadian Council of Animal Care.
References
Caillon, A., Mian, M. O. R., Fraulob-Aquino, J. C., Huo, K.-G., Barhoumi, T., Ouerd, S., Sinnaeve, P. R., Paradis, P. and Schiffrin, E. L. (2017). γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury. Circulation 135(22): 2155-2162.
Caillon, A., Paradis, P. and Schiffrin, E. L. (2019). Role of immune cells in hypertension. Br J Pharmacol 176(12): 1818-1828.
Higaki, A., Caillon, A., Paradis, P. and Schiffrin, E. L. (2019). Innate and Innate-Like Immune System in Hypertension and Vascular Injury. Curr Hypertens Rep 21(1): 4.
Higaki, A., Mahmoud, A. U. M., Paradis, P. and Schiffrin, E. L. (2021). Role of interleukin-23/interleukin-17 axis in T-cell-mediated actions in hypertension. Cardiovasc Res 117(5): 1274-1283.
Itani, H. A., Xiao, L., Saleh, M. A., Wu, J., Pilkinton, M. A., Dale, B. L., Barbaro, N. R., Foss, J. D., Kirabo, A., Montaniel, K. R., et al. (2016). CD70 Exacerbates Blood Pressure Elevation and Renal Damage in Response to Repeated Hypertensive Stimuli. Circ Res 118(8): 1233-1243.
Madhur, M. S., Lob, H. E., McCann, L. A., Iwakura, Y., Blinder, Y., Guzik, T. J. and Harrison, D. G. (2010). Interleukin 17 Promotes Angiotensin II–Induced Hypertension and Vascular Dysfunction. Hypertension 55(2): 500-507.
Shokoples, B. G., Comeau, K., Higaki, A., Ferreira, N. S., Caillon, A., Berillo, O., Oukka, M., Paradis, P. and Schiffrin, E. L. (2022). Angiotensin II-induced a steeper blood pressure elevation in IL-23 receptor-deficient mice: Role of interferon-γ-producing T cells. Hypertens Res 46(1): 40-49.
Telford, W. G., Babin, S. A., Khorev, S. V. and Rowe, S. H. (2009). Green fiber lasers: An alternative to traditional DPSS green lasers for flow cytometry. Cytometry A 75(12): 1031-1039.
UWCCC_Flow_Lab. (2016). FlowJo for Antibody Titrations: Separation Index and Concatenation. Available: https://cancer.wisc.edu/research/documents/flowjo-for-antibody-titrations/
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Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF)
ML Macy Lozen *
YC Yue Chen *
RB Rebecca A. Boisvert
Katharina Schlacher
(*contributed equally to this work)
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4680 Views: 753
Reviewed by: Gal HaimovichDavid PaulVaibhav B. Shah
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Original Research Article:
The authors used this protocol in Science Advances Dec 2021
Abstract
Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5′-bromo-2′-deoxyuridine (BrdU) or 5′-ethynyl-2′-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF).
Graphical overview
Schematic overview of Mitochondrial Replication Assay (MIRA). 5′-ethynyl-2′-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.
Keywords: MIRA Mitochondria Mitochondrial DNA mtDNA replication mtDNA instability Proximity ligation assay BRCA2 Fanconi anemia
Background
Mitochondria, the organelles best known for their orchestration of cellular bioenergetics, operate semi-autonomously within eukaryotic cells and are key signaling hubs for intracellular communication (Friedman and Nunnari, 2014). Each mitochondrion harbors multiple copies of its ~16 kb circular genome, which encodes essential subunits of the electron transport chain as well as a set of transfer and ribosomal RNAs (Falkenberg et al., 2007). Mitochondrial DNA (mtDNA) is replicated separately from nuclear DNA (nDNA): the mitochondrial genome is replicated by its organelle-specific replisome, comprised of DNA polymerase POLγ and other nuclear-encoded proteins (Holt and Reyes, 2012; Gustafsson et al., 2016; Bailey and Doherty, 2017). mtDNA replication can occur at any cell cycle stage. Because of its proximity to events during oxidative phosphorylation, mtDNA is constantly exposed to reactive oxygen species, rendering it highly susceptible to oxidative DNA damage (Kang and Hamasaki, 2002). Failure to repair damage of the mitochondrial genome additionally results in instability of mtDNA and mitochondrial dysfunction, which is associated with premature aging, tumorigenesis, and neurological disorders (Trifunovic et al., 2004; Chatterjee et al., 2006; Ishikawa et al., 2008; Park and Larsson, 2011). Apart from mtDNA repair pathways, mtDNA stability is also regulated at the level of mtDNA replication with the exonuclease activity of POLγ polymerase and MGME1 (mitochondrial genome maintenance exonuclease 1) degrading damaged mtDNA and generating new mtDNA (Torregrosa-Munumer et al., 2019).
To date, detailed mechanisms of mtDNA replication linking to stability are incompletely understood. A comprehensive understanding of these mechanisms is imperative to allow the development of effective and specific agents targeting disease outcomes. For this purpose, efficient tools to specifically monitor and quantifiably measure nascent mtDNA replication are needed. mtDNA is replicated primarily by POLγ within one to two hours, yet methods for labeling mtDNA have historically relied on prolonged exposures (up to 24 h) of 5′-bromo-2′-deoxyuridine (BrdU) or 5′-ethynyl-2′-deoxyuridine (EdU) (Clayton, 1982; Korhonen et al., 2004). Incorporation of these nucleoside analogs for labeling times sufficiently short to monitor nascent mtDNA replication before completion of the entire mtDNA genome, e.g. less than two hours, does not produce signals suited for efficient quantitative analysis (Luzwick et al., 2021). The assay system described here, termed Mitochondrial replication assay (MIRA), utilizes proximity ligation assay (PLA) as a means of signal amplification of the mtDNA-incorporated nucleoside analogs to overcome this limitation. Specifically, mtDNA is labeled with the thymidine analog EdU, which is then biotinylated using Click-IT chemistry (Moses and Moorhouse, 2007). The PLA technology was developed by Soderberg et al. (2008) for single-molecule protein–protein interaction studies, utilizing antibodies with oligonucleotide conjugates that can be ligated into a circular DNA molecule when two antibodies are in close proximity to each other (<40 nm). Once ligated, the DNA circle can be amplified by rolling circle DNA polymerase. After annealing of fluorescent DNA probes that are complementary to the DNA circle, this procedure results in a highly amplified fluorescent signal, readily detectable by conventional immunofluorescence (IF) microscopy. In MIRA, PLA is adapted to signal-amplify the nascent mtDNA signals by utilizing complementary antibodies against biotin (detecting biotinylated EdU). As with conventional EdU and BrdU labeling, MIRA also detects and visualizes nDNA. Nonetheless, only cytoplasmic MIRA signals are considered for analysis, which are present irrespective of the cell cycle state and do not form without EdU or in mtDNA-depleted Rho zero cells (Luzwick et al., 2021). With amplification, the nascent mtDNA can be readily detected and quantified with a 1 h EdU pulse, which is less time than needed to replicate the entire genome. This assays system enables the monitorization of ongoing mtDNA replication prior to completion of mtDNA genome replication. It therefore differs from most common mtDNA label schemes, which use prolonged nucleoside analog incorporation for extended time over multiple hours. Since the mtDNA is replicated within one to two hours, prolonged labeling will predominantly result in post-replicative signals. Moreover, as an adaptation from our single-cell assay for in situ protein Interactions with nascent DNA Replication Forks (SIRF) protocol (Roy et al., 2018; Roy and Schlacher, 2019), MIRA can be combined with a primary antibody against a protein of interest to detect protein mtDNA interactions (mitoSIRF). The MIRA and mitoSIRF assay systems allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection (Luzwick et al., 2021). Specifically, BRCA and FANC tumor-suppressor proteins protect nascent mtDNA from instability during ongoing mtDNA replication; this would not have been detectable by previous assay systems that visualize post-replicative events. The instability is caused by MRE11-dependent nuclease degradation, which results in cGAS-mediated inflammatory signaling (Luzwick et al., 2021).
The MIRA method requires a minimal number of cells (~100–1,000), preserves the single-cell resolution as seen with IF, which provides valuable information regarding cell morphology, and can be readily and accurately quantified. The MIRA assay can furthermore be combined with conventional IF for multi-parameter biomarker analysis, including cell cycle stage or cell identity. If combined with other biomarkers, primary and secondary antibody staining for conventional IF is performed following the Click-IT and PLA reaction to avoid interference of PLA secondary antibodies from cross-reacting with IF primary antibodies.
Materials and Reagents
8-well chamber microscope slides (Thermo Scientific, Nunc, catalog number: 177402)
Plastic coverslips (Electron Microscopy Sciences, catalog number: 72261-50)
Glass coverslips (Fisher Scientific, catalog number: 12-548-5M)
Kimwipes (Fisher Scientific, catalog number: NC9855580)
Aluminum foil (Fisher Scientific, catalog number: 01-213-105)
Paper towel (Envision, catalog number: 23304)
0.22 μm bottle top filter (Corning, catalog number: 430758)
Rabbit anti-biotin antibody (Cell Signaling, catalog number: 5597S)
Mouse anti-biotin antibody (Sigma-Aldrich, catalog number: B7653)
5′-ethylene-2′-deoxyuridine (EdU) (Invitrogen, catalog number: A10044)
Paraformaldehyde 32% solution, EM grade (PFA) (EMS, catalog number: 15714)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Biotin azide (Invitrogen, catalog number: B10184)
Alexa Fluor 488 azide (Invitrogen, catalog number: A10266)
Copper sulfate solution (Fluka Analytical, catalog number: 35185)
Sodium ascorbate (Sigma-Aldrich, catalog number: 11140)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417)
Duolink® mouse plus PLA probe (Sigma-Aldrich, catalog number: DUO92001-100RXN)
Duolink® rabbit minus PLA probe (Sigma-Aldrich, catalog number: DUO92005-100RXN)
Duolink® blocking solution (Sigma-Aldrich, catalog number: DUO82007-8ML)
Duolink® antibody diluent (Sigma-Aldrich, catalog number: DUO82008-8ML)
Duolink® PLA detection reagent red (Sigma-Aldrich, catalog number: DUO92008-100RXN)
Duolink® in situ wash buffers (Sigma-Aldrich, catalog number: DUO82049-20L)
4’,6-diamidino-2-phenylindole (DAPI) (Life Technologies, catalog number: 62248)
Prolong Gold antifade reagent (Invitrogen, catalog number: P36934)
Cell-specific culturing media
Dideoxycytidine (ddC, 20 μM) (Sigma-Aldrich, catalog number: D5782)
MitoPQ (10 μM) (Abcam, catalog number: 1821370-28-8)
mtOX (Wisnovsky et al., 2016)
Sodium chloride (NaCl) (Fisher Scientific, catalog number: 7647-14-5)
Tween 20 (Sigma-Aldrich, catalog number: P7949)
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 7647-01-0)
Trizma hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: T5941)
Tris base (Fisher Scientific, catalog number: BP152-1)
Dimethyl sulfoxide (DMSO) (Santa Cruz Biotechnology, catalog number: sc-358801)
Fixation solution (see Recipes)
Permeabilization solution (see Recipes)
Wash buffer A (see Recipes)
Wash buffer B (see Recipes)
EdU stock solution (see Recipes)
Biotin-azide stock solution (see Recipes)
Alexa488-azide stock solution (see Recipes)
Equipment
Autoflow IR water jacketed CO2 incubator (NUAIRE, model: NU-4750)
Nikon eclipse Ti-U inverted microscope (Nikon, model: Ti-U)
Colin jar (Thermo ScientificTM E94)
Fine curved forceps (Fine Science Tools, Dumont #7, catalog number: 11271-30)
Slide box (VWR, catalog number: 82003-414)
Vortexer (VWR analog vortex mixer, catalog number: 10153-838)
4 °C refrigerator (BSI, model: SCGP21OW1AREF)
-20 °C freezer (BSI, model: ABT-2020MB)
Software
NIS-elements (Nikon, https://www.nikoninstruments.com/Products/Software)
ImageJ (ImageJ, https://imagej.net/Welcome)
Microsoft Excel (Microsoft, https://products.office.com/en-us/excel)
GraphPad Prism (GraphPad, https://www.graphpad.com/scientific-software/prism/)
Procedure
Cell labeling
One day before the experiment, plate 1 × 104–2 × 104 cells growing in log-phase in 300 µL of appropriate growth medium in each well of an 8-well chamber microscope slide. Cells should reach 50%–60% confluency the day of the experiment.
Note: We have only used this procedure with adherent cells. However, it should be possible to use suspension cells, as they can be treated with EdU in solution, deposited on microscope slides using a cytospin, and then fixed to proceed with the assay.
The next day, aspirate growth medium from chamber wells and add 200 µL of prewarmed growth medium containing 20 μM EdU. Incubate the slide for 1 h at 37 °C in a tissue culture incubator. Note that the length of incubation may need to be optimized for the cell line/type. This condition, E only, allows for detection of unperturbed newly replicated mtDNA.
Note: Prewarm growth medium to 37 °C prior to treating cells, to avoid replication disruption of cells during treatments. For the same reason, be succinct with washes and treatments to limit exposure to room temperature (RT), which may disrupt replication.
(Optional) For assaying mtDNA fork protection, aspirate the media containing EdU after 1 h, wash chamber wells swiftly but gently with PBS (pH 7.4) at RT, aspirate, and add growth medium containing a mtDNA replication stalling agent of choice for a respective amount of time.
Note: Typical mtDNA-specific replication stalling agents are ddC (20 μM), MitoPQ (10 μM), or mtOX (4 μM) added for 1–3 h at 37 °C before fixation. For assaying replication restart, which is the capacity of resuming mtDNA replication after replication stalling, treat with mtDNA stalling agent first, followed by swift PBS washes and incubation of EdU media.
Always include a well with no EdU treatments as a negative control—No EdU.
Cell fixation
Aspirate growth medium and gently wash wells twice with PBS at RT. Carefully add 200 μL of freshly prepared fixation solution (2% PFA diluted in PBS) to each well. Incubate at RT for 15 min without disturbing the slide.
Note: Fixation time may vary with cell type. Handle PFA with caution inside a chemical safety cabinet. Discard PFA waste according to institutional biosafety guidelines. Other fixation methods may be possible but need to be tested.
After fixation, discard PFA and wash wells with PBS twice for 5 min each at RT. Disassemble chambers from the slide and remove silicone gasket using fine curved forceps (Figure 1). Ensure that the wells do not dry out by carefully placing them in a Coplin jar containing PBS as needed.
Note: Fixed slides can be stored in PBS at 4 °C for up to one week.
Figure 1. Silicone gasket removal. Removal of silicone gasket using fine curved forceps. Take caution to avoid scratching slide with the forceps.
Cell permeabilization
Permeabilize cells by placing slides in a Coplin jar containing 60 mL of permeabilization solution (0.25% Triton X-100 in PBS), enough to completely cover the slides for 15 min at RT.
Next, wash slides three times in a Coplin jar containing PBS for 5 min at RT.
Click-IT reaction
Prepare a humidified chamber by placing wet paper towels or Kimwipes inside a slide box at RT (Figure 2). Let the chamber equilibrate for 5 min while closed.
Figure 2. Humidified slide chamber. A. Preparation of a humidified chamber by placing folded paper towels or Kimwipes wetted with distilled water or PBS in an empty slide box. B. Slides are laid flat, facing up during incubations, and are carefully covered with plastic coverslips. Air bubbles can be avoided by applying the coverslip one end at a time.
Prepare fresh Click-IT reaction cocktail. In the following order, add 2 mM copper sulfate to PBS, 10 μM biotin azide, and 100 mM sodium ascorbate. Mix well by vortexing after adding each component.
Notes:
Make 1 M sodium ascorbate fresh before each use.
Protect biotin azide from light while handling.
A mixture of Alexa 488 azide and biotin azide (1:10, total 10 μM) can be used rather than biotin azide alone, to enable determination of cell-cycle phase (Figure 3). Protect Alexa 488 azide from light while handling.
Figure 3. Representative image to distinguish MIRA signals at various stages of the cell cycle and controls. A. Example of MIRA in UWB1.289 cells treated with 20 μM EdU for 1 h. Note that the green nuclear signal for Alexa 488 azide co-click enables distinction of S-phase from non-S-phase cells [no green nuclear signal, DAPI signal (blue) only]. PLA signal (red) is detected inside the nucleus during S-phase and outside the nucleus in mitochondria during all cell cycle phases. Scale bars = 20 μm. B. Representative images of MIRA assay (red) in Rho zero SH2038 cells that are depleted of mtDNA as seen by PicoGreen DNA stain (green), with and without EdU as negative control. Scale bars = 10 μm.
Place slides in the humidified chamber facing up (Figure 2). Add 40–50 μL/well of Click-IT reaction cocktail. Carefully cover each slide with a clean plastic coverslip, avoiding the formation of air bubbles.
Note: Do not allow drying of the wells at any point during the protocol.
Close the chamber lid to maintain humidified conditions in the chamber and incubate at RT for 1 h.
Blocking and primary antibody incubation
Gently remove plastic coverslips and wash slides in a Coplin jar containing PBS three times for 5 min at RT.
Place slides back in the humidified chamber. Add 50 μL/well of Duolink blocking solution. Carefully cover each slide with a clean plastic coverslip, avoiding air bubbles.
Close the humidified chamber and incubate for 1 h at RT.
Dilute primary antibodies [mouse anti-biotin (1:100) and rabbit anti-biotin (1:100)] in Duolink antibody diluent.
Note: For mitoSIRF, replace either rabbit anti-biotin or mouse anti-biotin with a primary antibody against a protein of interest. Primary antibody concentrations and length of incubation can be adjusted for optimization.
After 1 h, gently remove plastic coverslips and tap slides to remove blocking solution.
Place slides in the humidified chamber and directly add 35 μL/well of the diluted primary antibody solution. Apply new plastic coverslips, avoiding formation of air bubbles.
Close humidified chamber and incubate at 4 °C overnight.
Proximity ligation assay (PLA)
Wash slides in a Coplin jar containing 60 mL of Duolink wash buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% Tween 20) three times for 5 min each at RT to remove primary antibody.
Prewarm the humidified chamber by placing it at a 37 °C incubator during washes.
Using the Duolink PLA detection kit, prepare Duolink in situ PLA probes (anti-mouse plus and anti-rabbit minus) by diluting them 1:5 in antibody diluent.
Tap off excess Duolink wash buffer A and place slides in the prewarmed humidified chamber. Add 30 μL/well of PLA probes.
Apply a new plastic coverslip to each slide and incubate at 37 °C for 1 h in the humidified chamber.
Next, wash slides three times for 5 min each at RT in a Coplin jar containing Duolink wash buffer A to remove PLA probes.
Using the Duolink PLA detection kit, prepare DNA ligation mix by diluting the 5× Duolink ligation buffer (1:5) and Duolink 1× ligase (1:40) in autoclaved water.
Note: Vortex the ligation stock buffer during and after thawing, making sure to dissolve any precipitate. Keep ligase in a freezing block at -20 °C and add to ligation buffer.
Tap off excess wash buffer and place slides back in the humidified chamber. Add 30 μL/well of ligation mix.
Put a new plastic coverslip on each slide and incubate in the humidified chamber at 37 °C for 30 min.
Wash slides in a Coplin jar with Duolink wash buffer A two times for 5 min each at RT.
Prepare amplification mix by diluting the 5× Duolink amplification buffer (1:5) and Duolink 1× polymerase (1:80) in autoclaved water.
Note: Limit the exposure of the amplification buffer to light, e.g., by preparing the solution in an amber light-protected tube, or in a tube covered with aluminum foil. Keep polymerase in a freezing block at -20 °C.
Tap off excess wash buffer and place slides back in the humidified chamber. Add 30 μL/well of amplification mix.
Cover each slide with a new plastic coverslip and incubate in the humidified chamber at 37 °C for exactly 100 min.
Wash slides in a Coplin jar containing Duolink wash buffer B (200 mM Tris-HCl pH 7.5 and 100 mM NaCl) three times for 10 min each at RT and in the dark.
Wash slides in a Coplin jar containing 0.01× diluted Duolink wash buffer B for 1 min in the dark to remove excess salt.
DAPI staining and mounting
Prepare DAPI solution (1:1,000, 1 μg/mL end concentration) in PBS.
Note: Protect from light.
Tap off excess wash buffer B and place slides back in the humidified chamber. Add 30 μL/well of DAPI solution.
Cover with new plastic coverslips and incubate in the humidified chamber at RT for 5 min.
Wash slides in a Coplin jar containing PBS two times for 5 min each at RT.
Tap off excess PBS from slides and place them on a paper towel for mounting.
Add ~20 μL of Prolong Gold antifade reagent to each well of the slide and mount with glass coverslips (1.5 mm). Avoid air bubbles while mounting and wipe off excess mounting reagent from the edges of the coverslip. Keep slides in a dry slide box at RT overnight to cure and avoid exposure to light.
Note: Should be imaged within two days. If not feasible, store slides at -20 °C, allow slides to come to RT before imaging, and do not freeze slides repeatedly.
Data analysis
Image slides using a fluorescent microscope (such as Nikon Eclipse Ti) at a magnification of 40×/0.95 numerical aperture (N.A.) (20×/0.75 N.A. may also be sufficient for larger cells). PLA signals are captured in the TXRED channel (Ex 594 nm, Em 624 nm, as recommended by Sigma Duolink for Duolink detection reagent red). The GFP filter is used to image cells when co-clicked with Alexa 488 azide (Ex 495 nm, Em 519 nm). DAPI filter is used to visualize cell nuclei (Ex 358 nm, Em 461 nm). The merged image file is used for subsequent analysis (Figure 3). Typically, 4–8 image fields in different areas of the well are obtained for a total of 50–150 cells per condition. The microscope intrinsic quantitation software, such as Nikon NIS-elements software (bright spot detection) or ImageJ (https://imagej.net/Welcome), can be used to determine the number of MIRA PLA or mitoSIRF signals per cell. Hereby, only signals outside the nucleus are considered. Alternatively, when signals are very abundant and indistinguishable by the software, the mean fluorescent intensity of the TXRED channel is measured using the microscope software, such as Nikon NIS-elements software.
Counting signals outside of the nucleus of cells containing Alexa 488 allows distinction between mitochondrial events in S-phase from those in non-S-phase.
A student t-test is used to determine the significance of the difference between conditions and/or cell lines. If the data follows a non-normal distribution as measured by the D'Agostino & Pearson normality test, a Mann-Whitney t-test is used to determine the statistical significance (Luzwick et al., 2021).
Recipes
Fixation solution
Add 10 mL of 32% PFA solution stock in 150 mL of PBS pH 7.4 to make 2% PFA and mix well.
Store leftover diluted PFA in air-tight bottles, protected from light at either RT (for up to one week) or 4 °C for up to one month.
Permeabilization solution
Add 12.5 mL of 10% Triton X-100 solution in 487.5 mL of PBS (pH 7.4) to make 0.25% Triton X-100 solution.
Mix well and store at RT.
Wash buffer A
Dissolve 8.8 g of NaCl, 1.2 g of Tris base, and 0.5 mL of Tween 20 in 800 mL of autoclaved water.
Adjust pH to 7.4 using HCl.
Add autoclaved water to 1,000 mL (final concentrations 0.01 M Tris, 0.15 M NaCl, and 0.05% Tween 20).
Filter the solution (0.22 μm filter) and store at 4 °C. Bring the solutions to RT before use.
Wash buffer B
Dissolve 5.84 g of NaCl, 4.24 g of Tris base, and 26 g of Tris-HCl in 500 mL of autoclaved water.
Adjust pH to 7.5 using HCl.
Add autoclaved water to 1,000 mL (final concentrations 0.2 M Tris and 0.1 M NaCl).
Filter the solution (0.22 μm filter) and store at 4 °C. Bring the solutions to RT before use.
EdU stock solution
Add 2 mL of DMSO directly into the vial containing 50 mg of EdU to make 100 mM stocks. Dissolve powder by vortexing. Store aliquots of stock solution at -20 °C protected from light.
Biotin-azide stock solution
Add 1.623 mL of DMSO directly into the vial containing 1 mg of biotin azide to make 1 mM stocks. Dissolve powder by vortexing. Store aliquots of stock solution at -20 °C, desiccate, and protect from light.
Alexa488-azide stock solution
Add 0.58 mL of DMSO directly into the vial containing 0.5 mg of Alexa 488 azide to make 1 mM stocks. Dissolve powder by vortexing. Store aliquots of stock solution at -20 °C, desiccate, and protect from light.
Acknowledgments
The work was supported by the NIEHS under award 1R01ES029680, and by CPRIT RP180463, R1312 and RP180813 (K.S.). K.S. is a Rita Allen Foundation Fellow and a CPRIT scholar in Cancer Biology (previous award R1312). The protocol is derived from the assay systems described in Luzwick et al. (2021).
Competing interests
The authors have no competing interests.
References
Bailey, L. J. and Doherty, A. J. (2017). Mitochondrial DNA replication: a PrimPol perspective. Biochem Soc Trans 45(2): 513-529.
Chatterjee, A., Mambo, E. and Sidransky, D. (2006). Mitochondrial DNA mutations in human cancer. Oncogene 25(34): 4663-4674.
Clayton, D. A. (1982). Replication of animal mitochondrial DNA. Cell 28(4): 693-705.
Falkenberg, M., Larsson, N. G. and Gustafsson, C. M. (2007). DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem 76: 679-699.
Friedman, J. R. and Nunnari, J. (2014). Mitochondrial form and function. Nature 505(7483): 335-343.
Gustafsson, C. M., Falkenberg, M. and Larsson, N. G. (2016). Maintenance and Expression of Mammalian Mitochondrial DNA. Annu Rev Biochem 85: 133-160.
Holt, I. J. and Reyes, A. (2012). Human mitochondrial DNA replication. Cold Spring Harb Perspect Biol 4(12).
Ishikawa, K., Takenaga, K., Akimoto, M., Koshikawa, N., Yamaguchi, A., Imanishi, H., Nakada, K., Honma, Y. and Hayashi, J. (2008). ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320(5876): 661-664.
Kang, D. and Hamasaki, N. (2002). Maintenance of mitochondrial DNA integrity: repair and degradation. Curr Genet 41(5): 311-322.
Korhonen, J. A., Pham, X. H., Pellegrini, M. and Falkenberg, M. (2004). Reconstitution of a minimal mtDNA replisome in vitro. EMBO J 23(12): 2423-2429.
Luzwick, J. W., Dombi, E., Boisvert, R. A., Roy, S., Park, S., Kunnimalaiyaan, S., Goffart, S., Schindler, D. and Schlacher, K. (2021). MRE11-dependent instability in mitochondrial DNA fork protection activates a cGAS immune signaling pathway. Sci Adv 7(51): eabf9441.
Moses, J. E. and Moorhouse, A. D. (2007). The growing applications of click chemistry. Chem Soc Rev 36(8): 1249-1262.
Park, C. B. and Larsson, N. G. (2011). Mitochondrial DNA mutations in disease and aging. J Cell Biol 193(5): 809-818.
Roy, S., Luzwick, J. W. and Schlacher, K. (2018). SIRF: Quantitative in situ analysis of protein interactions at DNA replication forks. J Cell Biol 217(4): 1521-1536.
Roy, S. and Schlacher, K. (2019). SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks. Bio Protoc 9(18): e3377.
Soderberg, 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.
Torregrosa-Munumer, R., Hangas, A., Goffart, S., Blei, D., Zsurka, G., Griffith, J., Kunz, W. S. and Pohjoismaki, J. L. O. (2019). Replication fork rescue in mammalian mitochondria. Sci Rep 9(1): 8785.
Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J. N., Rovio, A. T., Bruder, C. E., Bohlooly, Y. M., Gidlof, S., Oldfors, A., Wibom, R., et al. (2004). Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429(6990): 417-423.
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4,681 | https://bio-protocol.org/en/bpdetail?id=4681&type=0 | # Bio-Protocol Content
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Rapid Identification of Pathogens in Severe Pneumonia by Species-specific Bacterial Detector (SSBD)
CZ Cong Zhang *
XL Xiaohui Liang *
YQ Yali Qin
WY Wenkui Yu
QC Qihan Chen
(*contributed equally to this work)
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4681 Views: 484
Reviewed by: Alka MehraLionel Schiavolin Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in eLIFE Oct 2022
Abstract
Fast and accurate detection of pathogenic bacterial infection in patients with severe pneumonia is significant to its treatment. The traditional culture method currently used by most medical institutions relies on a time-consuming culture process (over two days) that is unable to meet clinical needs. Rapid, accurate, and convenient species-specific bacterial detector (SSBD) has been developed to provide timely information on pathogenic bacteria. The SSBD was designed based on the fact that Cas12a indiscriminately cleaves any DNA following the binding of the crRNA-Cas12a complex to the target DNA molecule. SSBD involves two processes, starting with PCR of the target DNA using primers specific for the pathogen, followed by detection of the existence of pathogen target DNA in the PCR product using the corresponding crRNA and Cas12a protein. Compared to the culture test, the SSBD can obtain accurate pathogenic information in only a few hours, dramatically shortening the detection time and allowing more patients to benefit from timely clinical treatment.
Keywords: Severe pneumonia Pathogen detection Cas12a Species-specific bacterial detector Bacterial infections
Background
Severe pneumonia is a progressive inflammation of the lungs caused by pathogenic bacterial infection, leading to systemic infection if not treated promptly. The types of pathogens that cause severe pneumonia are complex and diverse, and antibacterial drugs used for different pathogen infections have great differences (De Pascale et al., 2012; Hansen et al., 2016). Therefore, the detection of early pathogenesis is not only the basis for precise diagnosis, but also necessary for accurate treatment and rational drug selection. However, the traditional culture method used by most medical institutions often relies on the process of pathogen culture, which takes several days (Lazcka et al., 2007). Inability to provide timely information about pathogen infection affects the clinical need to treat critically infected patients. Therefore, it is critical to accurately and rapidly identify the pathogens that cause severe pneumonia to enhance the diagnosis and treatment of such severe diseases to the greatest degree feasible.
The CRISPR/Cas system, widely used in the gene editing field, performs endonuclease functions on target nucleic acid molecules by Cas proteins and the guide RNA complementary to the target sequence. Recently, some Cas proteins (e.g., Cas12a and Cas13a) have been found to exhibit nuclease activity that indiscriminately cleaves arbitrary nucleic acids following specific binding to target molecules (Gootenberg et al., 2017; Chen et al., 2018; Li et al., 2018). Thus, by introducing single-stranded reporter nucleic acids that can generate fluorescent signals after cleavage, the detection of target nucleic acid molecules can be enabled by detecting fluorescent signals. Based on this principle, we have developed an accurate and rapid species-specific bacterial detector (SSBD) system (Wang et al., 2022). In previous studies, we have proven through clinical trials that SSBD can accurately detect ten of the most common clinical pathogens, namely Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Stenotrophomonas maltophilia, and Staphylococcus capitis.
The detection target for SSBD needs to be selected for each species. In summary, we identify the detection target through two processes. The first process utilizes bioinformatics to align the 1,791 high-quality genomes of 232 microorganism species (each species includes multiple isolated strains) and screen species-specific DNA fragments of each species. In the second process, the species-specific DNA fragments are manually selected and experimentally validated to identify the optimal DNA targets. For the first bioinformatics analysis process (Figure 1), we first performed genome sequence alignment of multiple strains belonging to the same species to filter the common DNA fragments of the species. Then, these common DNA fragments were aligned with other species' genomes to remove similar DNA fragments present in other species, with the remaining DNA fragments being the species-specific DNA fragments. The screened species-specific DNA fragments of each pathogen had the following features: (1) intra-species conservation (these DNA fragments are present in multiple isolated strains belonging to the same species), and (2) inter-species specificity (these DNA fragments are not present in other species). For each species, dozens to thousands of specific DNA fragments were screened, but not all of these fragments were ideal targets for Cas12a detection. Therefore, the bioinformatics data were then artificially filtered based on the following three principles: DNA length preferably longer than 200 bp to facilitate primer design, absence of repetitive sequences, and the existence of PAM sequences (5′-TTTN-3′) of Cas12a. Then, for 10 common clinical pathogens, several species-specific DNA fragments were randomly selected as the candidate target for detection. Primers and crRNAs were designed according to the selected candidate target DNA fragments sequence of each pathogen. Finally, the specificity and reliability of these primers and crRNAs were verified by crossover validation experiments, and the best-performing primer and crRNA for each species were selected based on the experimental results.
Figure 1. Schematic diagram of screening species-specific DNA fragments by bioinformatics. Reference from our published articles (Wang et al., 2022).
The advantage of the SSBD system is mainly reflected on detection accuracy and speed. The accuracy of the SSBD is reflected by the fact that the target of the detection is the species-specific DNA fragments (intra-species conserved and inter-species specificity) of each pathogen (DNA targets of the 10 pathogens in this protocol are shown in Table 5), as screened by the bioinformatics analysis in our previous study (Wang et al., 2022). The corresponding PCR amplification primers and crRNAs guiding Cas12a for targeted cleavage were designed according to the species-specific DNA fragments. SSBD can be completed in just a few hours, as it does not include a culture process and only involves three steps of sample DNA extraction, PCR amplification, and Cas12a testing. Meanwhile, the method is easy to be replicated, as DNA extraction and PCR amplification are the fundamental experimental assays in many molecular testing departments. Additionally, Cas12a protein and crRNA can be ordered from biotech companies.
In conclusion, SSBD has great advantages as a new rapid detection method for pathogens, showing potential to even replace traditional culture methods, making it well worth to be extended and applied in the clinical diagnosis of patients with severe pneumonia. Meanwhile, the SSBD is based on species-specific DNA targets of pathogenic microbes, which means it can be expanded to any species other than bacteria.
Materials and Reagents
384-well microplate (Corning, catalog number: 3701)
1.5 mL nuclease-free tube (Eppendorf, catalog number: 0030125150)
0.2 mL PCR tube (Eppendorf, catalog number: 0030124332)
10 μL tips (SANFICO, catalog number: TFR810)
10 μL extended tips (SANFICO, catalog number: TFR810-L)
200 μL tips (SANFICO, catalog number: TFR8200)
1,000 μL tips (SANFICO, catalog number: TFR81000)
Quick-DNA/RNA Pathogen Miniprep kit (ZYMO RESEARCH, catalog number: R1042)
TIANSeq HiFi Amplification Mix (TIANGEN, catalog number: NG219)
NEBufferTM 3.1 (New England Biolabs, catalog number: B7203)
Nuclease-free water (Thermo Fisher, catalog number: 10977015)
Cas12a protein
Note: In our experiments, the Cas12a protein expression and purification was performed as described (Li et al., 2018). We also recommend purchasing Cas12a from a biological company (e.g., New England Biolabs).
Primers for PCR amplification (primer sequences are shown in Table 1)
Note: The sequence of primers was published in our previous article in Appendix 1, Table 1 (Wang et al., 2022). The primers can be ordered as PAGE-purified DNA oligonucleotide from a DNA synthesis company (e.g., GenScript Biotech or Sangon Biotech).
Table 1. Primers sequences
Species Primers sequences (5′ > 3′)
A. baumannii forward primer CACAGCGTTTAACCCATGCC
reverse primer TATCGCCACCTGCACAGAAG
E. coli forward primer GTTCCTGACTATCTGGCGGG
reverse primer GCTTCCTGACTCCAGACACC
K. pneumoniae forward primer CATGGGCATATCGACGGTCA
reverse primer CCTGCAACATAGGCCAGTGA
S. aureus forward primer AGGTGCAGTAGACGCATAGC
reverse primer CATTCGCTGCGCCAATACAA
P. aeruginosa forward primer TCTCTCTATCACGCCGGTCA
reverse primer TCGCATCGAGGTATTCCAGC
S. epidermidis forward primer CACGCATGGCACTAGGTACA
reverse primer CGAAAAAGAGTTGTCCTTGTTGA
S. capitis forward primer GGTTCAGTCATCCCCACGTT
reverse primer CAGCTGCGACAACTGCTTAC
E. faecalis forward primer CGGCAAGTTTGGAAGCAGAC
reverse primer CAGCGCCTAGTCCTTGTGAT
E. faecium forward primer ATCGGAAATCGGTGTGGCTT
reverse primer TCAAATGCATCCCTGTGCCT
S. maltophilia forward primer CGCCTCCCGTTTACAGATTA
reverse primer TCGGCTCCACCACATACAC
crRNA used in Cas12a reaction (crRNA sequences are shown in Table 2)
Note: The RNA can be ordered as a desalted RNA oligonucleotide or as PAGE-purified RNA oligonucleotide from an RNA synthesis company (e.g., GenScript Biotech or Sangon Biotech).
Table 2. crRNA sequences
Species crRNA sequences (5′ > 3′)
A. baumannii UAAUUUCUACUAAGUGUAGAUGUAAAGAAGAAUUACUUGAA
E. coli UAAUUUCUACUAAGUGUAGAUACGGUCGCUAUGAUGGCAAG
K. pneumoniae UAAUUUCUACUAAGUGUAGAUACUGCGGUAAUCGCCAUCUU
S. aureus UAAUUUCUACUAAGUGUAGAUACAACGUGAACUUGCUGAGG
P. aeruginosa UAAUUUCUACUAAGUGUAGAUUUGUAAACACACAUGAGGAG
S. epidermidis UAAUUUCUACUAAGUGUAGAUUAAUAUUCAUCAAUUAGAGA
S. capitis UAAUUUCUACUAAGUGUAGAUAAUCACCUUAUUAAUCAAUA
E. faecalis UAAUUUCUACUAAGUGUAGAUGCACCAAAUAACACAGCUGA
E. faecium UAAUUUCUACUAAGUGUAGAUACUACCUGAACUUAUAUACA
S. maltophilia UAAUUUCUACUAAGUGUAGAUGCAUGACACCUGCGACCAGG
Note: The backbone sequence of crRNA is 5′-UAAUUUCUACUAAGUGUAGAU-3′, and the following sequence is used to recognize the target DNA.
ssDNA-reporter (synthesized by Sangon Biotech)
Note: Sequence of ssDNA-reporter: 5′-FAM-TTATT-BHQ1-3′. The feature of the ssDNA-reporter is that a fluorescent molecule that can generate a fluorescent signal (e.g., FAM) is coupled at one DNA end, and a quencher molecule (e.g., BHQ1) that can block the fluorescent molecule producing a fluorescent signal is coupled at the opposite DNA end.
Ethanol (Sangon Biotech, catalog number: A500737-0500)
Equipment
Centrifuge (Eppendorf, Centrifuge 5418 R)
Centrifuge for 0.2 mL PCR tube (Scilogex, catalog number: 914041419999)
PCR thermocycler (LongGene, T20D)
Fluorescence plate reader (Austria Tecan, Infinite M200 Pro)
NanoDrop One (Thermo Scientific, catalog number: ND-ONE-W)
Biological safety cabinet (Thermo Scientific, catalog number: 51028226)
Vortexer (Scilogex, catalog number: 821200049999)
1–10 μL pipettes (Eppendorf, catalog number: 3123000020)
2–20 μL pipettes (Eppendorf, catalog number: 3123000098)
20–200 μL pipettes (Eppendorf, catalog number: 3123000055)
100–1,000 μL pipettes (Eppendorf, catalog number: 3123000063)
Software
Primer design software, NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)
CrRNA design software, CHOPCHOP (https://chopchop.cbu.uib.no/)
Software to control the fluorescence plate reader, Tecan i-control (Austria Tecan)
Software for data analysis, Microsoft Excel
Procedure
Detection targets design
Note: The primers and crRNAs used in this protocol for 10 pathogens (Table 1) have been experimentally and clinically validated with excellent effectiveness.
Select detection targets. Manual screening of species-specific DNA data from bioinformatics; the screening principles are:
To facilitate primer design, DNA length is preferably longer than 200 bp.
DNA fragment without repetitive sequences.
DNA fragment contains PAM sequences (5′-TTTN-3′) of Cas12a.
Design crRNA.
Find PAM sequences (5′-TTTN-3′) within the DNA target region.
Select the sequence 20 bp downstream of the PAM sequence as the target of crRNA. Replace the T-base in the 20 bp target sequence with a U-base. Place the target sequence downstream of the crRNA backbone sequence (5′-UAAUUUCUACUAAGUGUAGAU-3′).
Note: The base distribution of the target sequence should be balanced, with a GC content of 40%–80%. We also recommend using crRNA design software, e.g., CHOPCHOP (https://chopchop.cbu.uib.no/).
Use NCBI Primer-BLAST primer design software to select primers.
The input sequence is the upstream and downstream sequence of the crRNA target site.
Set the Primer parameters as default parameters.
Set Primer Pair Specificity Checking Parameters Database as Refseq representative genomes, and Organism as Homo sapiens (taxid: 9606).
Pick a primer pair predicted without non-specific amplification.
Experimental validation
For example, for the 10 sets of primers and crRNAs used in this protocol, each set of primers and crRNAs needs to ensure that no non-specific signals are detected for the remaining pathogens and human genomic DNA.
DNA extraction from bronchoalveolar lavage fluid (BALF) sample
Biosafety notes: DNA extraction procedure should be performed in a biological safety cabinet to ensure user safety. Wear biological protecting clothing, gloves, and masks during operation.
DNA extraction was performed referring to the Quick-DNA/RNA Pathogen Miniprep kit instructions. The procedure is as follows:
Add 800 μL of DNA/RNA shield to each 200 μL of BALF sample and mix well by using vortexer.
Centrifuge at 16,000× g for 1 min. Transfer up to 200 μL of the supernatant into a 1.5 mL nuclease-free tube.
Add 2 μL of proteinase K and mix well.
Add 400 μL of pathogen DNA/RNA buffer. Mix well and incubate at room temperature for 5 min.
Transfer the mixture into a Zymo-Spin IIC column in a collection tube and centrifuge at 16,000× g for 30 s. Discard the flowthrough.
Add 500 μL of pathogen DNA/RNA wash buffer to the Zymo-Spin IIC column and centrifuge at 16,000× g for 30 s. Discard the flowthrough.
Repeat the previous step.
Add 500 μL of ethanol (95%–100%) to the Zymo-SpinTM IIC column and centrifuge at 16,000× g for 1 min to ensure that the ethanol is removed. Discard the collection tube and transfer the Zymo-SpinTM IIC column into a new nuclease-free 1.5 mL tube.
Add 25–50 μL of nuclease-free water at 65 °C directly to the matrix of the column and centrifuge at 16,000× g for 1 min to elute the DNA.
Measure DNA concentration using a NanoDrop and adjust DNA concentration to 100 ng/μL.
Note: The DNA yield extracted from 200 μL of BALF is 8–43 μg. Depending on the patient's situation and operating procedure, typically 200 μL of BALF is sufficient for subsequent analysis. If the DNA yield is low, centrifuge the BALF sample at 12,000× g for 5 min, discard the supernatant, and retain 200 μL of bottom liquid.
PCR amplification enrichment of target DNA sequences to be tested
For one BALF sample DNA, prepare 10 PCR reactions using amplification primers, one for each of the 10 pathogenic bacteria.
Setting up the positive and negative controls: the templates for the positive and negative controls are the genomic DNA of the corresponding pathogen and nuclease-free water, respectively.
Prepare the PCR reactions in a 0.2 mL PCR tube.
Component 50 μL reaction
2× HiFi amplification mix 25 μL
Forward and reverse primers (10 μM) 2 μL of each
Template DNA (100 ng/μL) 1 μL
Nuclease-free water 20 μL
Mix well and pulse-spin in a microcentrifuge.
Place a 0.2 mL PCR tube in a PCR thermocycler.
PCR procedure:
98 °C
2 min
98 °C
10 s
38 cycles
52 °C
10 s
72 °C
15 s
72 °C
5 min
4 °C
Forever
After the PCR reaction, without the PCR purification step, directly use the PCR reaction solution as the detection substrate for the Cas12a reaction.
Detection of PCR products using Cas12a
Prepare the reactions in a 384-well microplate on ice:
Component 20 μL reaction
ssDNA-reporter (10 μM) 1 μL
PCR reaction solution 2 μL
10× NEBuffer 3.1 2 μL
Cas12a (10 μM) 1 μL
crRNA (10 μM) 1 μL
Nuclease-free water 13 μL
Note: The PCR reaction solution for each pathogen requires the use of the corresponding crRNA. Preparation of premixes is recommended to improve experimental efficiency.
With a pipette set at 15 μL and keeping the plate on ice, mix the reaction by pipetting the entire solution up and down 10 times.
Note: Pipetting 10 times can ensure sufficient mixing of the reaction solution. Avoid excessive bubbles in the reaction solution; any trace bubbles will clear after 30 min of incubation at 37 °C in the next step.
Place the plate in a fluorescence plate reader. Incubate at 37 °C for 30 min, then measure the fluorescence values of each reaction well (excitation wavelength: 485 nm; emission wavelength: 535 nm).
Data analysis
Note: The data analysis of this protocol is applied to the detection of pathogenic bacteria in patients with severe pneumonia in the ICU. For other studies, the analysis standards require adjustments accordingly.
For each sample, analyze the fluorescence values for each pathogen separately.
For each pathogen detected, the positive control (PC) is the genomic DNA of corresponding pathogen, and the negative control (NC) is nuclease-free water.
The fluorescence value of BALF sample (shown as F) is two-fold larger than the negative control, indicating that the SSBD method detected the corresponding pathogen.
In our previous article, the application of SSBD was for pathogen detection in ICU patients, so the degree of pathogenic infection is required to guide drug usage. The pathogen strength is determined to some extent by the fluorescence signal (Table 3). Set I (interval) as the point of distinction. I = (PC - 2NC)/3. When 2NC < F < 2NC+I, the pathogen strength level is defined as level I. When 2NC+I < F < 2NC+2I, the pathogen strength level is defined as level II. When F > 2NC+2I, the pathogen strength level is defined as level III.
Table 3. Determining the presence of bacteria by fluorescence signal
Fluorescence value Test results Bacterial DNA molarity
F < 2NC Undetected N.A.
2NC < F < 2NC+I Detected level I 10-15 M–10-14 M
2NC+I < F < 2NC+2I Detected level II 10-14 M–10-13 M
F > 2NC+2I Detected level III over 10-13 M
NC: Valuenegative control; F: ValueSample; I = (Valuepositive control - 2 Valuenegative control)/3
Example results
Using the data of two samples from our previously published article, we calculated the values of 2NC, 2NC+I, and 2NC+2I according to the fluorescence values of PC and NC, respectively. The fluorescence values of the samples are shown in Table 4.
Table 4. Fluorescence values of two samples
Species Fluorescence value Detection result
PC NC Sample 1 Sample 2 2NC 2NC+I 2NC+2I Sample 1 Sample 2
A. baumannii 9788 903 9300 9398 1806 4467 7127 Detected Level III Detected Level III
E. coli 8435 821 657 630 1642 3906 6171 Undetected Undetected
K. pneumoniae 8285 1791 1718 1730 3582 5150 6717 Undetected Undetected
S. aureus 8316 1008 1018 1003 2016 4116 6216 Undetected Undetected
P. aeruginosa 9323 1170 1164 1069 2340 4668 6995 Undetected Undetected
S. epidermidis 9309 1237 1210 1183 2474 4752 7031 Undetected Undetected
S. capitis 8832 1633 1949 1772 3266 5121 6977 Undetected Undetected
E. faecalis 8241 614 512 505 1228 3566 5903 Undetected Undetected
E. faecium 8432 1936 1692 1700 3872 5392 6912 Undetected Undetected
S. maltophilia 8194 1620 1549 1579 3240 4891 6543 Undetected Undetected
The fluorescence value of A. baumannii in these two samples was higher than 2NC+2I. Therefore, the presence of A. baumannii in these two samples was detected, with a level III bacterial strength. For the rest of the pathogens, the fluorescence values of the samples were lower than 2NC, which indicates that the rest of the pathogens were not detected.
Notes
Optimizing PCR reactions can significantly avoid the generation of non-specific signals. When designing primers, BLAST the primers and select primers with excellent specificity. The suitable PCR reaction program is confirmed by experiment. Moreover, it is basically impossible for crRNA recognition sequences to exist in the non-specific amplification products generated during PCR. Therefore, combining the crRNA-Cas12a reaction with the PCR reaction greatly ensures the specificity of the detection. It is noteworthy that if crRNA is synthesized by in vitro transcription with RNA polymerase, the DNA used as in vitro transcription template needs to be removed to avoid contaminating crRNA (since the DNA templates can bind to crRNA to activate Cas12a).
Features considered important while selecting target:
Target sequences are present in multiple strains of this species' genomes.
Target sequences are not found in other species' genomes.
Target without repetitive sequences.
Target including PAM sequences.
Table 5. Information of target DNA region used in the protocol
Species GenBank of genome Target sequences (5′ > 3′) Corresponding gene
A. baumannii CP115629.1 CACAGCGTTTAACCCATGCCATTGGTAAAGCTAAAGCAATGGAAATGTGCCTTACCGCTCGTCAAATGGGTGCAGTAGAGGCAGAACAAAGTGGTTTGGTTGCACGTGTGTTTAGTAAAGAAGAATTACTTGAACAAACCTTGCAAGCCGCTGAAAAAATCGCAGCACGGTCTTTAACTGCAAACATGATGCTCAAAGAAACCATTAATCGAGCTTTTGAGGTGAATCTCACCGAAGGTTTACGTTTTGAACGCCGCATGTTCCACTCCATTTTTGCAACTGCGGACCAAAAAGAAGGCATGCAGGCATTTGTTGAAAAACGGCAGGCAAATTTTAAAAATCAGTAAGAGATCAATAAAATGAATACAGAAAATCACTTAATGATTGAGCGACAAGGTAAGCTTGGCGTGATCACCTTAGATCGTGTGACTCATCTCAATGCATTGTCGCTAGATATGATTGAAGGAATTGGCGCCCAATTGGAGTTGTGGCGAAATGATGCTGCTGTTCAGGCGATCTTAATCAAATCAAATAGCCCAAAAGCCTTCTGTGCAGGTGGCGATA enoyl-CoA hydratase/isomerase family protein
E. coli CP115173.1 GTTCCTGACTATCTGGCGGGGAATGGTGTGGTTTATCAAACCAGTGATGTGAAGTATGTGATTGCCAACAACAACTTGTGGGCCAGCCCGTTGGATCAACAGTTGCGCAACACCCTGGTTGCCAACCTGAGTACGCAACTGCCCGGCTGGGTGGTTGCCTCCCAGCCTCTGGGAAGCGCCCAGGACACGCTCAATGTTACCGTAACGGAGTTTAACGGTCGCTATGATGGCAAGGTCATTGTCAGTGGTGAGTGGCTGTTGAACCACCAGGGACAACTGATCAAACGTCCGTTCCGTCTGGAAGGAGTGCAAACTCAGGATGGTTACGATGAGATGGTTAAAGTGCTGGCCGGTGTCTGGAGTCAGGAAGC membrane integrity-associated transporter subunit PqiC
K. pneumoniae CP115714.1 CATGGGCATATCGACGGTCACCGATTCGCCCGGACGCAGGCGAACATCTTTTTCCGCCACCCGGTCCGCCTGCAGCGCCTGGCCATCGCCGGCAAACAGCGACGGGTAGTCAGTATTGTCGAACGCTTGCCGGTCCTTCAGCTGATAAATACGCACCACCGTCGCCAGGGAGGCGCCTTTGGCGTTGTTATTCACGCCTTCCCGGGCGCGAAGATCCAGATGCAGGGTTTTCACCTGGGGGTAAAAAATGGACTGGGTCACGGAAACGGCGCCGTCTTTCACGGTCTGCGTCAGGCCGCAGCCGGTTAAGACGGTAACCATGAGGAATGCCAGCAACCTGGCGGAGGGTTTAACTGCGGTAATCGCCATCTTCATCGCTCTCCCTGCGGTGGATATTTTCCCGGACGCGCTGATAGCGCCCCAGATAAATCGTGACTCTGTCGTCAGCCTGTTTCTGCGCATCCAGAGGACGCAGCACAGCGGTACGGCCGAGCTGGACGGCATGCTCCTGCTGGCAGCAAAGTTGCGCATCCGGCAGTAAATGGCGGGCGACGCAAAGCTGCAGCCGCACGTCAAGATGAGATCCGAGCCAGACGTGCAGAAGCGCCATCAGGTCGCTGAACAGTTCGCCGCCGGGCAGCCAGCCGCGAACCTCATCAGGCTTTTCCGTGGCCAGCTGCAGCAATACCTGACCGTTCACATCCGTGGCGTGGGTTCCCATCACTGGCCTATGTTGCAGG type VI secretion system baseplate subunit TssG; type VI secretion system lipoprotein TssJ
S. aureus CP063802.1 AGGTGCAGTAGACGCATAGCATCATCATATTGAATAGTAAAAACAAATAAAACATAGTAACGTGATTCAGTCGATGTAACAGTCGATAATGAGTCACGTTTTTTTATAGAAAAATATAAGACATAAAAATGTCATAATTTATAGTCGACAAATATCATACTGTATAAACATTTATCATTTTCTCAAGTGCCTTTTACACGATGGAATGAACTTACTTTTTACGAAATTATGCGTATTTTATAAACAAATATCATTGATATAACGGTAAATGTAAGCGTTTACAACAGAAATAACTGCATGCTACGATATTTTTGTAAATTCACTGATTCAAGTATTTTAAGTCAATATGAGGAGGGATGTTATGAGCGATTCTGAGAAAGAAATTTTAAAAAGAATTAAAGATAATCCGTTTATTTCACAACGTGAACTTGCTGAGGCAATTGGATTATCTAGACCCAGCGTAGCAAACATTATTTCAGGATTAATACAAAAGGAATATGTTATGGGAAAGGCATATGTTTTAAATGAAGATTATCCTATTGTTTGTATTGGCGCAGCGAATG winged helix-turn-helix transcriptional regulator
P. aeruginosa CP080405.1 TCAGTGACATGAAGCCCGTCCCCGCGGCCAGGCTGCGCAGTTGCGCCTACGAGATGGGTTTTTCTCTCTATCACGCCGGTCAATGCTTGCGCGTGGTTCTCTAGGTTCGACCGAATCAAGAGTGTGTTTTCAGCAAAATAATGATAGTTTGTTGTAAACACACATGAGGAGGTCGTCATGAGCGCTCTCATCAAGGAACGTCCCAGCGCCGATGCCGTCCTGGCCAAGGCCGTCCTGGCCGCGCGCGAGCAATTGGGGCTGACGCAGCTCGAACTGGCCGGCATCGTCGGCGTCGATCGCAGCGCCATCAGCCGCTGGAAGACCCAGGGCCTGCGGGTGGACAGCAAGACCGGCGAGCTGGCTCTGCTGCTGGTGCGAGTCTATCGCGCACTGTATGCCCTGTTCGGCGGGCAGCAGGAGGACATGCGCCACTTCCTGCGCACTCCCAACCATCACCTGGCGGGCGAGCCGCTGGCACTGATGGGACAGGTGCAGGGCCTGGTTCATGTGCTGGAATACCTCGATGCGA DUF2384 domain-containing protein; FAD-dependent oxidoreductase
S. epidermidis CP106834.1 CACGCATGGCACTAGGTACAAATCCAGTATAAACAGCGTAGATTAATAATAGTAAAATAGTTACACCTTTAATGAGAACTAGAGGTATATTTAAACGTACTAACAGTTGATAAACGAGGACAATAATTGTACCAACATGTGTACCACTTATAGCTAGTAAATGGTAAATACCTATATCTTTGATGTTAGATTTGTAATATTCATCAATTAGAGAGGTATCACCTGTTATCAACGCTAATATTCTTTCAGGATGCGTGACCCCAGATTTATGAATTATTTTGGTGATATAATTTTGATGATGATATATAGGAGTGAAAATATTGTTTTCTTGGCAAGACTTAAATTGAACTGTTGAAATATTCAACAAGGACAACTCTTTTTCG DNA internalization-related competence protein ComEC/Rec2
S. capitis AP014956.1 GGTTCAGTCATCCCCACGTTTTGATCTTCAATATTCATGTTATAATTTTCTCCTTTTTAAAAGGTTTCATTTACCATTGTTTACTACAGTAAAGGTTGTTTTAAACATATTCGTTTTAATATTTTTAGAGTGAAGATTCTAAAACGTGTGATAAAGGTGGAATATACACATAAAAATTTCCGATATAGAAAAATAAAAAGCTTTCAATCACCTTATTAATCAATAAAAAGTGATTAAAAGCTTTAAATTAAAAGAGGATAAACATCAATTCATATCTCAAACGCTTCTAACATTTTTGAGTTAGATGACTGAATTTGAACTGTGTTTATATCAAGCTTTTGTCTATTCTAGCCATCTTAGAAAACTATATAGAATGAATTCTTATTTAAGAAATCAATGCAGTATTTCTTTAAACTTCTGTTACTTCGACTTCGATGTGTTCGATGCGATGATATGCACCATGATCAACAGGTCCTACAAATTCATTCATTTTCCAACCGTTTTTGATTGCAGATGCTACGAAAGCTTTAGCAGCGATGACTGCTTCTCTTGGAGATTTGCCGTTAGCTAGGTAAGCAGTTGTCGCAGCTG putative pyridoxine kinase; FAD binding superfamily protein
E. faecalis CP046113.1 CGGCAAGTTTGGAAGCAGACAAAGGTATTTTTGCAGTATTGAATGTCATCATTGGGATTAATGGTTTTGTTTGGAATGGTACATTGGCTATCGCTGGTTTGATTTTCGCTTTTTCTTGGGGCTATAACTTAGCGAAAGCATACAATGTGAATGAGCTAGCTGGTGGGATTGTCAGTTTAGCTACCTTAATTTCAGGTGTTGCTTTTGCACCAAATAACACAGCTGAATTAGCTGTAAAAGTGCCAGAAAAGATTGCCAACGCAATTAATGGGGCAGAAATTGGTGCGTCAATTGCTAATAATCAATTGACTGTCAATCCATGGGGTTGGTTAAATCTCAACCATCTAAATGGTAACGCTTTCTTTACGGTGATGATTATGGGCGCTTTATCAACCATTATTTTTTGTAAATTAATGCAAGCGGATTTAACAATAAAAATGCCTGATTCTGTACCACCAGCAGTTTCAAAAGCGTTTGCGGCAATTTTACCAGCAACAATCGCATTGTATGTCGTAGCAATTATTAACTTTACGGTGTCAAAACTTTCAGGCGGTCAATTATTAATTGACTTAATTCAAAAATATATCGCGGAACCGTTCCTTGGGTTATCACAAGGACTAGGCGCT PTS sugar transporter subunit IIC
E. faecium CP112862.1 ATCGGAAATCGGTGTGGCTTATGCGAAAGGCATCCCAGTGATCGGCTTATATACTGATACTCGACAGCAAGGCGGAACCCACCCGAAAAAAATCGCTGCACTACAAGAAACAGCTGAAAATCAATTTCACTACCTGAACTTATATACAATAGGTTTGATCAAATTGAACGGAAAAGTGGTTTCTTCAGAAATTGAGTTGCTTTCAGAAGTAAAAAGATTTTTAGATGGAGGGACTTTCAGTGATTAAAGAAATCAAAAAGGTGCAAATAGCTTTACTTGCTTTTGGTGTATTCGTTGTCTGCTATAATTTTTATGAATTTATCACACAAAAATATTCGACTTCTCAAGGAATCACATTTATCGTGGAATCTTTATTAGGGATTGCATTGATTTTCATGCCACAAGTCATTTTGAAAGTTTTCAAACTTAAAATACCAGCAGCAATCGTTTTGTTTTACTGGTTTTTCTTGTTCATCTCTGTCTTTTTAGGCACAGGGATGCATTTGA nucleoside 2-deoxyribosyltransferase; hypothetical protein
S. maltophilia CP040440.1 CCCGCGAGAAGCTGGATGCGCTGGAAACCGCCGTGCGCGAGCTGGAAGGGCGCGGCGCCGCAGAATGAAAAAAGGAGGCGCACCGCGCCTCCCGTTTACAGATTATTTCCCGGATGGGGCCTTGCAGCAAGGCCCACCGGGCGCCCTAGACTCGCCGGCCTGTCGATCCGGCAGCACAGAGAGGGGCAGTGCCGATGGCGCAGGACACCGAATGGACACCCGTTTCGCATGACACCTGCGACCAGGTCGCCGGGCCGTTCTATCACGGCACCCGCGCTGACCTGGCGGTGGGCGAGCTGCTGAGCGCGGGCTTCCGCTCCAACTATCGCGACAGCGTGGTGATGAACCACATCTACTTCACCACGATTGCCAAGGGCGCCGGGTTGGCTGCGGAGATGGCCCGGGGTGAAGGGCGACCGCGCGTGTATGTGGTGGAGCCGA accessory factor UbiK family protein; NAD(+)--rifampin ADP-ribosyltransferase
Acknowledgments
This protocol was adapted from our previously published work (Wang et al., 2022). This work was supported by National Natural Science Foundation of China (81927808).
Competing interests
The authors declare no competing interests.
Ethics
This study was registered in English at https://clinicaltrials.gov/ (NCT04178382) in November 2019.
References
Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M. and Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360(6387): 436-439.
De Pascale, G., Bello, G., Tumbarello, M. and Antonelli, M. (2012). Severe pneumonia in intensive care: cause, diagnosis, treatment and management: a review of the literature. Curr Opin Pulm Med 18(3): 213-221.
Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., Verdine, V., Donghia, N., Daringer, N. M., Freije, C. A., et al. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356(6336): 438-442.
Hansen, V., Oren, E., Dennis, L. K. and Brown, H. E. (2016). Infectious Disease Mortality Trends in the United States, 1980-2014. JAMA 316(20): 2149-2151.
Lazcka, O., Del Campo, F. J. and Munoz, F. X. (2007). Pathogen detection: a perspective of traditional methods and biosensors. Biosens Bioelectron 22(7): 1205-1217.
Li, S.-Y., Cheng, Q.-X., Liu, J.-K., Nie, X.-Q., Zhao, G.-P. and Wang, J. (2018). CRISPR-Cas12a has both cis-and trans-cleavage activities on single-stranded DNA. Cell Res 28(4): 491-493.
Wang, Y., Liang, X., Jiang, Y., Dong, D., Zhang, C., Song, T., Chen, M., You, Y., Liu, H., Ge, M., et al. (2022). Novel fast pathogen diagnosis method for severe pneumonia patients in the intensive care unit: randomized clinical trial. eLife 11: e79014.
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© 2023 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 > PCR
Molecular Biology > RNA
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why can't the reverse primers match?
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作者你好。请问可以详细描述一下有关于体外检测的引物设计方法吗?
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4,682 | https://bio-protocol.org/en/bpdetail?id=4682&type=0 | # Bio-Protocol Content
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Peer-reviewed
Expression and Purification of scFv2H7-P18F3, a Bi-Modular Fusion Protein (BMFP) Targeting Human CD20
CB Carine Brousse
NR Nathan E. Rainey
AD Alexandra Desrames
JT Jean-Luc Teillaud
BG Benoît Gamain
AC Arnaud Chêne
Published: Vol 13, Iss 10, May 20, 2023
DOI: 10.21769/BioProtoc.4682 Views: 669
Reviewed by: Victor TseRan ChenKuo-Ching MeiSuresh Kumar
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Cited by
Original Research Article:
The authors used this protocol in Science Advances Feb 2022
Abstract
P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.
Keywords: BMFP Anti-EBV antibodies Fusion protein Immunotherapy CD20 scFv B lymphoma Escherichia coli SHuffle®
Background
During the last decades, monoclonal antibody therapy has proven to be very effective for the treatment of many diseases such as cancers (Zahavi and Weiner, 2020), chronic inflammatory diseases (Voge and Alvarez, 2019), and certain diseases of infectious origin such as COVID-19 (Hwang et al., 2022). Nevertheless, the complex nature of immunoglobulins (Ig) (molecules of high molecular weight, with multiple chains and post-translational modifications impacting their effector functions) requires the use of demanding heterologous expression systems (mostly eukaryotic cell-based systems) for industrial production, which results in high manufacturing costs. To overcome this limitation, we recently conceptualized a novel immunotherapeutic approach and generated bi-modular fusion proteins (BMFPs), able to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, leading to their clearance. As a proof-of-concept, we have established that this strategy is efficient against cancer cells (Gamain et al., 2022). We have engineered and expressed, in Escherichia coli, a BMFP comprising a Fc-deficient binding moiety (scFv originating from the mouse IgG2b monoclonal antibody 2H7) targeting the human CD20 (huCD20) B-cell marker fused to a fragment of the EBV-P18 antigen (P18F3), which recruits circulating endogenous anti-P18F3 IgG in EBV+ individuals. We have shown that in vitro treatment of huCD20+ Burkitt’s lymphoma cells, in presence of plasma containing anti-P18F3 antibodies, elicits a significant activation of the antibody-dependent complement cascade and trigger FcγRIII-mediated activation of cellular pathways, leading to antibody-dependent cell-mediated cytotoxicity (Gamain et al., 2022). Furthermore, we have also demonstrated in a mouse tumor model that therapy performed with scFv2H7-P18F3 significantly leads to increased mice survival and full cancer remission in some animals (Gamain et al., 2022).
As P18F3-based BMFPs display efficient biological activity, they could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing and purifying scFv2H7-P18F3 in Escherichia coli (SHuffle®). It can also be used for expression and purification of other BMFPs with alternative binding specificities (Figure 1).
Figure 1. Standardized construct backbone for rapid and easy modulation of the binding moiety. The DNA sequence of scFv2H7-P18F3 was cloned into the pET-28a expression plasmid. The resulting amino acid sequence of scFv2H7 is displayed in dark gray, and the amino acid sequence of P18F3 is displayed in green. The recombinant protein possesses a C-terminal Histidine tag for easy purification and detection in functional assays. An alanine followed by a serine (AS) link scFv2H7 to the P18F3 polypeptide. When alternative binding specificity is desired, a new DNA sequence encoding for another binding moiety (devoid of Fc part) can be inserted between the NcoI and NheI restriction sites. We recommend using DNA sequences recoded for optimal E. coli codon usage, as it might ease the expression of the recombinant protein in SHuffle®.
Materials and Reagents
We describe here the exact materials and reagents that we use. Any equivalent materials and reagents may also be suitable for the procedure.
Materials
Filter tips PIPETMAN® DIAMOND 0.1–10 μL, 2–30 μL, 20–200 μL, 200–1,000 μL (Gilson, catalog numbers: F171203, F171303, F171503, F171703)
Sterile serology pipette; 2, 5, 10, 25, and 50 mL (Greiner, catalog numbers: 710160, 606180, 607107, 760180, 768160)
Expanded polystyrene ice bucket
Laboratory bottles with screw cap; 100 and 500 mL and 1 and 2 L (VWR, catalog numbers: 215-1592, 215-1594, 215-1595, 215-1596)
Petri dishes, polystyrene, 100 × 15 mm (Corning-Falcon, catalog number: 351029)
1.5 mL safe-lock Eppendorf microtubes (Eppendorf, catalog number: 0030121872)
Greiner culture tubes, 14 mL round bottom with snap vent cap (Merck, catalog number: Z617954)
Tube racks that can accommodate 1.5 (ø 11 mm), 15 (ø 17 mm), and 50 mL tubes (ø 30 mm)
L-shaped cell spreaders (Thermo Fisher Scientific, catalog number: SPCS01)
Tube with conical bottom; 15 and 50 mL (Corning-Falcon, catalog numbers: 352096, 352070)
Minisart high-flow syringe filter 0.2 μm pore size (Sartorius, catalog number: 16532)
Sterile syringes for single use; 1, 5, 10, and 60 mL (FisherbrandTM, Thermo Fisher Scientific, catalog numbers: 17161936, 15809152, 15819152, 15839152)
Cryotube 1.8 mL round bottom (Nunc, catalog number: 368632)
Aluminum foil
PES 0.2 μm filtration unit (Nalgene, catalog number: 564-0020)
High-resolution gel filtration Superdex® 200 Increase 10/300 GL column (Cytiva, catalog number: 28990944)
Reagents
Double-distilled water (ddH2O)
Ice
Super optimal broth with catabolite repression (SOC) medium (Thermo Fisher Scientific, catalog number: 15544034). Alternatively, SOC medium could be prepared according to the recipe provided in the Recipe section
SHuffle® T7 express competent E. coli (New England BioLabs, catalog number: C3029J)
NucleoSpin Plasmid QuickPureTM Kit (Macherey-Nagel, catalog number: 740615)
YPD (YEPD) agar powder (MP Biomedicals, catalog number: 114001222)
Lysogeny broth (LB) powder (MP Biomedicals, catalog number: 113002022)
Kanamycin sulphate powder (Thermo Fisher Scientific, catalog number: 15160054)
Imidazole (Merck, catalog number: I2399)
Phosphate buffer saline (PBS) pH 7.2 (Merck, catalog number: D8537). Alternatively, PBS could be prepared according to the recipe provided in the Recipe section
Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Merck, catalog number: I5502)
Sodium chloride (NaCl) (Merck, catalog number: S7653)
Trizma® hydrochloride (Tris-HCl) (Merck, catalog number: 93363)
Lysozyme from chicken egg white (Merck, catalog number: L6876)
Protease inhibitor cocktail tablets (cOmpleteTM) (Merck, catalog number: 11697498001)
HisTrap FF 1 mL (prepacked with pre-charged Ni Sepharose® 6 fast flow) (Cytiva, catalog number: 17531901)
Laemmli sample buffer 4× (Bio-Rad, catalog number: 161-0747)
XT reducing agent (Bio-Rad, catalog number: 161-0792)
4%–15% 10-well mini-protean TGX stain-free gel (Bio-Rad, catalog number: 4568084)
10× Tris/Glycine/SDS (TGS) buffer (Bio-Rad, catalog number: 161-0772)
Nitrocellulose membrane 0.2 μm (Trans-Blot Turbo Transfer Pack) (Bio-Rad, catalog number: 1704158)
Western blotting detection reagents (ECLTM Prime) (Cytiva, catalog number: RPN2232)
Tryptone (Merck, catalog number: T2559)
Yeast extract (Merck, catalog number: Y1625)
KCl (Merck, catalog number: P9333)
MgCl2 (Merck, catalog number: M2670)
MgSO4 (Merck, catalog number: 63138)
Glucose (Merck, catalog number: G7528)
Centrifugal concentrator Amicon Ultra-4 (10 kDa MWCO) (Merck, catalog number: UFC8010)
Glycerol ≥99% (Merck, catalog number: G6279)
LB medium (1×) (see Recipes)
SOC medium (1×) (see Recipes)
Kanamycin stock solution (50 mg/mL) (see Recipes)
50% glycerol solution (see Recipes)
Bacteria resuspension solution/IMAC running buffer (see Recipes)
IMAC elution buffer (see Recipes)
Tris buffer saline (TBS) (see Recipes)
Tris buffer saline tween (TBST) (see Recipes)
Western blot blocking solution (TBS-5% milk) (see Recipes)
Western blot antibody dilution solution (TBST-5% milk) (see Recipes)
Phosphate buffer saline (PBS) (see Recipes)
Equipment
Pipette PIPETMAN® 0.1–10 μL, 2–20 μL, 20–200 μL, 100–1,000 μL (Gilson, catalog numbers: FA10002M, FA10003M, FA10005M, FA10006M)
Macroman® pipettor (Gilson, catalog number: F110120)
Borosilicate glass narrow neck Erlenmeyer flasks, 50 and 500 mL and 3 L (FisherbrandTM, Thermo Fisher Scientific, catalog numbers: 15499093, 15439103, 15469103)
Measuring glass cylinders, 100 and 500 mL and 1 and 2 L (VWR, catalog numbers: 612-3836, 612-3838, 612-3839; 612-3840)
Vertical gel electrophoresis system (PROTEAN Tetra cell) (Bio-Rad, catalog number: 1658004)
Autoclave (to be run at 120 °C for 20 min, under saturated steam ≥ 15 psi)
Water bath allowing precise temperature adjustment (range: 37–42 °C)
Shaking incubator allowing precise temperature adjustment (range: 20–37 °C), such as Multitron Standard incubator (Infors HT)
Incubator chamber allowing precise temperature adjustment (range: 30–37 °C)
Spectrophotometer allowing OD monitoring at 600 nm, such as BioSpectrometer® Basic (Eppendorf)
Standard fridge and freezer (-20 °C)
Ultra-low temperature freezer (-80 °C)
Refrigerated centrifuge, such as versatile 5810 series centrifuge (Eppendorf)
pH meter
Magnetic stirrer with magnetic stir bars (VWR, catalog number: 442-1271)
EmulsiFlex-C5 Homogenizer (Avestin) with air supply (recommended incoming pressure 120 psi/8 bar)
Vacuum pump
Power supply (Power PAC 300) (Bio-Rad, catalog number: 164-5050)
ChemiDocTM MP imaging system (Bio-Rad)
Rocking platform
Trans-Blot Turbo transfer system (Bio-Rad)
Automated liquid chromatography system, such as ÄKTA purifier 10 (Cytiva)
Chromatography refrigerator, such as Mediline LKPv 6522 (Liebherr)
Procedure
Expression of scFv2H7-P18F3
Antibody fragments such as scFv or VHHs (also known as nanobodies) possess structuring disulfide bonds mandatory for their functionality. In bacteria, disulfide bound formation requires translocation of proteins to the periplasmic compartment of the cell via an N-terminal signal peptide (Denoncin and Collet, 2013). Therefore, expression of disulfide-bonded recombinant proteins in standard E. coli–based systems usually leads to low production yields. Technological advances now enable overcoming this constraint by using mutant E. coli strains (such as SHuffle®), capable of promoting disulfide bound formation in the cytoplasm, leading to more efficient folding of recombinant proteins, improved activities, and increased production yields (Lobstein et al. 2012).
Day 1. Preparation of selection plates containing LB and agar supplemented with kanamycin (LB–agar K+) and of LB K+ expression medium
For 10 plates, weigh 2.25 g of agar powder (15 g/L) and 3.75 g of LB powder (25 g/L). Dissolve in 150 mL of ddH2O in a 250 mL autoclavable glass bottle. Put the lid back on the bottle without fully screwing it to allow pressure released during autoclaving.
Autoclave at 120 °C for 20 min, under saturated steam ≥ 15 psi.
Thaw a tube of kanamycin (stock solution at 50 mg/mL) on ice.
Once the autoclave cycle is done, let the bottle cool down to approximately 50 °C (at this temperature, you should be able to maintain your hand on the glass bottle for a few seconds).
Add 150 μL of kanamycin into the bottle and mix well to obtain LB–agar K+ (final concentration: 50 μg/mL).
Distribute 15 mL of LB–agar K+ into 10 Petri dishes (100 × 15 mm) (Figure 2A).
Let the plates cool down with the lid slightly open, until the LB–agar K+ is totally solidified and dried (Figure 2B).
Once the plates are dried, they can be stored at 4 °C (with the LB–agar K+ layer facing the top) for up to two weeks (Figure 2C).
Figure 2. Casting LB–agar K+. A. 15 mL of warm LB–agar K+ are poured into a Petri dish using a serological pipette. B. The plate is then let to dry. C. Once fully dried, the plate is turned upside down and can be stored at 4 °C for up to two weeks.
Day 2. Transformation of SHuffle® T7 express competent E. coli with pET-28a-NC-(scFv2H7-P18F3)
Thaw pET-28a-NC-(scFv2H7-P18F3) DNA on ice.
Thaw a tube of SHuffle® T7 express competent E. coli (50 μL) on ice.
Add 50 ng of pET-28a-NC-(scFv2H7-P18F3) DNA (5 μL from the 10 ng/μL stock solution) to the bacteria suspension and gently mix (without vortexing).
Leave the tube on ice for 30 min. Meanwhile, equilibrate a water bath at 42 °C.
Remove the tube from the ice and dip it for 30 s in the water bath (42 °C heat shock).
Place the tube back on ice for 5 min.
Add 950 μL of room temperature SOC medium to the tube and transfer the whole volume (1 mL) to a new Greiner culture tube. Put the lid back on the tube without clipping it to allow air to go in.
Place the tube in a suitable rack and shake vigorously for 1 h at 30 °C. Here, we use a Multitron Standard incubator and set the tray revolution at 250 rpm. Meanwhile, equilibrate five LB–agar K+ selection plates at 30 °C in an incubator chamber.
Stop the incubation of the transformation mixture and perform five 10-fold serial dilutions in 1.5 mL safe-lock Eppendorf tubes using SOC medium (Figure 3).
Spread 100 μL of each dilution onto LB K+ selection plates using a L-shaped cell spreader.
Incubate the plates [plates should be inverted (Figure 2C)] at 30 °C in an incubator chamber overnight (18–20 h) for SHuffle® optimal growth.
Figure 3. Plating procedure of competent E. coli transformed with pET-28a-NC-(scFv2H7-P18F3). The transformation mixture is subjected to serial dilutions in 1.5 mL tubes. 100 μL of each dilution is then used to seed five different LB K+ selection plates.
Day 3. Liquid bacteria culture from a single colony
Remove plates (plates 1–5) from the incubator and store them at 4 °C.
At the end of the day, thaw a tube of kanamycin (stock solution at 50 mg/mL) on ice.
Add 1 mL of the kanamycin stock solution to 1 L of LB medium.
Prepare a Greiner culture tube containing 1 mL of LB medium supplemented with kanamycin at 50 μg/mL (LB K+) and store the remaining LB K+ medium at 4 °C.
Pick up one single, isolated colony from one of the plates (plates 1–5) using a 200 μL clean tip (Figure 4A).
Place the tip inside the tube containing 1 mL of LB K+, put the lid back on the tube without clipping it, and incubate overnight at 30 °C with vigorous shaking (Figure 4B).
Figure 4. Picking up a single bacteria colony to inoculate an overnight liquid culture. A. A single bacteria colony is touched with a 200 μL pipette tip. B. The tip is then introduced in a tube containing 1 mL of LB K+. The tip can either be removed after a few seconds of swirling or left in the tube during overnight incubation at 30 °C.
Day 4. Bacteria culture scale up, induction of recombinant protein expression, glycerol stock preparation
Bacteria culture scaling up, induction of recombinant protein expression
Equilibrate 1 L of LB K+ medium at 30 °C in an incubator chamber.
Transfer 1 mL of the overnight culture into a 50 mL Erlenmeyer culture flask containing 9 mL of LB K+ medium. Cover the neck of the flask with aluminum foil (Figure 5A).
Incubate the flask with vigorous shaking at 30 °C for 3 h.
Transfer 10 mL of the culture into a 500 mL Erlenmeyer culture flask containing 90 mL of LB K+ medium. Cover the neck of the flask with aluminum foil (Figure 5B).
Incubate the flask with vigorous shaking at 30 °C for 3 h.
Transfer 100 mL of the culture into a 3 L Erlenmeyer culture flask containing 900 mL of LB K+ medium. Cover the neck of the flask with aluminum foil (Figure 5C).
Incubate the flask with vigorous shaking at 30 °C and check frequently (every 30 min) the optical density (OD600nm) of the culture using a spectrophotometer.
Thaw a tube of IPTG stock solution at 1 M on ice.
When OD reaches 0.8, collect 10 mL of the culture in a 15 mL Falcon tube (pre-induced sample) and keep it at room temperature until processing (see procedure in Figure 6).
Add 200 μL of IPTG from the stock solution into the flask (the final IPTG concentration is 0.2 mM) (Figure 5D).
Incubate the flask with vigorous shaking (250 rpm) at 20 °C overnight (14–16 h).
Figure 5. Culture scaling up and induction of scFv2H7-P18F3 expression. A–C. The overnight culture is expended following three consecutive 1:10 dilutions in LB K+ medium. D. A first aliquot of culture is sampled (pre-induced culture) before introduction of IPTG. A second aliquot of culture is sampled (IPTG-induced culture) after 16 h incubation at 20 °C.
Glycerol stock preparation
A glycerol stock of bacteria containing pET-28a-NC-(scFv2H7-P18F3) can be established to avoid cell transformation/plating steps each time a protein expression process is undertaken. This procedure should be performed right after sampling of the pre-induced culture aliquot.
Transfer 1 mL of the pre-induced culture sample (from Day 4, step 9) into a 1.5 mL safe-lock Eppendorf tube, centrifuge at 500× g for 20 min, remove the supernatant, and freeze the cell pellet at -20 °C until further analysis (Figure 6A).
Transfer 4 mL of culture from the pre-induced culture sample to a new 15 mL Falcon tube (Figure 6B).
Add 4 mL of 50% glycerol solution (see Recipes) into the tube and mix well by inverting up to 10 times.
Distribute the culture/glycerol mixture to eight cryotubes (1 mL per tube) and freeze them at -80 °C.
Sequencing of the plasmid at this stage is highly recommended.
Centrifuge the remaining 5 mL of culture sample (from Day 4, step 9) at 500× g for 10 min, discard the supernatant, and perform plasmid DNA isolation from the pelleted cells using a NucleoSpin Plasmid kit following the manufacturer’s instructions (Figure 6C).
The isolated plasmid DNA could then be sequenced to ensure suitability of the bacteria glycerol stock for new protein expression processes (glycerol stock validation).
Figure 6. Glycerol stock preparation and validation. Ten milliliters of pre-induced sample are collected at Day 4, step 9. A. A first aliquot is frozen at -20 °C to serve as the non-induced culture sample during protein expression analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). B. The pre-induced bacteria culture is mixed with 50% glycerol (v/v) and distributed into cryotubes. The cryotubes are then frozen at -80 °C to constitute a glycerol stock of bacteria. C. The leftover of the pre-induced sample is used to isolate plasmid DNA and ensure its correct sequence.
Addendum: Performing protein expression from a glycerol stock
A new protein expression process can be undertaken from a validated glycerol stock.
Prepare a Greiner culture tube containing 1 mL of LB medium supplemented with kanamycin (LB K+).
Remove a glycerol stock cryotube from -80 °C (do not let it thaw completely).
Immediately scrap the top of the frozen culture/glycerol mixture with a 200 μL clean tip.
Place the tip inside the tube containing 1 mL of LB K+, put the lid back on the tube without clipping it, and incubate with vigorous shaking overnight.
Put the glycerol stock cryotube back at -80 °C.
The following day, proceed with bacteria culture scale up and induction of recombinant protein expression as previously described (steps 1–11 of Day 4).
Day 5. Harvest of the IPTG-induced bacteria culture and assessment of protein expression
Culture harvest
Transfer 1 mL of the IPTG-induced culture sample into a 1.5 mL safe-lock Eppendorf tube, centrifuge at 500× g for 20 min, remove the supernatant, and freeze the cell pellet at -20 °C until further analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
Transfer the induced bacteria culture from the 3 L flask to four 500 mL centrifugation tubes.
Centrifuge the tubes at 500× g for 20 min at room temperature.
Discard the supernatant and resuspend each of the four bacteria pellets in 10 mL of bacteria resuspension solution (see Recipes).
Pool bacteria suspensions (4 × 10 mL) into a 50 mL Falcon tube and freeze at -80 °C until further use.
Assessment of protein expression
An assessment of protein expression is recommended before starting the purification processes. The following protocol describes how to analyze protein expression in whole culture samples by SDS-PAGE followed by western blotting. A finer analysis could also be undertaken on lysed bacteria by detecting the recombinant protein in the soluble as well as in the insoluble fractions*.
*Note: To prepare these fractions, the whole culture sample is centrifuged at 500× g for 20 min (4 °C) and the supernatant is discarded. The bacteria pellet is resuspended in PBS (same volume as discarded supernatant). Cells are lysed by repeated freeze/thaw cycles. The bacteria tube is put at -80 °C for 20 min, quickly thawed at 37 °C, and immediately put back at -80 °C for 20 min. Four to five freeze/thaw cycles might be needed for efficient cell lysis. The sample is then centrifuged at 15,000× g for 20 min (4 °C) and the supernatant, representing the soluble fraction is saved. PBS (same volume as the saved supernatant) is added to the insoluble material. Resuspension of the insoluble fraction can be achieved by dynamic pipetting followed by vigorous vortexing.
Thaw on ice the 1.5 mL safe-lock Eppendorf tubes containing the cell pellets of the pre-induced culture sample and of the IPTG-induced culture sample.
Resuspend cell pellets in 1 mL of bacteria resuspension solution.
Prepare two tubes, each containing 5 μL of 4× Laemmli sample buffer, 1 μL of XT reducing agent, and 14 μL of resuspended bacteria (non-induced and IPTG-induced).
Incubate tubes at 90 °C for 5 min.
Prepare a vertical electrophoresis system using a 4%–15% 10-well mini-protean TGX stain-free gel and TGS buffer as running buffer.
Load 20 μL of each sample into the wells and run the gel at 100 V until the blue migration front goes through the gel cast (approximately 2 h).
Uncast the gel and assess protein migration using the Stain Free technology on a ChemiDocTM MP imaging system (activation for 45 s) (Figure 7–left panel).
Transfer the gel onto a 0.2 μm nitrocellulose membrane using a Trans-Blot Turbo Transfer Pack and a Trans-Blot Turbo Transfer system (1.3 A, 25 V, 7 min).
Incubate the membrane in western blot blocking solution (TBS-5% milk; see Recipes) for 1 h at room temperature on a rocking platform.
Discard the blocking solution and incubate the membrane with an HRP-conjugated anti-His antibody (Penta His) diluted 1:10,000 in TBST-5% milk (see Recipes) for 1 h at room temperature on a rocking platform.
Discard the antibody solution and proceed with three consecutive membrane washes of 10 min each in TBST.
Perform a last membrane wash of 10 min in TBS.
Prepare 1 mL of western blotting detection solution (ECLTM Prime) by mixing 500 μL of Prime Luminol Enhancer Solution with 500 μL of Prime Peroxide Solution.
Drain excess of wash buffer from the membrane.
Add 1 mL of western blotting detection solution onto the membrane and incubate at room temperature for 5 min.
Drain excess of detection solution from the membrane.
Capture chemiluminescence signal using a ChemiDocTM MP imaging system (Figure 7–right panel).
Figure 7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein expression in whole bacteria culture. (Left panel) Migration profile of the non-induced and IPTG-induced culture samples. There is no apparent over-expression of scFv2H7-P18F3 (33.4 kDa) in the induced sample when the gel is subjected to Stain Free protein detection (artificial colors). (Right panel) Western blot analysis, performed using an anti-His antibody, revealed that scFv2H7-P18F3 is exclusively expressed in the IPTG-induced sample. MW: molecular weight. kDa: kilo Dalton.
Purification of scFv2H7-P18F3
Sample preparation
Thaw the bacteria suspension at room temperature.
Add 40 mg of lysozyme (1 mg/mL), mix well by vortexing, and keep the tube on ice.
Prime an EmulsiFlex-C5 homogenizer with bacteria resuspension solution at 4 °C.
Load 40 mL of bacteria suspension onto the homogenizer, perform bacteria lysis at a pressure of 14,500 psi, and collect the lysate. Repeat the lysis procedure two more times (total of three passages).
Dissolve one tablet of protease inhibitor in 1 mL of PBS.
Add 1 mL of protease inhibitor to the lysate.
Centrifuge the cell lysate at 20,000× g for 20 min at 4 °C in suitable centrifugation tubes.
Collect the supernatant in a 50 mL tube and filter it at 0.22 using a 50 mL filtering unit connected to a vacuum pump.
Let the vacuum pump on for a few minutes until the filtered solution is fully degassed (no more visible bubbles in the liquid). Indeed, lowering the pressure in the container will result in dissolving gas from the liquid. The sample may then be applied to a column without the risk of air bubbles forming in the column.
Purification by immobilized metal affinity chromatography (IMAC)
The following purification procedures (IMAC and size exclusion chromatography) are performed using a ÄKTA purifier 10 (Cytiva). The process can be easily adapted to other liquid chromatography systems. Working at temperatures ≤ 15 °C is highly recommended to minimize proteolysis.
Prime pump A (inlet A1) and pump B (inlet B1) with ddH2O.
Run the system without any column attached (flow: 1 mL/min) until pressure, conductivity, OD280nm, and OD215nm values are stable.
Pause the system and connect a HisTrap FF (1 mL) column to the system [set up the pressure alarm at total system pressure (~0.1 MPa) + column pressure limit (0.5 MPa)].
Wash the column with 5 mL of ddH2O (flow: 1 mL/min).
Pause the system and place the inlet A1 into a bottle containing 250 mL of IMAC running buffer (see Recipes) and the inlet B1 into a bottle containing 250 mL of IMAC elution buffer (see Recipes).
Equilibrate the column with 5 mL of IMAC running buffer (flow: 1 mL/min; gradient: 0% B).
Pause the system and place the inlet A1 into a 50 mL tube containing the sample to be processed.
Here, pump A is used as a sample pump. Using an independent, dedicated sample pump on newer systems is recommended.
Run the sample through the column in IMAC running buffer, 10 mM imidazole (flow: 1 mL/min; gradient: 2% B) (Figure 8).
Save the flowthrough using a fraction collector.
Pause the system and place the inlet A1 into the bottle containing IMAC running buffer (clean inlet with ddH2O beforehand).
Wash the column with 15 mL of IMAC running buffer, 40 mM imidazole (flow: 1 mL/min; gradient: 8% B) (Figure 8). When purifying a novel type of BMFP, we recommend performing a scouting run prior to large scale purification to establish the best percentage of B to be used during the washing step (the protein of interest should not elute from the column).
Save the wash fraction using the collector.
Elute the column-bound material with 5–7 mL of IMAC running buffer, 250 mM imidazole (flow: 1 mL/min; gradient: 50% B) (Figure 8).
Collect the eluted fraction.
The column can be cleaned/stored following the instructions of the manufacturer and re-used for purification of the same recombinant protein at least three times.
Concentrate the eluted fraction down to 500 μL using a centrifugal concentrator (cut-off: 10 kDa) at 4 °C.
Homogenize the concentrated sample by gently pipetting up and down and transfer it to a pre-chilled 1.5 mL safe-lock Eppendorf tube. The sample is now ready to be further purified by size exclusion chromatography. It is highly recommended to proceed with size exclusion chromatography without delay, to minimize the time the protein spends in imidazole buffer.
Figure 8. Elution profile resulting from immobilized metal affinity chromatography (IMAC). The adjustment of imidazole concentration in the running buffer is performed by mixing the solutions from pump A and B. OD: optical density; AU: arbitrary units; E: elution.
Purification by size exclusion chromatography (gel filtration)
Prime pump A (inlet A1) with PBS (pH 7.2).
Run the system without any column attached (flow: 0.7 mL/min) until pressure, conductivity, OD280nm, and OD215nm values are stable.
Pause the system and connect a Superdex 200 Increase 10/300 GL column to the system [set up the pressure alarm at total system pressure (~0.1 MPa) + column pressure limit (3 MPa)].
Equilibrate the column with PBS (flow: 0.7 mL/min).
Load the sample loop with 500 μL of sample to be processed. Do not pause the system.
Monitor protein elution at OD280nm and collect the fraction corresponding to the peak of interest (elution volume between 14 and 16.5 mL) (Figure 9).
The column is then cleaned/stored following the manufacturer’s instructions.
Check protein concentration.
When measurement of OD280nm is used to estimate protein concentration, the molar extinction coefficient ϵ (M-1cm-1) can be calculated from the protein amino acid sequence using the ProtParam tool (Expasy, Swiss Bioinformatics Resource Portal; https://web.expasy.org/protparam/). Protein concentration can then be calculated using the following formula: [protein]=OD280nm / (ϵ.L), where L is the distance that light travels through the solution (usually 1).
Concentrate the eluted fraction to the desired final concentration using a centrifugal concentrator (cut-off: 10 kDa) at 4 °C (check protein concentration on a regular basis during the concentration process).
Homogenize the concentrated sample, transfer it into pre-chilled 1.5 mL safe-lock Eppendorf tubes, and snap freeze in liquid nitrogen. Protein can then be stored at -80 °C.
The commonly obtained protein yield following this two-step purification process is 0.7–1 mg per liter of bacteria culture. Protein identity and purification quality can also be assessed by SDS-PAGE as previously described in this protocol (Figure 10).
Figure 9. Elution profile resulting from size exclusion chromatography purification. Red bars delimit the peak of interest. To assess the quality of the purification, 100 μL of the eluted fraction containing scFv2H7-P18F3 was re-injected on the Superdex column (analytical sample). OD: optical density; AU: arbitrary units; E: elution.
Figure 10. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified scFv2H7-P18F3. (Left panel) The migration profile of the eluted fraction of interest from size exclusion chromatography revealed a main band between 25 and 37 kDa with very limited amounts of visible contaminants. The gel was subjected to Stain Free protein detection (artificial colors). (Right panel) Western blot analysis performed using an anti-His antibody confirmed the identity of the purified protein. MW: molecular weight; kDa: kilo Dalton.
Recipes
LB medium (1×)
Favour the use of LB powder if possible. LB medium can alternatively be prepared according to the following recipe: 1% tryptone, 0.5% yeast extract, and 1% NaCl. Adjust to pH 7.0 and sterilize by autoclaving. Store at room temperature.
SOC medium (1×)
Favour the use of super optimal broth with catabolite repression (SOC) medium if possible. SOC medium can alternatively be prepared according to the following recipe: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose. Adjust to pH 7.0 and sterilize by autoclaving. Store at room temperature.
Kanamycin stock solution (50 mg/mL)
For 10 aliquots of 1 mL, weigh 500 mg of kanamycin sulfate powder and dissolve in 10 mL of ddH2O. Filter-sterilize using a syringe filter (0.2 μm pore size). Distribute 1 mL in ten 1.5 mL safe-lock Eppendorf tubes and freeze the aliquots at -20 °C.
50% glycerol solution
For 100 mL, mix 50 mL of sterile glycerol with 50 mL of sterile ddH20. Store at room temperature.
Bacteria resuspension solution/IMAC running buffer
50 mM Tris and 500 mM NaCl. Adjust to pH 7.2*. Store at room temperature.
IMAC elution buffer
50 mM Tris, 500 mM NaCl, and 500 mM imidazole. Adjust to pH 7.2*. Filter at 0.2 μm and degas solution. Store at room temperature.
*Note: The pH of the solution should be at least 1 pH unit lower or higher than the theoretical isoelectric point (pI) of the protein of interest to minimize protein aggregation during the purification process. The theoretical pI can be calculated from the protein amino acid sequence using the ProtParam tool (Expasy, Swiss Bioinformatics Resource Portal;https://web.expasy.org/protparam/). The theoretical pI of the BMFP scFv2H7-P18F3 is 9.23.
Tris buffer saline (TBS)
50 mM Tris-Cl and 150 mM NaCl. Adjust to pH 7.6. Store at room temperature.
Tris buffer saline tween (TBST)
50 mM Tris-Cl and 150 mM NaCl. 0.5% Tween 20. Adjust to pH 7.6. Store at room temperature.
Western blot blocking solution (TBS-5% milk)
TBS supplemented with 5 g per 100 mL of low-fat milk. Filter at 0.2 μm.
Western blot antibody dilution solution (TBST-5% milk)
TBST supplemented with 5 g/100 mL of low-fat milk. Filter using a syringe filter (0.2 μm pore size).
Phosphate buffer saline (PBS)
2.7 mM KCl, 138 mM NaCl, 1.5 mM KH2PO4, and 8 mM Na2HPO4. Adjust to pH 7.2. Filter at 0.2 μm. Store at room temperature.
Acknowledgments
This work was supported by SATT IDF-Innov, Paris, France (to B.G. and A.C.); Institut National de la Santé et de la Recherche Médicale (INSERM), France (to B.G., J.L.T. and A.C.); Institut National de la Transfusion Sanguine (INTS), Paris, France (to B.G. and A.C.).
Competing interests
BG and AC are inventors on a patent application related to this protocol filed by Institut National de la Transfusion Sanguine, Centre National de la Recherche Scientifique (CNRS), Inserm, Université de Paris (now universtité Paris Cité) (no. WO2017103020A1, filed 25 December 2015, published 22 June 2017). The authors declare that they have no other competing interests.
References
Denoncin, K. and Collet, J. F. (2013). Disulfide bond formation in the bacterial periplasm: major achievements and challenges ahead. Antioxid Redox Signal 19(1): 63-71.
Gamain, B., Brousse, C., Rainey, N. E., Diallo, B. K., Paquereau, C. E., Desrames, A., Ceputyte, J., Semblat, J. P., Bertrand, O., Gangnard, S., et al. (2022). BMFPs, a versatile therapeutic tool for redirecting a preexisting Epstein-Barr virus antibody response toward defined target cells. Sci Adv 8(6): eabl4363.
Hwang, Y. C., Lu, R. M., Su, S. C., Chiang, P. Y., Ko, S. H., Ke, F. Y., Liang, K. H., Hsieh, T. Y. and Wu, H. C. (2022). Monoclonal antibodies for COVID-19 therapy and SARS-CoV-2 detection. J Biomed Sci 29(1): 1.
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: 56.
Voge, N. V. and Alvarez, E. (2019). Monoclonal Antibodies in Multiple Sclerosis: Present and Future. Biomedicines 7(1).
Zahavi, D. and Weiner, L. (2020). Monoclonal Antibodies in Cancer Therapy. Antibodies 9(3): 34.
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Abstract
Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons.
Key features
Preparation of ex vivo paired cultured hippocampal neurons.
Bi-directional electrophysiological recordings of synaptic transmission and plasticity.
Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel α2δ-2 subunit).
Graphical overview
Keywords: Primary hippocampal culture Paired patch clamp recording Synaptic transmission Calcium channel α2δ subunit Viral infection Electrophysiology Short-term plasticity Postsynaptic currents Paired pulse facilitation and depression.
Background
Signal transmission between neurons occurs via neurotransmitter release into the synaptic cleft. The temporal and spatial relation of pre- and post-synaptic firing modulates the strength of synaptic connections between neurons (Deperrois and Graupner, 2020). Two types of synaptic activity can be registered at the single cell level. First, miniature excitatory or inhibitory postsynaptic currents (mEPSC or mIPSC) appear as result of spontaneous local fusion of single synaptic vesicles. Second, excitatory and inhibitory postsynaptic currents (EPSC or IPSC) can be recorded in response to action potential firing by presynaptic glutamatergic or GABAergic neurons, respectively. Analysis of miniature postsynaptic potentials provides information on the amount and density of synaptic connections (frequency) as well as the postsynaptic receptor abundance (amplitude) and hence helps to study elementary synapse properties. However, higher levels of synaptic function, including the responses of synapses in regard to action potential firing as well as short- and long-term adaptations of synaptic strength, require the analysis of evoked synaptic transmission. For example, paired-pulse stimulation protocols can serve as a basic model for studying short-term synaptic plasticity ex vivo (Bouteiller et al., 2010). Evoked synaptic transmission and plasticity in specific neuronal pathways typically can be studied in brain slices (Wang and Baudry, 2019). Alternatively, synaptic transmission and plasticity can be studied in cultured neurons, such as by employing optogenetic activation of neuronal cell populations (Barral and Reyes, 2017). In an acute brain slice, which is the standard model for the electrophysiological analysis of synaptic functions, presynaptic stimulation and postsynaptic responses can only be analyzed in one direction. However, as synaptic plasticity involves the possibility of changes in both pre- and post-synaptic components, one-directional measurements limit the study of mechanisms involved in modulating plasticity.
Here we describe a protocol for culturing simple networks of paired hippocampal neurons for the be-directional electrophysiological analysis of synaptic functions. This cellular ex vivo model has the following advantages: first, it allows the easy identification of the innervated cells. In classical neuronal cell cultures employing dispersed neurons, this is inherently difficult due to excessive branching of the axons and the possibility of hetero-synaptic innervation of the target neuron. Second, due to the defined simple network all synapses are formed between the paired neurons. This results in increased amplitudes of postsynaptic responses and hence allows the reliable detection of changes in postsynaptic receptor function. Third, both cells of the cultured paired network can function as presynaptic (stimulated) and postsynaptic (innervated) neurons. Hence, this method allows recording synaptic transmission bi-directionally. This is particularly relevant in the context of genetic manipulations of one of the two paired neurons: as one of the paired neurons can be genetically modified by overexpression or knockdown of specific proteins, bi-directional stimulation-protocols allow analyzing pre- and post-synaptic consequences in comparison with wild-type synaptic connections in the same neuronal network. As a proof of principle, we altered the expression of α2δ proteins, which on the one hand act as auxiliary subunits of voltage-gated calcium channels (Geisler et al., 2015; Ablinger et al., 2020; Dolphin and Obermair, 2022), and on the other hand as critical synaptic organizers (Eroglu et al., 2009; Geisler et al., 2019; Schöpf et al., 2021; Ablinger et al., 2022). Hence, we employed cultured paired hippocampal neurons to investigate the role of a splice variant of the α2δ-2 isoform in the trans-synaptic regulation of synapse formation and synaptic transmission, including short-term synaptic plasticity.
Materials and reagents
Animals
Timed-pregnant wild-type mice (BALB/c, gestational age 16–17 days; Charles River Laboratories, Sulzfeld, Germany).
Biological materials
Lentiviral particles, carrying RNA encoding for the α2δ-2_ΔE23 splice variant and soluble eGFP as fluorescent marker (Geisler et al., 2019). Lentiviral particles were generated as previously described (Nasri et al., 2014; Benskey and Manfredsson, 2016).
Critical: Lentiviruses are classified as a biosafety level 2 (BSL-2) organisms.
Materials
Surgical Scissors - Sharp-Blunt, Straight 14.5 cm (Fine Science Tools, catalog number: 14001-14)
Tissue Forceps - Slim1x2 Teeth 10 cm (Fine Science Tools, catalog number: 11023-10)
Fine Scissors - Sharp, Curved 10.5 cm (Fine Science Tools, catalog number: 14061-10)
Fine Scissors - Sharp, Straight 10.5 cm (Fine Science Tools, catalog number: 14060-10)
Dumont #5 Standard forceps (Fine Science Tools, catalog number: 11251-30)
Dumont #5 Biology forceps (Fine Science Tools, catalog number: 11252-30)
Vannas-Tübingen Spring Scissors (Fine Science Tools, catalog number: 15004-08)
18 mm glass coverslips (Marienfeld Superior, catalog number: 0111580)
Rack for coverslips (custom build, Institute of Physiology, Medical University Innsbruck, Austria)
PTFE dish (Carl Roth, catalog number: K837.1)
12.5 cm filter paper (Carl Roth, catalog number: AP86.1)
Hemacytometer (Neubauer, catalog number: Brand 717805)
72 μm nylon mesh (Falcon, catalog number: 352350)
T75 flask (Falcon, catalog number: 353810)
Transfer pipette 3.5 mL (Sarstedt, catalog number: 86.1171.001)
15 mL centrifuge tube (Falcon, catalog number: 352070)
50 mL centrifuge tube (Falcon, catalog number: 352096)
60 mm plastic Petri dish (Falcon, catalog number: 353802)
60 mm Primaria plastic Petri dish (Falcon, catalog number: 353004)
15 cm glass Petri dish (Duran, catalog number: 237555201)
Pasteur pipette (Assistent, catalog number: 40567002)
5 mL serological pipette (Sarstedt, catalog number: 86.1253.001)
10 mL serological pipette (Sarstedt, catalog number: 86.1254.001)
25 mL serological pipette (Sarstedt, catalog number: 86.1685.001)
1.5 mL miniature spray (Rene Lezard)
Borosilicate glass with filament (Sutter Instrument, model: BF150-75-10)
2.5% trypsin (10×) (Gibco, catalog number: 15090-046)
0.5% Trypsin-EDTA (10×) (Gibco, catalog number: 15400-054)
B-27 supplement (50×) (Gibco, catalog number: 17504-044)
Glutamax (Gibco, catalog number: 35050-038)
Horse serum (Gibco, catalog number: 16050-122)
PenStrep (Penicillin-Streptomycin) (Gibco, catalog number: 15140-122)
MEM (Gibco, catalog number: 41090-028)
Neurobasal medium (Gibco, catalog number: 21103-049)
HBSS (10×) (Gibco, catalog number: 14180-046)
HEPES 1M solution (Gibco, catalog number: 15630-056)
Poly-L-lysine (Sigma, catalog number: P2636)
Ara-C (Sigma, catalog number: C6645)
DNase (Sigma, catalog number: DN-25)
Sodium pyruvate (Sigma, catalog number: P2256)
Paraffin (Carl Roth, catalog number: X880.1)
Gelatine (Fluka, catalog number: 48722)
Nitric acid (Carl Roth, catalog number: 4625.2)
Glucose (Carl Roth, catalog number: HN06.3)
Boric acid (Sigma, catalog number: B6768)
Sodium tetraborate decahydrate (Borax) (Sigma, catalog number: B9876)
Sodium chloride (NaCl)
Potassium chloride (KCl)
Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239.2)
Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma, catalog number: M0250)
Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.3)
Potassium hydroxide (KOH) (Carl Roth, catalog number: 6751.1)
Gluconic acid, potassium salt (K-gluconate) (Carl Roth, catalog number: 4621.1)
HEPES (Carl Roth, catalog number: 6763.1)
EGTA (Sigma, catalog number: E3889)
ATP, magnesium salt (Sigma, catalog number: A9187)
GTP, sodium salt (Sigma, catalog number: G8877)
Solutions
Pyruvate solution, 100 mM, 50 mL (see Recipes)
1% gelatine solution, 50 mL (see Recipes)
HBSS, 500 mL (see Recipes)
Glia medium, 500 mL (see Recipes)
Neuronal maintenance medium, 200 mL (see Recipes)
Neuronal plating medium, 200 mL (see Recipes)
1% DNase solution, 100 mL (see Recipes)
Sodium borate buffer, 500 mL (see Recipes)
Poly-L-lysine solution, 1 mg/mL (see Recipes)
EGTA solution, 0.5M, 1 mL (see Recipes)
Extracellular solution, 100 mL, adjust pH 7.4 with NaOH (see Recipes)
Intracellular solution, 20 mL, adjust to pH 7.2 with KOH (see Recipes)
Software
Anvi’o v7 (https://merenlab.org/software/anvio/)
Snakemake (https://snakemake.readthedocs.io/en/stable/)
Installing Anvi’o
Conda setup: If the conda is not installed in the system, it is necessary to open a terminal such as iTerm.
Command:
conda install
To verify whether you already have conda installed, copy and paste the following command into your terminal:
Command:
conda --version
Always make sure that you work in an up-to-date conda environment by using the following command:
Command:
conda update conda
Anvi’o environment setup
Create a new conda environment using the command:
Command:
conda create -y --name anvio-7.1 python=3.6
Then, activate it using the command:
Command:
conda activate anvio-7.1
Installing Anvi’o
The first step is to download the python source package for the Anvi’o release using the following command:
Command:
curl -L https://github.com/merenlab/anvio/releases/download/v7.1/anvio-7.1.tar.gz \
--output anvio-7.1.tar.gz
Then, use the following command to install Anvi’o:
Command:
pip install anvio-7.1.tar.gz
Users should note that the installation of Anvi’o is user friendly but may take a long time to finish and is computationally intensive.
Data availability
The data can be accessed at https://figshare.com/s/35ea294e2671d75f1d5c and https://github.com/Bio-protocol/bioprotocol_2104071.
Case study
Input data
Anvi’o workflows help users to:
Streamlining standard repetitive steps of ‘omics data analysis like assembly, mapping, mapping results profiling, annotation of functions/taxonomy, and generating Anvi’o databases in a scalable form.
Ask biological questions about the data.
Describe the data and the results easily to the scientific community.
Anvi’o uses the program anvi-run-workflow to run the workflows. For a particular workflow, the program will help users to prepare the config file.
The following code asks the program what workflows it knows:
Command
anvi-run-workflow --list-workflows
Available workflows ....: contigs, metagenomics, pangenomics, phylogenomics, trnaseq
After you have decided the process you wish to use, the config file allows you to change the parameters and order of steps associated with that process. Even if you are satisfied with all the default parameters, the config file is required for all workflows. Users should ensure that the config files are proper, and preparing a config file for a particular workflow could be challenging. The following code will help users to generate a config file:
Command
anvi-run-workflow -w WORKFLOW-NAME \
--get-default-config OUTPUT-FILE-NAME
The --get-default-config will generate a default config file for a workflow that you can modify. Configurable flags and parameters will be contained in this file. You can either leave as is any parameters that you do not intend to change, or you can remove those that you do want to change from your config file to make it shorter and cleaner.
There are three configurations in the config file:
General workflow parameters: You will need a name for your project and the workflow mode you want to employ.
Parameters: Parameters that are exclusively applicable to a single rule, such as the Anvi'o profiling steps’ minimum contig length. Each program has unique parameters. Users should make sure to use parameters that appear in the config file that would be identical to the names used in the particular program. For instance, if there are multiple ways to use adjustable parameters or arguments, users should use the longer one. As an example,anvi-run-hmmsare able to accept with-Hor--hmm-profile-dirparameters that specify the directory path of HMM profile. However, users are only allowed to use--hmm-profile-dirin the config file.
Names of the output directory: This regards how Anvi’o will deal with output directories and files.
Samples.txt
The samples.txt file is for associating sample names with raw sequencing reads. There should be three or four columns (plus the optional groups column) in the samples.txt, with each column separated from the others by a TAB character. The following column names should be included in the header:
Sample: A name for each of your metagenomic samples.
r1 and r2: These two columns include the path (which could be relative or absolute; absolute paths are always preferred) to the FASTQ files corresponding to the sample.
It may additionally include the following column as an option:
Group: While binning genomes from metagenomic assemblies, one of the strategies is to combine numerous samples. This column's function is to specify which samples will be co-assembled. This is an optional column; if it is not present in the samples.txt file, each sample will be assembled independently. Only the samples utilized for the co-assembly would be mapped to the resultant assembly by default. You can co-assemble groups of samples, but you must then map all samples to each assembly.
Workflow
Raw paired-end sequencing reads for shotgun metagenomes are the default entry point into the metagenomics workflow. The workflow's default endpoint is a merged profile database ready for bin refinement, as well as an annotated Anvi'o contigs database. The steps in the workflow are as follows:
Using illumina-utils, quality-check metagenomic short reads and generate a thorough report for the outcomes of this step: quality-check to be performed by removing the low quality reads according to the criteria mentioned in Minoche et al. (2011), the combination of B-tail trimming and passed chastity filter to be used, and reads to be removed that contained uncalled bases.
Select programs for generating taxonomic profiles of short reads. These profiles are imported to individual databases of profiles that are available in the merged profile database of Anvi’o.
Using megahit and/or idba_ud and/or metaspades, assemble quality-filtered metagenomic reads.
Using anvi-gen-contigs-database, create an Anvi'o contigs database using assembled contigs. The contigs database should have annotations of the functions, taxonomy, and HMMs.
Using bowtie2, map short reads from metagenomes to contigs and then generate indexed and sorted BAM files.
Using anvi-profile to produce single Anvi'o profiles from individual BAM files.
Using anvi-merge, merge to generate single Anvi'o profiles.
You will only need a samples.txt file and a few FASTQ files. We will go through a mock example with three small metagenomes in this section. A limited number of reads were selected to create these metagenomes. The following samples.txt file can be found in your working directory:
samplegroupr1r2
P1G01M-1_S21_L001_R1_001.fastq.gzM-1_S21_L001_R2_001.fastq.gz
P2G02M-2_S22_L001_R1_001.fastq.gzM-2_S22_L001_R2_001.fastq.gz
P3G03M-3_S23_L001_R1_001.fastq.gzM-3_S23_L001_R2_001.fastq.gz
This file details the raw paired-end reads locations for the samples and 'groups'. The default name is samples.txt for the samples_txt file, but you can change it in the config file.
Let's have a look at the config file config-megahit.json in the working directory.
{
"workflow_name": "metagenomics",
"config_version": “2”,
"samples_txt": "samples.txt",
"megahit": {
"--min-contig-len": 1000,
"--memory": 0.4,
"threads": 7,
"run": true,
}
}
Every customizable parameter will be given a default value. We normally start with a default config file and delete every line that we do not want to keep. We have everything now to start. Let’s generate a workflow graph at this stage. The following code will generate a workflow graph:
Command
anvi-run-workflow -w metagenomics \
-c config-megahit.json \
--save-workflow-graph
We can now start the workflow:
Command
anvi-run-workflow -w metagenomics \
-c config-megahit.json
After completing all the steps in this pipeline, users will be able to utilize these generated profiles for downstream work like manual refining and functional analyses.
Result interpretation
This workflow explained the steps from quality control to mapping for generation of metagenome-assembled genomes. A general introduction was provided about the config and samples.txt files to connect raw sequencing reads with sample details. Anvi’o was coupled with snakemake workflows to generate profiles that could be used for downstream analyses.
Discussion
The concept of using snakemake with Anvi’o was to have better documentation and reproducibility of the entire work. Snakemake also allows the Anvi’o workflow to be more specific using config files. There are opportunities to repurpose this existing workflow to user’s own projects.
Acknowledgments
Soumyadev Sarkar acknowledges the National Science Foundation EPSCoR for his research grant. This protocol is based on using Anvi’o (Eren et al., 2015 and 2021). This material is based upon work supported by the National Science Foundation under Award No. OIA‐1656006 and matching support from the State of Kansas through the Kansas Board of Regents.
Competing interests
The authors declare no competing interests.
References
Eren, A. M., Esen, O. C., Quince, C., Vineis, J. H., Morrison, H. G., Sogin, M. L. and Delmont, T. O. (2015). Anvi'o: an advanced analysis and visualization platform for 'omics data. PeerJ 3: e1319.
Eren, A. M., Kiefl, E., Shaiber, A., Veseli, I., Miller, S. E., Schechter, M. S., Fink, I., Pan, J. N., Yousef, M., Fogarty, E. C., et al. (2021). Community-led, integrated, reproducible multi-omics with anvi'o. Nat Microbiol 6(1): 3-6.
Köster, J. and Rahmann, S. (2012). Snakemake--a scalable bioinformatics workflow engine. Bioinformatics 28(19): 2520-2522.
Minoche, A. E., Dohm, J. C. and Himmelbauer, H. (2011). Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome Biol 12(11): R112.
Mölder, F., Jablonski, K. P., Letcher, B., Hall, M. B., Tomkins-Tinch, C. H., Sochat, V., Forster, J., Lee, S., Twardziok, S. O., Kanitz, A., et al. (2021). Sustainable data analysis with Snakemake. F1000Res 10: 33.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Plant Science > Plant molecular biology > DNA
Systems Biology > Genomics > Functional genomics
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4,684 | https://bio-protocol.org/en/bpdetail?id=4684&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Visualizing, Binning, and Refining of Metagenome-assembled Genomes (MAGs) with Anvi’o
SS Soumyadev Sarkar
TR Tanner Richie
SL Sonny T. M. Lee
Published: May 20, 2023
DOI: 10.21769/BioProtoc.4684 Views: 327
Reviewed by: Varun KesherwaniHassan Rasouli Anonymous reviewer(s)
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Abstract
High throughput ‘omics technologies generate huge datasets that need to be properly analyzed in order to decipher the biological implications. The workflow of handling such datasets must be user friendly to facilitate rapid analysis. Here, we demonstrate the use of the Anvi’o workflow, which is a visualization platform that allows for advanced analysis of metagenomics data. In this protocol, we provide the pre-packaged plant-microbiome dataset. Then, we use the dataset to visualize and perform manual binning and refinement of metagenome-assembled genomes (MAGs). Anvi’o works with an easy-to-use interface and also helps users to test and implement research ideas in a timely manner.
Keywords: MAGs Anvi’o Metagenomics Visualization Binning Refining Microbiome
Background
Shotgun metagenomics is a popular approach for studying microbial community, diversity, and functional potential (Handelsman et al., 1998; Sogin et al., 2006). These types of high throughput sequencing technologies generate a huge amount of metagenomic data that need to be analyzed properly to understand the biological implications (D'Argenio, 2018). Assembling short reads into contigs is essential to improve annotations, and the compatible genomic binning technique can further link unconnected contigs into biologically meaningful units (Tyson et al., 2004; Venter et al., 2004). Metagenomic binning is the process by which metagenomic sequences can be grouped using the organisms of origin. This leads to reconstruction of genomes, which can be used in downstream analyses (Turaev and Rattei, 2016; Wang and Jia, 2016; Quince et al., 2017; Nissen et al., 2021). With existing automated software pipelines, studies have successfully implemented assembly and binning approaches to draft genomes that are near complete in nature (Hess et al., 2011; Alneberg et al., 2014; Wu et al., 2014; Kang et al., 2015; Raveh-Sadka et al., 2015). However, the existing pipelines do not provide the contigs distribution across samples (Sharon et al., 2013; Alneberg et al., 2014). At this juncture, there is a need for an easy visualization interface–based workflow that is competent for analyzing such large metagenomic datasets. Anvi’o (advanced analysis and visualization platform for ‘omics data) provides an easy management of contigs, where both manual or automatic genome bins identifications and curations are possible. This pipeline is also capable of generating a unified display of inferred taxonomy and GC-content with the contig numbers in different samples (Eren et al., 2015).
Software
Anvi’o v7 (https://merenlab.org/software/anvio/)
Installing Anvi’o:
Conda setup: If the conda is not installed in the system, it is necessary to open a terminal such as iTerm.
Command:
conda install
To verify whether you already have conda installed, copy and paste the following command into your terminal:
Command:
conda --version
Always make sure that you work in an up-to-date conda environment by using the following command:
Command:
conda update conda
Anvi’o environment setup
Create a new conda environment using the command:
Command:
conda create -y --name anvio-7.1 python=3.6
Then, activate it using the command:
Command:
conda activate anvio-7.1
Installing Anvi’o
The first step is to download the python source package for the Anvi’o release using the following command:
Command:
curl -L https://github.com/merenlab/anvio/releases/download/v7.1/anvio-7.1.tar.gz \
--output anvio-7.1.tar.gz
Then, use the following command to install Anvi’o:
Command:
pip install anvio-7.1.tar.gz
The users should note that the installation of Anvi’o is user friendly but may take a long time to finish and is computationally intensive.
Updating Anvi’o databases
If the Anvi’o databases are not compatible with the latest versions of Anvi’o, there are options to update the Anvi’o databases.
The safest option is to use:
Command:
anvi-migrate --migrate-dbs-safely *.db
When using this option, Anvi’o will generate a backup of a copy of each database. If there is an error during the migration, the Anvi’o will let the users know about what went wrong and will be able to restore the original database from the copy it made.
Another option is to use:
Command:
anvi-migrate --migrate-dbs-quickly *.db
This option does not create any backup files but might be useful when there are a lot of databases to migrate.
Data availability
The data can be accessed at https://figshare.com/s/acc45cb6fb5cbd819d69 and https://github.com/Bio-protocol/bioprotocol_2104072.git
Case study
Downloading the pre-packaged plant-microbiome dataset
The plant-microbiome data pack can be downloaded from https://figshare.com/s/acc45cb6fb5cbd819d69.
Following are some details about the plant-microbiome data pack:
In the dataset directory, you will see that the data pack contains an Anvi'o merged profile database (that describes six metagenomes: three from plants and three from fecal samples), an Anvi'o contigs database, and additional extra data that are required by various sections in this tutorial. Here are some basic descriptions of several of these files, as well as how they were created:
The profile and contigs databases. We produced the Anvi'o contigs database utilizing the program anvi-gen-contigs-database. This Anvi'o database keeps all the information that is associated with the contigs: k-mer frequencies for each contig, open reading frames positions, taxonomic and functional annotation of genes, among others. We also used the tool anvi-profile to create a merged Anvi'o profile database. Anvi'o profile databases store sample-specific information on contigs. Each profile database is linked to a contigs database, and Anvi'o uses the program anvi-merge to merge single profiles that are linked to the same contigs database into an Anvi'o merged profile.
Single-copy core genes in contigs. Among the contigs, we utilized the program anvi-run-hmms to find single-copy core genes for Bacteria, Archaea, and Eukarya, as well as ribosomal RNA sequences. The contigs database stores all these results as well. This data enables us to learn the completeness and redundancy estimations of freshly detected bins using the interactive interface. Note that if all single-copy core genes for a given domain are discovered once in the chosen bin, the completion rate is 100% and the redundancy rate is 0%. The redundancy score will rise if a few genes are discovered several times. In case a few genes are missing, the completion value will be reduced.
Assigning functions towards genes. We performed anvi-run-ncbi-cogs and anvi-run-kegg-kofams and stored gene functions results in the contigs database.
Genome-resolved metagenomics
This tutorial uses a plant-microbiome dataset to discuss genome-resolved metagenomics (with a focus on manual binning). You will be able to do the following by the end of this tutorial:
Learn how to use the interactive binning interface.
Examine contigs in relation to their metagenomic signal.
Perform manual binning to characterize bins.
Summarize the findings of manual binning for use in subsequent studies.
Curate bins manually for the purpose of quality control.
FASTA and BAM files of the contigs are used in a typical Anvi'o genome-resolved metagenomic approach. In this tutorial, we will start at the stage in the workflow where you have generated Anvi'o contigs and profile databases using your FASTA and BAM files (Figure 1).
Figure 1. Workflow for the users to follow to obtain the results for this protocol
Let us look at the merged profile database for the plant-microbiome dataset metagenome using the files in the data pack directory.
The following anti-interactive command on the merged profile database will initiate the Anvi’o interactive interface:
Command:
anvi-interactive -p PROFILE.db -c CONTIGS.db -C concoct
After you click “draw,” the Anvi’o interactive interface should greet you with the following display (Figure 2):
Figure 2. The Anvi’o interactive interface, showing the merged profile of three plant and fecal metagenomes
Close the window, return to the terminal, and hit CTRL + C to stop the server.
Taking a look at the binning findings
We are interested in putting our metagenomes into context with the genomes we have retrieved through binning. Comprehending the quantitative distribution patterns of the genomes in a collection, obtaining a table of function discovered, or summarizing our bins as separate FASTA files are all crucial for the downstream analysis of any binning workflow. We used anvi-summarize to summarize any collection saved in a Anvi'o profile database. You will have a static HTML page that you can visualize on any computer (Figure 3).
Command:
anvi-summarize -p PROFILE.db \
-c CONTIGS.db \
-C concoct \
-o SUMMARY
Figure 3. Summary of bins
Manual curation to refine individual MAGs
We can now go through a round of manual binning. A couple of binning pointers:
It is not necessary to bin all contigs. Instead, look for bins that correlate to a real genome with high completion values.
Avoid bins with redundancy higher than 10%. Those are most likely contaminated.
In this profile, we identified 69 bins after the auto-binning protocol. In Anvi’o, a collection describes one or more bins. Each bin describes single or multiple contigs. Please keep all the bins together as a collection. You can name your collection whatever you like. Individual bins can be visualized and refined if necessary to improve the quality of the MAGs collection. We used the program anvi-refine for this.
Command:
anvi-refine -p PROFILE.db \
-c CONTIGS.db \
-C concoct \
-b Bin_34
Contigs from a single bin are now displayed in the interactive interface (Figure 4A). During the curation step, several clustering algorithms can be used to detect outliers and determine if they are contaminants. We can select and remove the contigs that we do not want to keep in the bin, and then we can store the updated set of contigs in the database using the “bins” panel. In this example of MAG (Bin_34) we curated, we removed four contigs (Figure 4B):
Figure 4. Interactive interface displaying contigs from a single bin. A. Display of contigs from a single bin before manual curation. B. Curation of Bin_34, removing four contigs to achieve a higher level of completion and redundancy. Henceforth, we will rename Bin_34 as a metagenome-assembled genome (MAG).
Figure 4B can be obtained by clicking the branches to add into separate groups. After refining, the contamination reduces to much lower levels. The users can also refer to the video provided with the protocol to follow the steps required to curate the bin. The collection will be changed by storing the refined new bin in the database. This is a straightforward example. However, improving a given MAG can take hours in some circumstances.
Renaming bins in your collection
From the summary file, you can see that bin names are currently arbitrary, and we frequently find it helpful to impose some order at this step. This is a particularly beneficial method when the goal is to eventually merge numerous binning efforts. We use the tool anvi-rename-bins to rename bins:
Command:
anvi-rename-bins -p PROFILE.db \
-c CONTIGS.db \
--collection-to-read concoct \
--collection-to-write MAGs \
--call-MAGs \
--prefix Soil \
--report-file rename-bins-report.txt
With those parameters, a new MAG collection will be formed, in which (1) bins with a completion >70% are designated as MAGs (metagenome-assembled genomes), and (2) bins and MAGs are given a prefix and renamed based on the difference between redundancy and completion. The users can customize the parameters involving the completion and redundancy. We recommend bins with completions >70% and redundancy <10%.
At this point we can summarize the new collection using the program anvi-summarize
Command:
anvi-summarize -p PROFILE.db \
-c CONTIGS.db \
-C MAGs \
-o SUMMARY_AFTER_RENAME
Now, double-click on the file index.html to visualize the outputs that are currently in the newly created folder SUMMARY_MAGs (Figure 5).
We get the following MAGs:
Figure 5. Summary of the new collection of MAGs. The takeaway point here is that it is possible to enhance the results through manual refining when automatic binning techniques may produce poorly identified bins.
Result interpretation
Proper manual refinement of MAGs is necessary to elucidate meaningful biological implications. In this protocol, we used a plant-microbiome dataset to identify 69 bins and then curated the bins manually to remove the outliers and contaminants. It is recommended to perform manual refining after automatic binning to recover higher quality MAGs.
Discussion
Anvi’o is an easy-to-use interface that enables the user to visualize, perform binning, and refine MAGs. This protocol enables users to carry out the entire workflow and provides a scope to improvise the method for other datasets.
Acknowledgments
Soumyadev Sarkar acknowledges the National Science Foundation EPSCoR for his research grant. This protocol is based on using Anvi’o (Eren et al., 2015 and 2021). This material is based upon work supported by the National Science Foundation under Award No. OIA‐1656006 and matching support from the State of Kansas through the Kansas Board of Regents.
Competing interests
The authors declare no competing interest.
References
Alneberg, J., Bjarnason, B. S., de Bruijn, I., Schirmer, M., Quick, J., Ijaz, U. Z., Lahti, L., Loman, N. J., Andersson, A. F. and Quince, C. (2014). Binning metagenomic contigs by coverage and composition. Nat Methods 11(11): 1144-1146.
D'Argenio, V. (2018). The High-Throughput Analyses Era: Are We Ready for the Data Struggle? High Throughput 7(1).
Eren, A. M., Esen, O. C., Quince, C., Vineis, J. H., Morrison, H. G., Sogin, M. L. and Delmont, T. O. (2015). Anvi'o: an advanced analysis and visualization platform for 'omics data. PeerJ 3: e1319.
Eren, A. M., Kiefl, E., Shaiber, A., Veseli, I., Miller, S. E., Schechter, M. S., Fink, I., Pan, J. N., Yousef, M., Fogarty, E. C., et al. (2021). Community-led, integrated, reproducible multi-omics with anvi'o. Nat Microbiol 6(1): 3-6.
Handelsman, J., Rondon, M. R., Brady, S. F., Clardy, J. and Goodman, R. M. (1998). Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5(10): R245-249.
Hess, M., Sczyrba, A., Egan, R., Kim, T. W., Chokhawala, H., Schroth, G., Luo, S., Clark, D. S., Chen, F., Zhang, T., et al. (2011). Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331(6016): 463-467.
Kang, D. D., Froula, J., Egan, R. and Wang, Z. (2015). MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ 3: e1165.
Nissen, J. N., Johansen, J., Allesoe, R. L., Sonderby, C. K., Armenteros, J. J. A., Gronbech, C. H., Jensen, L. J., Nielsen, H. B., Petersen, T. N., Winther, O. et al. (2021). Improved metagenome binning and assembly using deep variational autoencoders.Nat Biotechnol 39(5): 555-560.
Quince, C., Walker, A. W., Simpson, J. T., Loman, N. J. and Segata, N. (2017). Shotgun metagenomics, from sampling to analysis. Nat Biotechnol 35(9): 833-844.
Raveh-Sadka, T., Thomas, B. C., Singh, A., Firek, B., Brooks, B., Castelle, C. J., Sharon, I., Baker, R., Good, M., Morowitz, M. J. et al. (2015). Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development. Elife 4: e05477.
Sogin, M. L., Morrison, H. G., Huber, J. A., Mark Welch, D., Huse, S. M., Neal, P. R., Arrieta, J. M. and Herndl, G. J. (2006). Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci U S A 103(32): 12115-12120.
Sharon, I., Morowitz, M. J., Thomas, B. C., Costello, E. K., Relman, D. A. and Banfield, J. F. (2013). Time series community genomics analysis reveals rapid shifts in bacterial species, strains, and phage during infant gut colonization. Genome Res 23(1): 111-120.
Turaev, D. and Rattei, T. (2016). High definition for systems biology of microbial communities: metagenomics gets genome-centric and strain-resolved.Curr Opin Biotechnol 39: 174-181.
Tyson, G. W., Chapman, J., Hugenholtz, P., Allen, E. E., Ram, R. J., Richardson, P. M., Solovyev, V. V., Rubin, E. M., Rokhsar, D. S. and Banfield, J. F. (2004). Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428(6978): 37-43.
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A., Wu, D., Paulsen, I., Nelson, K. E., Nelson, W., et al. (2004). Environmental genome shotgun sequencing of the Sargasso Sea. Science 304(5667): 66-74.
Wang, J., and Jia, H. (2016). Metagenome-Wide Association Studies: Fine-Mining the Microbiome. Nat Rev Microbiol 14(8): 508-522.
Wu, Y. W., Tang, Y. H., Tringe, S. G., Simmons, B. A. and Singer, S. W. (2014). MaxBin: an automated binning method to recover individual genomes from metagenomes using an expectation-maximization algorithm. Microbiome 2: 26.
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Systems Biology > Genomics > Functional genomics
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4,685 | https://bio-protocol.org/en/bpdetail?id=4685&type=0 | # Bio-Protocol Content
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Detecting Photoactivatable Cre-mediated Gene Deletion Efficiency in Escherichia coli
YK Yuta Koganezawa
YW Yuichi Wakamoto
MS Moritoshi Sato
MU Miki Umetani
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4685 Views: 863
Reviewed by: Chiara AmbrogioMatthew SwireAdriano Bolondi
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Original Research Article:
The authors used this protocol in eLIFE May 2022
Abstract
Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion.
Keywords: Gene deletion Genotype–phenotype mapping Optogenetics Escherichia coli Antibiotic resistance
Background
Analyzing the phenotypes of gene-deleted cell strains or multicellular organisms is the cornerstone of genetics studies. The functional roles of various genes in each organism can be deduced from the phenotypic changes caused by their deletion. For systematically analyzing the effects of gene deletion, gene knock-out libraries are available for several model organisms (Jorgensen et al., 2002; Baba et al., 2006; Kim et al., 2010; White et al., 2013). For example, the Keio collection (Baba et al., 2006) is a single-gene knock-out library of Escherichia coli that has been used to infer the roles of genes in various biological phenomena, such as cellular growth and morphogenesis (Campos et al., 2018), antibiotic sensitivity (Tamae et al., 2008), and evolution (Lukačišinová et al., 2020).
However, the significance of each gene might be context dependent. Gene expression levels change dynamically depending on environmental conditions (Schmidt et al., 2016; Bhatia et al., 2022). In Koganezawa et al. (2022), we induced a Cre-mediated antibiotic resistance gene deletion with blue-light illumination in the presence of the antibiotic. The microfluidic technique revealed that a small fraction of the gene-deleted cells could continue their growth without other genetic changes, and the fraction varied depending on the history of antibiotic administration. These results indicate that the impact of gene deletion on cellular physiology can depend on historical conditions (Koganezawa et al., 2022). Therefore, revealing dynamic genotype–phenotype relationships is crucial to understand the functional roles of each gene in given biological contexts.
Optogenetic techniques are useful for analyzing dynamic genotype–phenotype relationships. In particular, the photoactivatable Cre (PA-Cre) system allows us to induce gene deletion at desired timings by blue light exposure (Kawano et al., 2016). The PA-Cre system consists of split Cre recombinase, CreC, and CreN, fused to p-Magnet (p-Mag) and n-Magnet (n-Mag) protein fragments, respectively (Kawano et al., 2016). p-Mag and n-Mag are derived from a fungal photoreceptor Vivid and form a dimer upon blue light illumination (Kawano et al., 2015). Therefore, when these components of the PA-Cre system are expressed in the cytoplasm of bacterial cells, blue light illumination can induce the deletion of a target gene sandwiched between the two loxP sequences at desired timings. We constructed E. coli cell strains that harbor the PA-Cre system and the photocleavable fluorescently labeled chloramphenicol-resistance gene (Koganezawa et al., 2022). We observed the E. coli cells at the single-cell level in the microfluidic device and deleted the genes directly in the device by blue light illumination.
The duration of blue light illumination significantly influences the fraction of gene-deleted cells in the population. Therefore, by adjusting the illumination duration, we can simultaneously observe the dynamics of gene-deleted cells and non-deleted cells in a cell population. Since the target chloramphenicol resistance gene is tagged with the gene for the fluorescent protein mCherry, the fractions of gene-deleted cells are measurable simply by fluorescent microscopy. Here, we describe the protocol for measuring the fraction of gene-deleted cells based on batch-culture assay.
Materials and reagents
Glass test tube ϕ 16.5 × 165 mm (IWAKI, catalog number: B14-002-160)
Molton cap (AS ONE, catalog number: 6-352-07)
1.5 mL microtube (TreffLab, catalog number: 96.07246.9.01)
Minisart® syringe filter (Sartorius, catalog number: S7597-FXOSK)
Sterilized Petri dish ϕ90 × 15 mm (AS ONE, catalog number: 1-7484-01)
Aluminum foil
E. coli strain, carrying a fluorescently tagged target gene flanked with two loxP sequences and photoactivatable Cre (PA-Cre) system. Here, we used YK0083, which has a chloramphenicol resistance gene, cat, as a target gene for demonstration. The mCherry-tagged cat gene sandwiched with two loxP sequences was introduced into the intC locus on the genome. The genes for the PA-Cre system, creN-nmag and pmag-creC, are placed downstream of PLlacO1 promoter on a plasmid. These genes are inducible by IPTG. This strain is available by contacting the authors.
DifcoTM LB broth, Miller (Luria-Bertani) (BD, catalog number: 244620)
Agar (Wako, catalog number: 010-15815)
DifcoTM M9 minimal salts, 5× (BD, catalog number: 248510)
D(+)-glucose (Wako, catalog number: 049-31165)
MgSO4·7H2O (Wako, catalog number: 131-00405)
CaCl2·2H2O (Wako, catalog number: 31-00435)
MEM amino acids solution (50×) (SIGMA, catalog number: M5550)
Ampicillin sodium (Wako, catalog number: 016-23301)
Isopropyl-β-D(-)-thiogalactopyranoside (IPTG) (Wako, catalog number: 094-05144)
Elix® water produced by Milli-Q® Integral 3 (Merck, catalog number: ZRXQ003T0)
Milli-Q® water produced by Milli-Q® Integral 3 (Merck, catalog number: ZRXQ003T0)
LB broth (see Recipes)
LB agar containing 100 μg/mL of ampicillin (Amp) (see Recipes)
M9 medium with 0.2% glucose and amino acids (see Recipes)
1 M IPTG (see Recipes)
50 mg/mL Amp (see Recipes)
Equipment
Centrifuge (Hitachi, himac, CT 15RE)
Spectrometer (Shimadzu, UV-1800)
Blue light illuminator (Power Supply: CCS Inc., ISC-201-2; Blue light Illuminator: CCS Inc., SLM-150X150-BB)
BioShaker (TAITEC, BR-21FP)
Incubator (MITSUBISHI ELECTRIC ENGINEERING, SLC-25A)
Stereomicroscope (Stereomicroscope: Olympus, SZ61; LED source: NIGHTSEA, SFA-GR)
Portable photodiode-based laser power meter (Gentec-EO, PRONTO-Si)
Procedure
Sample preparation
Culture E. coli cells in LB broth with 50 μg/mL of Amp at 37 °C with shaking overnight.
Centrifuge 100 μL of the overnight culture at 21,500× g for 1 min at room temperature.
Remove the supernatant.
Resuspend the cellular pellet in 1 mL of M9 medium with 0.2% glucose and amino acids.
Measure the OD600 of the resuspended cells.
Adjust the OD600 to 0.001 in 2 mL of M9 medium with 0.2% glucose and amino acids containing 50 μg/mL of Amp and 0.1 mM IPTG.
Cover the test tube with the aluminum foil.
Cultivate the cells at 37 °C with shaking for 3 h.
Gene deletion induction
Remove the aluminum foil after the cultivation.
Expose the test tube to blue light using blue-light illuminator (light intensity: 6.8 mW) for the desired duration (Figures 1 and 2, see Notes).
Figure 1. Blue-light illumination inside a bio shaker with an illuminator. A blue-light illuminator was directly fastened onto a test tube spring holder inside a bio shaker. It casts a blue light downward on E. coli cultures. Light intensity was adjusted to 6.8 mW, measured with a laser power meter (λ = 470 nm).
Calculation of gene-deleted cells in batch
Dilute the cultures exposed to blue light to OD600 = 1.0 × 10-6, which is equivalent to approximately 7 × 102 cells/mL.
Spread 150 μL of the diluted cultures on LB agar plate containing 100 μg/mL of Amp.
Cover the agar plates with aluminum foil.
Incubate the plates at 37 °C for 18 h.
Count the number of colonies under ambient light and excitation light for examining mCherry fluorescence using a stereomicroscope (Figure 2A).
Calculate the fraction of gene-deleted cells as the number of non-fluorescent colonies divided by the number of total colonies.
Notes
The fraction of gene-deleted cells varies depending on the duration of blue-light illumination (Figure 2B). Therefore, an experimenter should determine the duration of blue-light exposure depending on the purpose of the experiment.
Cre-mediated gene deletion with blue-light illumination is detectable with loss of mCherry fluorescent signal.
Figure 2. Photoactivatable Cre-mediated gene deletion. (A) Stereomicroscopic pictures of YK0083 colonies after blue-light illumination under ambient light (left) and excitation light for detecting mCherry fluorescence (right). Cells were exposed to blue light for 1 h. Arrows show colonies generated from cells from which target genes were not deleted with blue-light illumination. (B) Blue-light illumination duration dependency of gene deletion efficiency. In case of less than 6 h blue-light illumination, test tubes were covered with aluminum foil and shaken at 37 °C, so that the time from the start of blue-light illumination to spreading on the LB agar was 6 h and consistent among the conditions. “no IPTG” indicates the condition where cells were cultured without IPTG and not exposed to blue light. Black points indicate the means. Error bars indicate standard errors (N = 239 for no IPTG, N = 327 for 0 h, N = 335 for 0.25 h, N = 373 for 0.5 h, N = 344 for 1 h, N = 350 for 2 h, N = 296 for 4 h, and N = 209 for 6 h). Reprinted/adapted from Koganezawa et al. (2022).
Recipes
LB broth
Mix the following:
25 g of DifcoTM LB broth, Miller (Luria-Bertani)
1 L of Elix® water
Autoclave at 121 °C for 15 min.
Store at room temperature.
At the time of use, add 50 mg/mL Amp on a clean bench as appropriate, if needed.
LB agar containing 100 μg/mL of Amp
Mix the following:
25 g of DifcoTM LB broth, Miller (Luria-Bertani)
15 g of agar
1 L of Elix® water
Autoclave at 121 °C for 15 min.
Add 2 mL of 50 mg/mL Amp on a clean bench.
Dispense 25 mL in each 90 mm dish on a clean bench.
Store at 4 °C.
M9 medium with 0.2% glucose and amino acids
Prepare the following reagents:
5× M9
1) Dissolve 56.4 g of DifcoTM M9 minimal salts, 5× in 1 L of Milli-Q® water.
2) Autoclave at 121 °C for 15 min.
3) Store at room temperature.
20% glucose
1) Dissolve 10 g of D(+)-glucose in Milli-Q® water to 50 mL.
2) Sterilize using a 0.2 μm pore filter.
3) Store at 4 °C.
1 M MgSO4
1) Dissolve 12.3 g of MgSO4·7H2O in Milli-Q® water to 50 mL.
2) Sterilize using a 0.2 μm pore filter.
3) Store at 4 °C.
1 M CaCl2
1) Dissolve 7.4 g of CaCl2·2H2O in Milli-Q® water to 50 mL.
2) Sterilize using a 0.2 μm pore filter.
3) Store at 4 °C.
Sterilized water
1) Autoclave Milli-Q® water at 121 °C for 15 min.
2) Store at room temperature.
Mix the following on a clean bench:
200 mL of 5× M9
10 mL of 20% glucose
10 mL of MEM amino acids solution (50×)
2 mL of 1 M MgSO4
100 μL of 1 M CaCl2
778 mL of sterilized water
Store at 4 °C.
At the time of use, add antibiotics or IPTG on a clean bench as appropriate, if needed.
1 M IPTG
Dissolve 2.383 g of IPTG in Milli-Q® water to 10 mL.
Sterilize using a 0.2 μm pore filter.
Dispense in microtubes on a clean bench.
Store dispensed tubes at -20 °C.
50 mg/mL Amp
Dissolve 500 mg of ampicillin sodium in 10 mL of Milli-Q® water.
Sterilize using a 0.2 μm pore filter.
Dispense in microtubes on a clean bench.
Store dispensed tubes at -20 °C.
Acknowledgments
This work was supported by JST CREST Grant Number JPMJCR1927 (Y.W.) and JPMJCR1653 (M.S.); JST ERATO Grant Number JPMJER1902 (Y.W.); Japan Society for the Promotion of Science KAKENHI Grant Number 17H06389 and 19H03216 (Y.W.); Project Grant from Kanagawa Institute of Industrial Science and Technology (KISTEC) (M. S.); and Grant-in-Aid for JSPS Fellows Grant Number JP19J22506 (Y.K.). This protocol was derived from Koganezawa et al. (2022).
Competing interests
The authors declare that no competing interests exist.
References
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L. and Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006 0008.
Bhatia, R. P., Kirit, H. A., Predeus, A. V. and Bollback, J. P. (2022). Transcriptomic profiling of Escherichia coli K-12 in response to a compendium of stressors. Sci Rep 12(1): 8788.
Campos, M., Govers, S. K., Irnov, I., Dobihal, G. S., Cornet, F. and Jacobs-Wagner, C. (2018). Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli. Mol Syst Biol 14(6): e7573.
Jorgensen, P., Nishikawa, J. L., Breitkreutz, B. J., and Tyers, M. (2002). Systematic identification of pathways that couple cell growth and division in yeast. Science 297(5580): 395-400.
Kawano, F., Okazaki, R., Yazawa, M., and Sato, M. (2016). A photoactivatable Cre–loxP recombination system for optogenetic genome engineering. Nat Chem Biol 12(12): 1059-1064.
Kawano, F., Suzuki, H., Furuya, A. and Sato, M. (2015). Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat Commun 6: 6256.
Kim, D. U., Hayles, J., Kim, D., Wood, V., Park, H. O., Won, M., Yoo, H. S., Duhig, T., Nam, M., Palmer, G., et al. (2010). Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol, 28(6): 617-623.
Koganezawa, Y., Umetani, M., Sato, M., and Wakamoto, Y. (2022). History-dependent physiological adaptation to lethal genetic modification under antibiotic exposure. eLife 11: e74486.
Lukačišinová, M., Fernando, B., and Bollenbach, T. (2020). Highly parallel lab evolution reveals that epistasis can curb the evolution of antibiotic resistance. Nat Commun 11(1): 3105.
Schmidt, A., Kochanowski, K., Vedelaar, S., Ahrne, E., Volkmer, B., Callipo, L., Knoops, K., Bauer, M., Aebersold, R. and Heinemann, M. (2016). The quantitative and condition-dependent Escherichia coli proteome. Nat Biotechnol 34(1): 104-110.
Tamae, C., Liu, A., Kim, K., Sitz, D., Hong, J., Becket, E., Bui, A., Solaimani, P., Tran, K. P., Yang, H., et al. (2008). Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli. J Bacteriol, 190(17): 5981–5988.
White, J. K., Gerdin, A. K., Karp, N. A., Ryder, E., Buljan, M., Bussell, J. N., Salisbury, J., Clare, S., Ingham, N. J., Podrini, C., et al. (2013). Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154(2): 452-464.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite
Category
Microbiology > Microbial genetics
Microbiology > Microbial physiology > Adaptation
Biological Sciences > Biological techniques > Microbiology techniques
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4,686 | https://bio-protocol.org/en/bpdetail?id=4686&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Polysome Profiling in Adult Mouse Testes
JK Jun-Yan Kang *
AZ Ai Zhong *
ZW Ze Wen
XY Xinghai Yu
YZ Yu Zhou
ML Mo-Fang Liu
(*contributed equally to this work)
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4686 Views: 908
Reviewed by: Gal HaimovichHeng SunMarion Hogg
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Original Research Article:
The authors used this protocol in Science Aug 2022
Abstract
Polysome profiling is widely used to isolate and analyze polysome fractions, which consist of actively translating mRNAs and ribosomes. Compared to ribosome profiling and translating ribosome affinity purification, polysome profiling is simpler and less time consuming in sample preparation and library constructions. Spermiogenesis, i.e., the post-meiotic phase of male germ cell development, is a highly coordinated developmental process in which transcription and translation are decoupled because of nuclear condensation, resulting in translation regulation as the major mode for the regulation of gene expression in post-meiotic spermatids. To understand the translation regulation during spermiogenesis, an overview of translational state of spermiogenic mRNAs is required. Here, we describe a protocol to identify translating mRNAs using polysome profiling. Briefly, mouse testes are gently homogenized to release polysomes containing translating mRNAs, following polysome-bound mRNAs isolated by sucrose density gradient purification and characterized by RNA-seq. This protocol allows to quickly isolate translating mRNAs from testes and analyze the discrepancy of translational efficiency in mouse testes from different mouse lines.
Key features
• Quickly obtain polysome RNAs from testes.
• Omit RNase digestion and RNA recovery from gel.
• High efficiency and robustness compared to ribo-seq.
Graphical overview
Schematic illustrating the experimental design for polysome profiling in mouse testes. Mouse testes are homogenized and lysed in Sample preparation, and polysome RNAs are enriched by sucrose gradient centrifugation and used to calculate translation efficiency in Sample analysis.
Keywords: Polysome profiling RNA extraction mRNA translation Translation efficiency Mouse testes Spermatid Germ cell Testicular lysate preparation Sucrose density gradient purification
Background
Gene expression is regulated at multiple levels, including epigenetic modifications, transcription, splicing, translation, etc. Among these, translation is the most important step in determining the level of protein expression (Schwanhäusser et al., 2011). An in-depth study of the translation process may connect the transcriptome with the proteome, allowing us to understand the regulatory mechanisms underlying the dynamic gene expression. Polysome profiling is a highly repeatable method used to identify mRNAs associated with polysomes, which can be isolated by subjecting cytosolic extracts to a continuous sucrose gradient (Chassé et al., 2017). Compared to polysome profiling, western blot is low throughput and should be coupled with RT-qPCR to qualitatively analyze the translation efficiency of specific genes; ribo-seq produces a precise snapshot of translatome by sequencing the RNA fragments protected by ribosomes from RNase-mediated degradation, which renders ribo-seq less efficient in identifying differences in translation efficiency. During male germ cell development, transcription and translation of spermiogenic mRNAs occur at distinct developmental stages, known as the uncoupling of transcription and translation (Steger, 1999; Sassone-Corsi, 2002). However, how those mRNAs transcribed in earlier developmental stages of spermatocytes and/or how round spermatids are translationally activated in late spermatids remain open questions. In our recently published work (Kang et al., 2022), we identified FXR1 as a potential translation activator of spermiogenic mRNAs. To verify the necessity of FXR1 for translation, we need to compare the translation efficiency in control and Fxr1 knockout mouse testes. To this end, we established the protocol for polysome profiling in adult mouse testes based on the method previously described (Ingolia et al., 2012).
Materials and reagents
1.5 mL microcentrifuge tube
2 mL Eppendorf tubes
20 mL syringe
C57BL/6N male mice
Cycloheximide (CHX) (MCE, catalog number: HY-12320)
cOmpleteTM EDTA-free protease inhibitor cocktail tablets (Roche, catalog number: 04693132001)
SUPERase•InTM RNase inhibitor (Invitrogen, catalog number: AM2694)
Triton X-100 (EMD Millipore, catalog number: 648466)
Sucrose, RNase- & DNase-free (AMRESCO, catalog number: 0335)
RNAiso Plus (Takara, catalog number: 9109)
Tris-HCl (1 M, pH 7.0, RNase-free) (Invitrogen, catalog number: AM9851)
NaCl (5 M) (Invitrogen, catalog number: AM9760G)
MgCl2 (1 M) (Invitrogen, catalog number: AM9530G)
Chloroform (Sinopharm Chemical Reagent, catalog number: 10006818)
Isopropanol (Sinopharm Chemical Reagent, catalog number: 80109218)
Ethanol (Sinopharm Chemical Reagent, catalog number: 10009218)
Polysome buffer (see Recipes)
Lysis buffer (see Recipes)
60% sucrose grading solution (see Recipes)
10% sucrose grading solution (see Recipes)
75% ethanol (see Recipes)
Equipment
Ultra-clear centrifuge tube, 14 × 89 mm (Beckman Coulter, catalog number: Z90805SCA)
Biocomp Gradient Maker and Fractionator (Biocomp)
Beckman Coulter Optima L-80 XP ultracentrifuge (with rotor SW41 Ti) (Beckman Coulter)
Dounce homogenizer, 2 mL (Sangon, catalog number: F519062)
Tweezers (Sangon Biotech, catalog number: F519022)
Scissor (Sangon Biotech, catalog number: F519234)
100 mm cell culture dish, DNase- & RNase-free (WHB-100)
Software
We perform all sequencing data analyses under the Linux system. We use the conda program to create the environment for polysome profiling data analysis and build computational pipelines using the snakemake program with the following software:
Sratoolkit (https://github.com/ncbi/sra-tools/wiki/01.-Downloading-SRA-Toolkit)
Fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
STAR (https://github.com/alexdobin/STAR)
Samtools (https://github.com/samtools/samtools)
RSeQC (https://rseqc.sourceforge.net/index.html)
R (https://www.r-project.org/)
Rsubread (https://bioconductor.org/packages/release/bioc/html/Rsubread.html)
DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
Xtail (https://github.com/xryanglab/xtail)
Databases
The genome sequences and gene annotation can be downloaded from the Genecode database (mm10 version 23), and tRNA and rRNA sequences can be downloaded using the UCSC Table Browser.
Genecode (https://www.gencodegenes.org/)
UCSC Genome Browser (https://genome.ucsc.edu/)
Procedure
Prepare mouse testicular lysate
C57BL/6N male mice were sacrificed by cervical dislocation and the testes were collected after abdominal anatomy (see also Note 4).
Use tweezers to peel off the tunica albuginea covering the surface of the testis.
Figure 1. Schematic diagram illustrating the tunica albuginea's location and shape and remaining testicular tissues
Weigh the testicular tissues and transfer them to a pre-cooled Dounce homogenizer (see Note 4).
Homogenize the testicular tissues with lysis buffer (1 mL per 100 mg of testes) by 10–15 strokes.
Transfer homogenate to a 1.5 mL microcentrifuge tube and incubate on ice for 15 min.
Centrifuge at 13,000× g for 2 min at 4 °C.
Transfer the supernatant to a fresh 1.5 mL microcentrifuge tube on ice.
Prepare sucrose gradient
Mark the ultra-clear centrifuge tube with the marker block provided by Biocomp and place it in the MagnaBase holder.
Add 10% sucrose up to the marker line with a 20 mL syringe.
Then, add an equal volume of 60% sucrose at the bottom of the tube with a fresh syringe.
Apply the short cap provided by Biocomp and remove the excess solution.
Turn on the Biocomp Gradient Station and level the rotating platform using a round bubble level.
Place the MagnaBase holder in the center of the plate.
Select the pre-programmed 10%–60% (w/v) gradients and press USE.
The station will beep when finished. Sucrose gradients should be used immediately.
Isolate polysome fractions from the total lysate
Take 10% of the lysates prepared in section A to a new 1.5 mL Eppendorf tube, add 500 μL of RNAiso Plus, and mix samples well. Label this sample as TOTAL and store it at -20 °C, or extract RNA following the procedure in section D.
Fill the tube prepared in section B with residual lysates.
Figure 2. Loading of lysates on sucrose gradients
Centrifuge the gradient at 38,000× g for 2 h at 4 °C in an SW41 Ti rotor.
After ultracentrifugation, gently remove tubes from the rotor and collect 12 fractions with Piston Gradient Fractionator.
Start the Gradient Station Fractionator and clean the upper valve assembly and the collection tubing.
Insert the centrifuge tube into the tube holder and lock it in.
Place the tube holder on the fractionator and turn it back 90° to lock it.
Cut out the cap of 2 mL Eppendorf tubes and place them onto the Fraction Collector.
Open the software, select the user profile, and zero the detector.
After blanking, press the AIR button according to the operation manual to force air through the entire system until no liquid comes out at the end of the collection tubing.
Run the fractionator and collect 12 fractions. The system will plot the absorbance in real time as the sample passes through the flow cell. Export data following software instructions.
Figure 3. An absorbance graph generated by Biocomp Gradient Maker and Fractionator
Transfer 700 μL of each fraction to a new 1.5 mL Eppendorf tube, add 500 μL of RNAiso Plus, and mix samples well. Label these samples as FRT#1–12 and store them at -20 °C, or extract RNA following the procedure in section D.
Extract polysome RNAs
Vortex for 10 s and keep the homogenate at room temperature for 5 min.
Centrifuge at 13,000× g for 5 min at 4 °C.
Transfer the supernatant to a new centrifuge tube and add 100 μL of chloroform.
Vortex for 10–15 s and keep at room temperature for 5 min.
Centrifuge at 13,000× g for 15 min at 4 °C.
Transfer the upper layer to a new centrifuge tube.
Add one volume of isopropanol and keep it at room temperature for at least 10 min.
Centrifuge at 13,000× g for 15 min at 4 °C.
Remove the supernatant and wash the RNA precipitate with 1 mL of pre-cooled 75% ethanol.
Centrifuge at 13,000× g for 5 min at 4 °C.
Remove the supernatant and keep the RNA precipitate air dried.
Dissolve the RNA precipitate with an appropriate amount of preheated nuclease-free water (see Note 5).
High-throughput sequencing
According to the absorbance graph, combine the RNA solutions extracted from polysome fractions (usually FRT#6–12 or FRT#7–12) and label it as POLY.
Subject comparable TOTAL and POLY RNAs to high-throughput sequencing with the Illumina platform (performed by Novogene Co, Ltd). Briefly, the concentration and purity of extracted RNAs were checked using Nanodrop and agarose gel electrophoresis, and integrity was further determined using Agilent 2100. Then, the libraries were constructed using NEBNext® UltraTM RNA Library Prep kit for Illumina®.
Data analysis
Make sure that conda is installed in your Linux system
Create a conda environment named PPIMT and install the snakemake and required software (sratoolkit, fastqc, STAR, samtools, rseqc, R, Rsubread, and DESeq2). Create directories named scripts and snakemake in your working directory.
Prepare a CSV file of sample information and the following scripts for downstream analysis (Table 1)
Table 1. Sample list
raw samples rep cond contrast
SRR14139008 Hetero_polysome_rep2 rep2 Hetero_polysome None
SRR14139009 Hetero_polysome_rep1 rep1 Hetero_polysome None
SRR14139016 cKO_polysome_rep2 rep2 cKO_polysome Hetero_polysome
SRR14139017 cKO_polysome_rep1 rep1 cKO_polysome Hetero_polysome
SRR14139014 cKO_total_rep2 rep2 cKO_total Hetero_total
SRR14139015 cKO_total_rep1 rep1 cKO_total Hetero_total
SRR14139018 Hetero_total_rep2 rep2 Hetero_total None
SRR14139019 Hetero_total_rep1 rep1 Hetero_total None
Sample information: PP_sample_info.csv
Scripts:
Meta.py
import os
import pandas as pd
## input
DATA_BASE = "../data"
GENOME_DIR = "../../anno"
G_INDEX_DIR = os.path.join(GENOME_DIR, "star.idx")
r_INDEX_DIR = os.path.join(GENOME_DIR, "star.idx_rRNA")
ANNO_DIR = GENOME_DIR
SCRIPT_DIR = "../scripts"
SAMPLE_INFO = "../PP_sample_info.csv"
REF_GTF = os.path.join(GENOME_DIR, "gencode.vM23.annotation.gtf")
REF_FA = os.path.join(GENOME_DIR, "mm10.fasta")
rRNA_FA = os.path.join(GENOME_DIR, "mm10_rRNA.fa")
SIZES = os.path.join(GENOME_DIR, "mm10.sizes")
Rscript = "Rscript"
sample_info = pd.read_csv(SAMPLE_INFO, sep=",")
samples = sample_info['samples']
cond = sample_info[sample_info['contrast']!="None"].cond.unique()
## output
RESULTS_BASE = "../results"
## RNAseq
def get_control(cond, samp_info):
contrast = samp_info[samp_info.cond == cond].contrast.unique()
contrast = "".join(list(contrast))
samp = samp_info[samp_info['cond']==contrast].samples
samp = list(samp)
return(samp)
def get_samp(cond, samp_info):
samp_info = samp_info[samp_info.cond == cond]
samp = samp_info.samples
samp = list(samp)
return(samp)
featurecount.R
#!/usr/bin/env Rscript
library("Rsubread")
args <- commandArgs(T)
## input
bam <- args[1]
gtf <- args[2]
strandspecf <- args[3]
name <- gsub("_uniq_sort.bam", "", basename(bam))
base <- dirname(dirname(dirname(bam)))
Nthread <- 8
## output
DESdir <- paste(base, "featurecount", sep = "/")
txt_feacount <- paste(DESdir, paste(name, "featureCounts.txt", sep = "_"), sep = "/")
log_feacount <- paste(DESdir, paste(name, "featureStat.log", sep = "_"), sep = "/")
##run
fc_SE <-featureCounts(
bam,
annot.ext = gtf,
isGTFAnnotationFile = T,
isPairedEnd = TRUE,
strandSpecific = as.integer(strandspecf),
nthreads = Nthread
)
write.table(fc_SE$counts,txt_feacount, quote = F,col.names = F,sep = "\t")
write.table(fc_SE$stat,log_feacount, quote = F,col.names = F,sep = "\t")
Distance.R
#!/usr/bin/env Rscript
## input
RAW_DIR <- "../results/featurecount"
## output
outDir <- "../results/plot_sample_cor"
args <- commandArgs(TRUE)
if (length(args) == 2) {
RAW_DIR <- args[1]
outDir <- args[2]
} else if (length(args) > 0) {
stop("
============================================================
Usage: 'Rscript 01_distance.R [/path/to/featurecounts_files_dir] [/out_dir]'
Or: 'Rscript 01_distance.R' (use default parameters)
============================================================
", call.=FALSE)
}
library(RColorBrewer)
library(ComplexHeatmap)
library(circlize)
library(dplyr)
samples <- list.files(RAW_DIR, pattern = "_featureCounts.txt")
t_samples <- samples[grep("total", samples)]
p_samples <- samples[grep("polysome", samples)]
pdf_poly <- file.path(outDir, "All_polysome_cor.pdf")
pdf_total <- file.path(outDir, "All_total_cor.pdf")
## run
plot_cor <- function(RAW_DIR, samples, pdf_cor) {
COLS <- rev(brewer.pal(n = 5, name = "RdYlBu"))
samp_base <- sapply(samples, function(x) strsplit(x, split = "_featureCounts")[[1]][1])
samp_cond <- sapply(as.vector(samp_base), function(x) strsplit(x, split = "_rep")[[1]][1])
full_name <- file.path(RAW_DIR, samples)
tsv <- lapply(full_name, function(x) read.csv(x, sep = "\t", header = F)$V2)
GENEID <- read.csv(full_name[1], sep = "\t", header = F)$V1
df <- do.call(cbind, tsv)
df <- df[apply(df, 1, sum) > 40, ]
colnames(df) <- as.vector(samp_base)
cor_mat <- cor(df, method = "spearman")
print(cor_mat)
p <- Heatmap(cor_mat, name = "Correlation", col = COLS, show_row_names = T,
show_column_names = T, cluster_columns = T, cluster_rows = T,
clustering_distance_rows = "spearman", clustering_distance_columns = "spearman")
pdf(pdf_cor, width = 5, height = 4)
print(p)
dev.off()
}
plot_cor(RAW_DIR, p_samples, pdf_poly)
plot_cor(RAW_DIR, t_samples, pdf_total)
DESeq.R
#!/usr/bin/env Rscript
library(DESeq2)
args <- commandArgs(T)
## input
samples <- args[1]
# samples <- gsub(" ","", samples)
samples <- strsplit(samples, split = ",")[[1]]
samp_cond <- strsplit(args[2], split = ",")[[1]]
## output
tsv_DE <- args[3]
## run
run_DESeq2 <- function(samples, samp_cond, tsv_DE) {
samples_base <- sapply(samples, basename)
samp_base <- sapply(samples_base, function(x) strsplit(x, split = "_featureCounts")[[1]][1])
tsv <- lapply(samples, function(x) read.csv(x, sep = "\t", header = F)$V2)
GENEID <- read.csv(samples[1], sep = "\t", header = F)$V1
df <- do.call(cbind, tsv)
rownames(df) <- GENEID
colnames(df) <- as.vector(samp_base)
colData <- data.frame(condition = samp_cond)
colData$condition <- factor(samp_cond, levels = unique(samp_cond))
dds <- DESeqDataSetFromMatrix(
countData = df,
colData = colData,
design = ~condition
)
dds <- DESeq(dds)
res <- results(dds)
print(head(res))
# write all expression gene name to a file
all_exp <- res[res$baseMean != 0, ]
all_exp <- as.data.frame(all_exp)
all_exp$name <- rownames(all_exp)
write.table(all_exp, tsv_DE, sep = "\t", quote = F, row.names = F)
}
run_DESeq2(samples, samp_cond, tsv_DE)
polysome_volcan.R
library(ggplot2)
library(readr)
## function
volcan_plot <- function (deg_p, name) {
library(ggplot2)
cutoff <- 0.58
PValue <- 0.05
Ylim <- 20
pdf_Vocan <- paste(name, "volcan.pdf", sep = "_")
deg_p <- deg_p[!is.na(deg_p$padj),]
deg_p$padj[(-log10(deg_p$padj) > Ylim)] <- 10 ^ (-Ylim)
##Construct the plot object
# with legend
deg_p$type <- ifelse((deg_p$log2FoldChange < (-cutoff)) & (deg_p$padj < 0.05) , "Down",
ifelse((deg_p$log2FoldChange >cutoff) & (deg_p$padj < 0.05), "Up", "NS"
))
label_tab <- table(deg_p$type)
label_stat <- paste(names(label_tab), label_tab, sep = ":")
p <- ggplot(data = deg_p,
aes(x = log2FoldChange,y = -log10(padj),colour = type)) +
geom_point(size = 0.2) +
scale_color_manual(labels=label_stat, values = c("blue", "grey", "red")) +
geom_vline( xintercept = c(-0.58, 0.58),lty = 4,col = "grey", lwd = 0.6 ) +
geom_hline( yintercept = -log10(0.05),lty = 4,col = "grey",lwd = 0.6) +
xlim(c(-6, 6)) +
ylim(c(0, Ylim)) +
theme_bw() +
theme(
legend.position = c(0.8, 0.8),
panel.grid = element_blank(),
legend.title = element_blank())+
labs(x = "log2(KO/WT)", y = "-log10(FDR)")
pdf(pdf_Vocan, width = 3.5, height = 4)
print(p)
dev.off()
}
## input
raw_DIR <- "../results/DESeq2"
OV <- file.path(raw_DIR, "cKO_polysome_DEG.tsv")
## output
OUTDIR <- "../results/figure"
pdf_volcan <- file.path(OUTDIR, "polysome")
##
deg_p <- read_tsv(OV)
volcan_plot(deg_p, pdf_volcan)
getExp_mRNA.R
#!/usr/bin/env Rscript
library(readr)
# args <- commandArgs(T)
## input
RAW_DIR <- "../results/featurecount/"
samples <- list.files(RAW_DIR, pattern = "_rpkm", full.names = T)[7:8]
## output
outDir <- "../results/figure "
tsv_mRNA <- file.path(outDir, "mRNAexp.tsv")
## run
samp_base <- sapply(samples, function(x) strsplit(basename(x), split = "_rpkm")[[1]][1])
samp_cond <- sapply(as.vector(samp_base), function(x) strsplit(x, split = "_rep")[[1]][1])
tsv <- lapply(samples, function(x) read.csv(x, sep = "\t", header = F)$V2)
GENEID <- read.csv(samples[1], sep = "\t", header = F)$V1
df <- do.call(cbind, tsv)
# df <- round(t(t(df/glt$length)))
rownames(df) <- GENEID
colnames(df) <- as.vector(samp_base)
df <- df[rowSums(df) > 0, ]
df_mean <- data.frame(
ID = rownames(df),
rpkm_mean = apply(df, 1, mean)
)
df_mean$ID <- sapply(df_mean$ID, function(x) strsplit(x, split = "\\.")[[1]][1])
write_tsv(df_mean, tsv_mRNA)
Xtail.R
## input
P_DEG <- ".. /DESeq2/cKO_polysome_DEG.tsv"
T_DEG <- ".. /DESeq2/cKO_total_DEG.tsv"
## output
outDir <- "../results/figure"
args <- commandArgs(TRUE)
if (length(args) == 3) {
P_DEG <- args[1]
T_DEG <- args[2]
outDir <- args[3]
} else if (length(args) > 0) {
stop("
=============================================================
Usage: 'Rscript 02_xtail.R [/path/to/polysome/DEG_file] [/path/to/total/DEG_file] [/out_dir]'
Or: 'Rscript 02_xtail.R' (use default parameters)
=============================================================
", call.=FALSE)
}
library(xtail)
DEtsv <- file.path(outDir, "DE_TE.tsv")
## run
## function
get_baseNmame <- function(x) {
strsplit(basename(x), split = "_featureCounts")[[1]][1]
}
get_mat <- function(x) {
count <- read.csv(x, sep = "\t", header = F)$V2
names(count) <- read.csv(x, sep = "\t", header = F)$V1
return(count)
}
## get data matrix
countDir <- "../results/featurecount/"
p_samps <- list.files(countDir, full.names = T, pattern = "polysome_rep[12]_featureCounts.txt")
t_samps <- list.files(countDir, full.names = T, pattern = "total_rep[12]_featureCounts.txt")
p_mat <- do.call(cbind, lapply(rev(p_samps), get_mat))
colnames(p_mat) <- sapply(rev(p_samps), get_baseNmame)
t_mat <- do.call(cbind, lapply(t_samps, get_mat))
colnames(t_mat) <- sapply(t_samps, get_baseNmame)
cond <- rep(c("control", "treat"), each = 2)
test.results <- xtail(t_mat, p_mat, cond, bins = 1000)
res_table <- test.results$resultsTable
res_table$ID <- row.names(res_table)
write.table(res_table, DEtsv, sep = "\t", row.names = F, quote = F)
DE_TE_scatter.R
library(readr)
library(ggplot2)
library(ggrepel)
library(clusterProfiler)
library(org.Mm.eg.db)
outDir <- "../results/figure"
## input
DEtsv <- file.path(outDir, "DE_TE.tsv")
P_DEG <- "../results/DESeq2/cKO_polysome_DEG.tsv"
T_DEG <- "../results/DESeq2/cKO_total_DEG.tsv"
## get data matrix
countDir <- "../results/featurecount/"
## output
pdf_volcan <- file.path(outDir, "DE_TE_scatter.pdf")
tsv_volcan <- file.path(outDir, "DE_TE_scatter.tsv")
## function
get_baseNmame <- function(x) {
strsplit(basename(x), split = "_featureCounts")[[1]][1]
}
get_mat <- function(x) {
count <- read.csv(x, sep = "\t", header = F)$V2
names(count) <- read.csv(x, sep = "\t", header = F)$V1
return(count)
}
###
## filter low exp genes
test.results <- read_tsv(DEtsv)
p_samps <- list.files(countDir, full.names = T, pattern = "polysome_rep[12]_featureCounts.txt")
t_samps <- list.files(countDir, full.names = T, pattern = "total_rep[12]_featureCounts.txt")
samps <- c(p_samps, t_samps)
fpkm_mat <- do.call(cbind, lapply(samps, get_mat))
colnames(fpkm_mat) <- sapply(samps, get_baseNmame)
ExpGenes_bool <- apply(fpkm_mat, 1, function(x) {
all(x > 1)
})
ExpGenes <- rownames(fpkm_mat)[ExpGenes_bool]
p_DEG <- read_tsv(P_DEG)
t_DEG <- read_tsv(T_DEG)
useGenes <- intersect(
p_DEG$name[!is.na(p_DEG$padj)],
t_DEG$name[!is.na(t_DEG$padj)]
)
useGenes <- intersect(useGenes, ExpGenes)
deg_p <- test.results[test.results$ID %in% useGenes, ]
cutoff <- 0.58
deg_p$type <- ifelse((deg_p$log2FC_TE_final < (-cutoff)) & (deg_p$pvalue.adjust < 0.05), "Down",
ifelse((deg_p$log2FC_TE_final > cutoff) & (deg_p$pvalue.adjust < 0.05), "Up", "NS")
)
deg_p$Gene <- sapply(
deg_p$ID,
function(x) strsplit(x, split = "\\.")[[1]][1]
)
label_tab <- table(deg_p$type)
label_stat <- paste(names(label_tab), label_tab, sep = ":")
HL_lst <- c(
"Gpd2", "Stat4",
"Hk1", "Akap3"
)
eg <- bitr(deg_p$Gene, fromType = "ENSEMBL", toType = "SYMBOL", OrgDb = "org.Mm.eg.db")
deg_p <- merge(deg_p, eg, by.x = "Gene", by.y = "ENSEMBL", all.x = T)
p <- ggplot(deg_p, aes(x = mRNA_log2FC, y = RPF_log2FC, color = type)) +
geom_point(size = 0.1) +
xlab(expression("log2FC(Transcriptome)")) + # x-axis label
ylab(expression("log2FC(Polysome)")) + # y-axis label
geom_vline(xintercept = c(-cutoff, cutoff), lty = 4, col = "grey", lwd = 0.6) +
geom_hline(yintercept = c(-cutoff, cutoff), lty = 4, col = "grey", lwd = 0.6) +
scale_color_manual(labels = label_stat, values = c("blue", "grey", "red")) +
geom_text_repel(
data = subset(deg_p, SYMBOL %in% HL_lst),
aes(mRNA_log2FC, RPF_log2FC, label = SYMBOL),
box.padding = 1.0, max.overlaps = 50,
col = "black"
) +
xlim(c(-3, 3)) +
ylim(c(-3, 3)) +
theme_bw() +
theme(
legend.position = c(0.8, 0.8),
panel.grid = element_blank(),
legend.title = element_blank()
)
pdf(pdf_volcan, width = 3.5, height = 3)
print(p)
dev.off()
write.table(deg_p, tsv_volcan, sep = "\t", row.names = F, quote = F)
DE_TE_scatter_0.01.R
library(readr)
library(clusterProfiler)
library(ggplot2)
library(ggrepel)
## input
outDir <- "../results/figure/"
DEtsv <- file.path(outDir, "DE_TE_scatter.tsv")
## output
pdf_volcan <- file.path(outDir, "DE_TE_scatter0.01.pdf")
tsv_volcan <- file.path(outDir, "DE_TE_scatter0.01.tsv")
## run
## function
deg_p <- read_tsv(DEtsv)
deg_p <- deg_p[!duplicated(deg_p$Gene), ]
cutoff <- 0.58
pcut <- 0.01
deg_p$type1 <- ifelse((deg_p$log2FC_TE_final < (-cutoff)) & (deg_p$pvalue.adjust < pcut), "Down",
ifelse((deg_p$log2FC_TE_final > cutoff) & (deg_p$pvalue.adjust < pcut), "Up", "NS"))
label_tab <- table(deg_p$type1)
label_stat <- paste(names(label_tab), label_tab, sep = ":")
HL_lst <- c( "Gpd2", "Stat4", "Hk1", "Akap3")
p <- ggplot(deg_p, aes(x = mRNA_log2FC, y = RPF_log2FC, color = type1)) +
geom_point(size = 0.1) +
xlab(expression("log2FC(Transcriptome)")) + # x-axis label
ylab(expression("log2FC(Polysome)")) + # y-axis label
geom_vline(xintercept = c(-cutoff, cutoff), lty = 4, col = "grey", lwd = 0.6) +
geom_hline(yintercept = c(-cutoff, cutoff), lty = 4, col = "grey", lwd = 0.6) +
scale_color_manual(labels = label_stat, values = c("blue", "grey", "red")) +
geom_text_repel(
data = subset(deg_p, SYMBOL %in% HL_lst),
aes(mRNA_log2FC, RPF_log2FC, label = SYMBOL),
box.padding = 1.0, max.overlaps = 50,
col = "black"
) +
xlim(c(-3, 3)) +
ylim(c(-3, 3)) +
theme_bw() +
theme(
legend.position = c(0.8, 0.8),
panel.grid = element_blank(),
legend.title = element_blank()
)
pdf(pdf_volcan, width = 3.5, height = 3)
print(p)
dev.off()
write_tsv(deg_p, tsv_volcan)
Prepare the references for alignment
Download the directory “anno” with the required Fasta files from our polysome_profiling project, including the mouse genome (mm10) (https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/GRCm38.p6.genome.fa.gz), tRNA and rRNA sequences (generated from https://genome.ucsc.edu/cgi-bin/hgTables), and genecode v23 gtf annotation file (https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M23/gencode.vM23.annotation.gtf.gz).
Build rRNA and tRNA index.
STAR --runThreadN 2 --runMode genomeGenerate \
--genomeDir r_INDEX_DIR \
--genomeFastaFiles mm10_rRNA.fa \
--outFileNamePrefix r_INDEX_DIR
Build genome index.
STAR --runThreadN 20 --runMode genomeGenerate \
--genomeDir g_INDEX_DIR \
--genomeFastaFiles mm10.fasta \
–sjdbGTFfile anno/gencode.vM23.annotation.gtf \
--outFileNamePrefix g_INDEX_DIR
Store the Fastq files of sequencing reads in a directory with the name data
Note: If you would like to download the data presented in our original article, use the sratoolkit program and the accession number list that you can find on our PPIMT project page.
mkdir data
while read line
do
name=$line
fastq-dump -O data --gzip $name
done < “SRR_Acc_List.txt”
Check the quality of raw sequencing data using fastqc as below
rule fastqc:
input:
fq1 = os.path.join(DATA_BASE, "{sample}_R1.fq.gz"),
fq2 = os.path.join(DATA_BASE, "{sample}_R2.fq.gz"),
output:
fq1 = os.path.join(RESULTS_BASE, "fastqc/{sample}/{sample}_R1_fastqc.html"),
fq2 = os.path.join(RESULTS_BASE, "fastqc/{sample}/{sample}_R2_fastqc.html"),
shell:
"""
fastqc -f fastq -t 2 --noextract -o {RESULTS_BASE}/fastqc/{wildcards.sample} {input.fq1} {input.fq2}
"""
Align the raw data to tRNA and rRNA, using the STAR program as below
rule map_rRNA:
input:
R1 = os.path.join(DATA_BASE, "{sample}_R1.fq.gz"),
R2 = os.path.join(DATA_BASE, "{sample}_R2.fq.gz"),
rRNA_index = "%s/SAindex" % r_INDEX_DIR,
output:
"%s/map_rRNA/{sample}/{sample}Unmapped.out.mate1" % RESULTS_BASE,
"%s/map_rRNA/{sample}/{sample}Unmapped.out.mate2" % RESULTS_BASE,
threads: 14
log:
"%s/map_rRNA/{sample}/{sample}.log" % RESULTS_BASE,
shell:
"""
STAR --genomeDir {r_INDEX_DIR} --readFilesIn {input.R1} {input.R2} \
--readFilesCommand gunzip -c --runThreadN {threads} \
--outFileNamePrefix {RESULTS_BASE}/map_rRNA/{wildcards.sample}/{wildcards.sample} \
--outReadsUnmapped Fastx --outFilterMatchNmin 40 --outFilterScoreMinOverLread 0 \
--outFilterMatchNminOverLread 0 1>{log} 2>&1
"""
Align the unmapped reads to genome, using the STAR program as below
rule map_genome:
input:
f_fastq1 = "%s/map_rRNA/{sample}/{sample}Unmapped.out.mate1" % RESULTS_BASE,
f_fastq2 = "%s/map_rRNA/{sample}/{sample}Unmapped.out.mate2" % RESULTS_BASE,
genome_index = "%s/SAindex" % G_INDEX_DIR,
output:
"%s/map_genome/{sample}/{sample}Aligned.out.bam" % RESULTS_BASE,
threads: 14
log:
"%s/map_genome/{sample}/{sample}.log" % RESULTS_BASE,
shell:
"""
STAR --genomeDir {G_INDEX_DIR} --readFilesIn {input.f_fastq1} {input.f_fastq2} \
--runThreadN {threads} \
--outFileNamePrefix {RESULTS_BASE}/map_genome/{wildcards.sample}/{wildcards.sample} \
--outSAMtype BAM Unsorted \
--outFilterMatchNmin 40 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 \
1>{log} 2>&1
"""
Filter out uniquely mapped reads to bam file and make an index using samtools program
rule sort_index_uniqbam:
input:
rules.map_genome.output
output:
unique_bam = temp("%s/sort_index_uniqbam/{sample}/{sample}_uniq.bam" % RESULTS_BASE),
sort_bam = "%s/sort_index_uniqbam/{sample}/{sample}_uniq_sort.bam" % RESULTS_BASE,
bai = "%s/sort_index_uniqbam/{sample}/{sample}_uniq_sort.bam.bai" % RESULTS_BASE,
threads: 8
log:
log1 = "%s/sort_index_uniqbam/{sample}/{sample}sort.log" % RESULTS_BASE,
log2 = "%s/sort_index_uniqbam/{sample}/{sample}index.log" % RESULTS_BASE,
shell:
"""
{samtools} view -@ {threads} -h -bq 255 {input} > {output.unique_bam}
{samtools} sort -@ {threads} {output.unique_bam} -o {output.sort_bam} 1>{log.log1} 2>&1
{samtools} index {output.sort_bam} 1>{log.log2} 2>&1
"""
Generate bigwig files from indexed bam files, using the bam2wig.py script from rseqc package
rule bam2bw:
input:
"%s/sort_index_uniqbam/{sample}/{sample}_uniq_sort.bam" % RESULTS_BASE,
output:
"%s/bam2bw/{sample}.bw" % RESULTS_BASE,
threads: 1
log:
"%s/bam2bw/{sample}.log" % RESULTS_BASE,
shell:
"""
bam2wig.py -i {input} -o {RESULTS_BASE}/bam2bw/{wildcards.sample} -t 1000000000 -s {SIZES} -u \
1>{log} 2>&1
"""
Count mapped reads for all annotated genes using the featureCounts program from Rsubreads package
rule featurecount:
input:
bam = "%s/sort_index_uniqbam/{sample}/{sample}_uniq_sort.bam" % RESULTS_BASE,
gtf = REF_GTF,
output:
"%s/featurecount/{sample}_featureCounts.txt" % RESULTS_BASE,
"%s/featurecount/{sample}_featureStat.log" % RESULTS_BASE,
threads: 8
params:
Rscript = Rscript,
log:
"%s/featurecount/{sample}.log" % RESULTS_BASE,
shell:
"""
{params.Rscript} {SCRIPT_DIR}/featurecount.R {input.bam} {input.gtf} 0 1>{log} 2>&1
"""
Analyze differential genes based on the reads counts of all genes in different samples with DESeq2 as below
rule DESeq2:
input:
control = lambda wildcards: ["%s/featurecount/%s_featureCounts.txt" % (RESULTS_BASE, samp)
for samp in get_control(wildcards.sample, sample_info)],
treat = lambda wildcards: ["%s/featurecount/%s_featureCounts.txt" % (RESULTS_BASE, samp)
for samp in get_samp(wildcards.sample, sample_info)]
output:
tsv = "%s/DESeq2/{sample}_DEG.tsv" % RESULTS_BASE,
params:
Rscript = Rscript,
run:
samples = ",".join(list(map(str, input.control))) + "," + ",".join(list(map(str,input.treat)))
group = ",".join(["WT"]*2+["TREAT"]*2)
cmd2 = " ".join([params.Rscript, SCRIPT_DIR + "/DESeq2.R", samples, group, output.tsv])
print(cmd2)
shell(cmd2)
Calculate sample correlation based on the reads counts of all genes in different samples with distance.R as below
rule sample_cor:
input:
"%s/featurecount/{sample}_featureCounts.txt" % RESULTS_BASE,
output:
poly= "%s/plot_sample_cor/All_polysome_cor.pdf" % RESULTS_BASE,
total= "%s/plot_sample_cor/All_total_cor.pdf" % RESULTS_BASE,
params:
Rscript = Rscript,
shell:
"""
{params.Rscript} {SCRIPT_DIR}/distance.R
"""
Figure 4. Correlation analysis. The Spearman correlation coefficient among four polysome profiling experiments for Fxr1cko and control testes with two replicates. The correlation coefficient was calculated based on reads counts of all genes between two different samples.
Visualize polysome profiling results with volcano plot based on the results of differential expression analysis between WT and FXR1 KO with polysome_volcan.R as below
rule plot_polysome_volcan:
input:
tsv = "%s/DESeq2/{sample}_DEG.tsv" % RESULTS_BASE,
output:
"%s/figure/polysome_volcan.pdf" % RESULTS_BASE,
params:
Rscript = Rscript,
shell:
"""
{params.Rscript} {SCRIPT_DIR}/polysome_volcan.R
"""
Figure 5. Volcano plot showing the significantly changed genes. The cutoff is FDR < 0.05 and fold-change > 1.5 between Fxr1cko and control testes.
Analyze differential translational efficiency based on the reads counts of expressed genes between WT and FXR1 KO using the R package Xtail between WT and FXR1 KO as below
rule plot_TE_scatter:
input:
total = "%s/DESeq2/cKO_total_DEG.tsv"" % RESULTS_BASE,
polysome = "%s/DESeq2/cKO_poly_DEG.tsv"" % RESULTS_BASE,
output:
"%s/figure/DE_TE_scatter0.01.pdf" % RESULTS_BASE,
params:
Rscript = Rscript,
shell:
"""
{params.Rscript} {SCRIPT_DIR}/getExp_mRNA.R
{params.Rscript} {SCRIPT_DIR}/Xtail.R
{params.Rscript} {SCRIPT_DIR}/DE_TE_scatter.R
{params.Rscript} {SCRIPT_DIR}/DE_TE_scatter_0.01.R
"""
Figure 6. Differential translational efficiency analysis. Scatter plot showing mRNA level changes (x-axis) against polysome profiling changes (y-axis) between adult Fxr1cko and control testes, with translation-deficient genes in Fxr1cko testes shown in blue.
Notes
Once testes are harvested, all manipulations should be performed on ice or at 4 °C.
Fill the ultra-clear centrifuge tube with lysate to approximately 2–3 mm from the border to prevent the tube from collapsing during centrifugation.
When collecting fractions with Piston Gradient Fractionator, the collection tube needs to be flushed with ddH2O. We recommend preheating the water to avoid clogging the tube with the sucrose solution.
Each sample requires two mice aged 25 days or one mouse older than 35 days. One testis of a 25-day-old mouse weighs approximately 30 mg and one testis of a 35-day-old mouse weighs approximately 40 mg.
The amount of water used to dissolve the RNA depends on the abundance of the RNA, or in other words, on the number of testes initially lysed. As a rule of thumb, 50–100 μL of water for INPUT yields approximately 1 μg/μL RNA, and 20–50 μL of water for FRT yields 80–800 ng/μL RNA for polysome fractions and 1–2 μg/μL RNA for RNP fractions. The volume of water used to dissolve 12 fractions should be consistent.
Recipes
Polysome buffer (50 mL)
50 mM Tris-HCl (pH 7.0)
100 mM NaCl
5 mM MgCl2
Nuclease free water
Lysis buffer (10 mL)
1% Triton X-100
100 μg/mL CHX
cOmpleteTM EDTA-free protease inhibitor cocktail
SUPERase•InTM RNase inhibitor
Polysome buffer (up to 10 mL)
60% (w/v) sucrose grading solution (10 mL)
60% (w/v) sucrose
100 μg/mL CHX
Cocktail protease inhibitor
RNase inhibitor
Polysome buffer (up to 10 mL)
10% (w/v) sucrose grading solution (10 mL)
10% (w/v) sucrose
100 μg/mL CHX
Cocktail protease inhibitor
RNase inhibitor
Polysome buffer (up to 10 mL)
75% (v/v) ethanol (50 mL)
75% (v/v) ethanol
Nuclease-free water
Acknowledgments
This protocol was originally applied in our study “LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs” published by Science (Kang et al., 2022). We are grateful for funding by the National Key R&D Program of China (2022YFA1303300, 2022YFC2702600 and 2021YFC2700200), National Natural Science Foundation of China (3191101352, 91940305, 31830109, and 31821004), Science and Technology Commission of Shanghai Municipality (2017SHZDZX01, 19JC1410200 and 17JC1420100), Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20210902), National Postdoctoral Program for Innovative Talent grant (BX20180331), China Postdoctoral Science Foundation grant (2018M642018), Open Fund of State Key Laboratory of Reproductive Medicine (SKLRM‐K202101), and the Foundation of Key Laboratory of Gene Engineering of the Ministry of Education. Furthermore, we would like to thank Ming-Shun Sun from the Cell Biology Core Facility in Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Chinese Academy of Sciences (CAS) for technical assistance.
Competing interests
The authors declare no competing interests.
Ethics
All animal studies were approved by the Institutional Animal Care and Research Advisory Committee in SIBCB, CAS.
References
Chassé, H., Boulben, S., Costache, V., Cormier, P., and Morales, J. (2017). Analysis of translation using polysome profiling. Nucleic Acids Res 45(3): e15.
Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. and Weissman, J. S. (2012). The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.Nat Protoc 7(8): 1534-1550.
Kang, J. Y., Wen, Z., Pan, D., Zhang, Y., Li, Q., Zhong, A., Yu, X., Wu, Y. C., Chen, Y., Zhang, X., et al. (2022). LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs. Science 377(6607): eabj6647.
Sassone-Corsi, P. (2002). Unique chromatin remodeling and transcriptional regulation in spermatogenesis. Science 296(5576): 2176-2178.
Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W. and Selbach, M. (2011). Global quantification of mammalian gene expression control. Nature 473(7347): 337-342.
Steger, K. (1999). Transcriptional and translational regulation of gene expression in haploid spermatids.Anat Embryol (Berl) 199(6): 471-487.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Developmental Biology > Reproduction
Molecular Biology > RNA > mRNA translation
Molecular Biology > RNA > RNA sequencing
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4,687 | https://bio-protocol.org/en/bpdetail?id=4687&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
An Improved System to Measure Leaf Gas Exchange on Adaxial and Abaxial Surfaces
DM D. A. Márquez
HS H. Stuart-Williams
SW S. C. Wong
GF G. D. Farquhar
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4687 Views: 505
Reviewed by: Ansul LokdarshiRaviraj Mahadeo Kalunke Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Plants Mar 2021
Abstract
Measurement of leaf carbon gain and water loss (gas exchange) in planta is a standard procedure in plant science research for attempting to understand physiological traits related to water use and photosynthesis. Leaves carry out gas exchange through the upper (adaxial) and lower (abaxial) surfaces at different magnitudes, depending on the stomatal density, stomatal aperture, cuticular permeability, etc., of each surface, which we account for in gas exchange parameters such as stomatal conductance. Most commercial devices measure leaf gas exchange by combining the adaxial and abaxial fluxes and calculating bulk gas exchange parameters, missing details of the plant's physiological response on each side. Additionally, the widely used equations to estimate gas exchange parameters neglect the contribution of small fluxes such as cuticular conductance, adding extra uncertainties to measurements performed in water-stress or low-light conditions. Accounting for the gas exchange fluxes from each side of the leaf allows us to better describe plants' physiological traits under different environmental conditions and account for genetic variability. Here, apparatus and materials are presented for adapting two LI-6800 Portable Photosynthesis Systems to work as one gas exchange system to measure adaxial and abaxial gas exchange simultaneously. The modification includes a template script with the equations to account for small fluxes. Instructions are provided for incorporating the add-on script into the device's computational sequence, display, variables, and spreadsheet results. We explain the method to obtain an equation to estimate boundary layer conductance to water for the new setup and how to embed this equation in the devices' calculations using the provided add-on script. The apparatus, methods, and protocols presented here provide a simple adaptation combining two LI-6800s to obtain an improved system to measure leaf gas exchange on adaxial and abaxial surfaces.
Graphical overview
Figure 1. Diagram of the connection of two LI-6800s. Figure adapted from Márquez et al. (2021).
Keywords: Leaf gas exchange Two-sides leaf gas exchange Adaxial surface Abaxial surface Gas exchange system Boundary layer Gas exchange parameters
Background
For simplicity, most common commercial gas exchange devices mix gases from the upper and lower cuvettes to estimate bulk gas exchange parameters from the mix. However, in some cases, it is desirable to analyse the characteristics of the leaf gas exchange on each leaf surface independently, such as when analysing adaxial and abaxial cuticular permeability in planta (Márquez et al., 2022), the stomatal contribution to the gas exchange of each surface (Wall et al., 2022), the estimation of water vapour (Wong et al., 2022) and CO2 (Márquez et al., 2023) gradients within the leaf, etc.
It has been common to use elaborate in-house built gas exchange systems to measure adaxial and abaxial gas exchange independently [see Jarvis and Slatyer (1966), Jones and Slatyer (1972) and Wong et al. (1985)]; however, it requires proficiency in electronics calibration, sensor tuning, environmental conditions control, and expertise in designing the tubing and manifolds. The LI-6800 incorporates automatic calibration protocols, well-tuned sensors, and its design permits modifying the chamber without major interference with the other parts of the system, allowing us to easily conjoin two instruments to analyse the adaxial and abaxial surfaces of a leaf.
The chamber modification demands re-measuring the boundary layer conductance to water for each cuvette to be accounted for in the computation of the gas exchange parameters. In this protocol, we walk the reader through the process of mounting the two LI-6800s together and measuring the boundary layer conductance following a method adapted from Parkinson (1985) to be used in LI-6800 devices. We provide an add-on script for the LI-6800 to include the new boundary layer conductance estimations for each cuvette in the calculations. The add-on script also includes an update to the latest theory for estimating gas exchange parameters presented by Márquez et al. (2021), which is a more precise physical approach to the electrical resistance analogy for gas exchange. The new boundary layer conductance and equations are included in the instrument's computation, display, variables, and spreadsheet results using the add-on code.
Materials and reagents
Joining two LI-6800
Two cuvette flanges (in-house designed, schematics in Supplement 1)
Lower chamber lid (in-house designed, schematics in Supplement 2)
Sealed top chamber lid (in-house designed)
Note: Sealed top lid design is similar to the lower chamber lid design except for the thermocouple holder apertures. The sealed top chamber lid does not have thermocouple holder apertures.
Sealed flange (in-house designed, schematics in Supplement 3)
Flexible stainless-steel tube, 12 mm diameter
O-rings and screws according to the schematics
Thermocouple extension cable with 3.5 mm Jack connectors
(Recommended) LI-6800 Custom Chambers Manifold (LI-COR, catalog number: 6800-19)
Bev-A-line IV tube
Before taking measurements
Polycarbonate sheet 2–3 mm thick, big enough to cover the surface of the chamber and seals
Boundary layer conductance estimation
Glass microfibre filters GF/A 9 cm (Whatman, catalog number: WHA1820090)
PEEK tube Orange 1/16 × 0.02 (IDEX, catalog number: 1532)
250 mL glass beaker
Pure water (reverse osmosis or distilled)
Equipment
Two photosynthesis systems LI-6800 (LI-COR Biosciences, Lincoln, Nebraska)
Software
Add-on script for LI-6800 to use double chamber (in-house coded, download from: https://github.com/PlantPhysiologist/MSF_UpperLower)
Procedure
Joining two LI-6800
Connect the flanges and tubing as shown in Figure 1 and 2. The upper and lower jaws holding the cuvettes should remain connected to LI-6800 #1 so that the lever for opening and closing the chamber remains operational. Place the thermocouple from the LI-6800 #1 to touch the leaf and position #2 to measure the air temperature of the lower cuvette. Keep the light of the LI-6800 #2 switched off to avoid extra heat in the system.
Figure 2. Flanges- and tubing-connected LI-6800 #1 (A) and #2 (B)
Alternatively, reference and sample gases from LI-6800 #2 (Figure 3) can be separated using a Custom Chambers Manifold from LI-COR (part number 6800-19, not included when buying an LI-6800), and then connecting the mixing volume and the reference gas in one tube going to the chamber and leaving the second tube connected to the sample gas inlet only. This last setup is recommended, though it is not used in Márquez et al. (2021) as it was developed after publication.
Figure 3. LI-6800 manifold in the head of the LI-6800 (A) and the connection for the Custom Chambers Manifold (B). Reference gas output (red), sample gas inlet (blue), and mixing fan volume (yellow). Connections are Bev-A-line IV tubes. Output and input tubing from the Custom Chamber Manifold are connected to Flexible stainless-steel tubes of 12 mm diameter.
Before taking measurements
Installing/loading the add-on script
Using a thumb drive or the Ethernet port, copy the add-on script file to the folder config.
Load the configuration Double cuvette from the display of your LI-6800 in the tab Startup/Configuration.
Warming up the instruments
As usual when using an LI-6800, put new chemicals in the water and CO2 scrub cylinders, refill the water in the humidifier, and install a new CO2 cartridge.
Turn off the light on both LI-6800s.
Place the polycarbonate sheet to isolate the gas from each cuvette and close the chamber.
Check for leaks by blowing CO2 (breath is a suitable mix) around the chamber and connections while checking the CO2 readings (they should not change).
Note: Correct any leak before continuing with measurements or warm up.
Run the automatic warm up of the instrument as usual.
Remove the polycarbonate sheet.
Boundary layer conductance estimation
Prepare the evaporative surface (Figure 4)
Fill the beaker with distilled water and submerge one end of the PEEK tube in it.
Suck through the PEEK tube to fill it with water.
Damp two microfibre filter sheets.
Put the PEEK tube between the two damped microfibre filter sheets.
Place the filter in the LI-6800 chamber and close it (the tube must sit in the middle of the chamber between the filters).
Place the beaker such that the water surface is a few centimetres higher than the filters, creating a siphon link, and taking care not to overflow the filter paper.
Ensure that the thermocouple in LI-6800 #1 is touching the microfibre filter and that #2 is not touching it.
Figure 4. Schematic of the setup to measure boundary layer conductance
Measuring boundary layer conductance at different fan speeds
Set the parameters:
Both LI-6800s to a flow rate of 600 μmol s-1; sample CO2 concentration at 400 μmol mol-1; and lights off.
LI-6800 #1 to an air temperature of 25 °C and air saturation deficit (ASD) at 1.2 kPa.
LI-6800 #2 to a leaf temperature (thermocouple) of 25 °C and vapour pressure difference (VPD) at 1.2 kPa.
Take measurements from both cuvettes varying the fan revolutions (rpm) from 5,000 up to 13,000 rpm in steps of 1,000 rpm (Figure 5).
Note: The flow rate also affects the estimated boundary layer conductance, but it is usually a minor effect for the measurements taken with an LI-6800.
Figure 5. Measurements of boundary layer conductance
Taking measurements
Placing a leaf in the chamber
The leaf must cover the whole area of the chamber.
Verify that only the thermocouple from LI-6800 #1 is touching the leaf.
Set the overpressure in both LI-6800s to 0.1 kPa. Always keep the same overpressure in both cuvettes to avoid mass flow across the leaf.
Start the protocol for your measurements and open a file to save your data.
Data analysis
Data analysis and adding the new boundary layer estimation to the LI-6800s
Extract the Excel log files from both LI-6800s.
In the data file from LI-6800 #2, replace the values given for leaf temperature with those given in the file from LI-6800 #1.
Calculate the boundary layer conductance to water (gbw).
where E is transpiration, wsat is vapour saturation mole fraction at filter temperature, and wa is water content in the cuvette air. Note that this is identical to the equation for total conductance (gtw) in the LI-6800 results spreadsheet.
Extract the gbw data and fit a quadratic equation (gbw = ax2 + bx + c) using fan speed per thousand (rpm/1,000) as the x input.
Polynomial of second order trendline from Excel is a suitable tool to fit the equation.
We use fan speed per thousand in the equation to adjust a, b, and c to four significant figures in the script.
Using the LI-6800 display, insert the values for the variables a, b, and c in the tab Constants/User.
If the tubing and manifolds in the connections are not modified, the equation for estimating gbw does not need to be recalculated.
Post-processing data
Replace the Tair values with the Tleaf from the LI-6800 #2 spreadsheet, and the Tleaf values of the #2 spreadsheet for those in #1. The new calculations included by the add-on will appear clustered in the first columns of the results spreadsheet but will not replace the normal ones. Thus, the user should be careful not to mix the data when retrieving it.
Notes
Work with flow rates of 500 μmol s-1 or higher on each LI-6800 when possible.
Set the same overpressure on both LI-6800s to avoid mass flow across the leaf.
The PEEK tube may be replaced by a piece of microfibre filter with one end in the beaker and the other between the two microfibre filters, but take extra precautions to prevent the microfibre outside the chamber from drying during the measurements.
Set the same air temperature on both cuvettes to keep the cuvette condition identical when modifying water parameters (notice that the thermocouple is measuring the air temperature in LI-6800 #2).
Acknowledgments
We thank P. Groeneveld for technical support and building the LI-6800 connector. We also thank ANID Doctorado, Becas Chile/2015 Folio 72160160 for funding part of the research and ARC support in the form of a Discovery Grant (no. DP210103186). The method here adapted was first published in Márquez et al. (2021) Nature Plants DOI: 10.1038/s41477-021-00861-w.
Competing interests
The authors declare no competing interests.
References
Jarvis, P. G. and Slatyer, R. O. (1966). A controlled-environment chamber for studies of gas exchange by each surface of a leaf. Commonwealth Scientific and Industrial Research Organization.
Jones, H. G and Slatyer, R. O. (1972). Effects of intercellular resistances on estimates of the intracellular resistance to CO2 uptake by plant leaves. Aust J Biol Sci 25(3): 443-454.
Márquez, D. A., Stuart-Williams, H., Cernusak, L. A. and Farquhar, G. D. (2023). Assessing the CO2 concentration at the surface of photosynthetic mesophyll cells. New Phytol. doi: 10.1111/nph.18784. Online ahead of print.
Márquez, D. A., Stuart-Williams, H. and Farquhar, G. D. (2021). An improved theory for calculating leaf gas exchange more precisely accounting for small fluxes. Nat Plants 7(3): 317-326.
Márquez, D. A., Stuart-Williams, H., Farquhar, G. D. and Busch, F. A. (2022). Cuticular conductance of adaxial and abaxial leaf surfaces and its relation to minimum leaf surface conductance. New Phytol 233(1): 156-168.
Parkinson, K. J. 1985. A simple method for determining the boundary layer resistance in leaf cuvettes. Plant Cell Environ 8(3): 223-226.
Wall, S., Vialet-Chabrand, S., Davey, P., Van Rie, J., Galle, A., Cockram, J. and Lawson, T. (2022). Stomata on the abaxial and adaxial leaf surfaces contribute differently to leaf gas exchange and photosynthesis in wheat. New Phytol 235(5): 1743-1756.
Wong, S. C., Canny, M. J., Holloway-Phillips, M., Stuart-Williams, H., Cernusak, L. A., Marquez, D. A. and Farquhar, G. D. (2022). Humidity gradients in the air spaces of leaves. Nat Plants 8(8): 971-978.
Wong, S. C., Cowan, I. R. and Farquhar, G. D. (1985). Leaf Conductance in Relation to Rate of CO(2) Assimilation: II. Effects of Short-Term Exposures to Different Photon Flux Densities. Plant Physiol 78(4): 826-829.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Plant Science > Plant physiology
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4,688 | https://bio-protocol.org/en/bpdetail?id=4688&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
Revised iCLIP-seq Protocol for Profiling RNA–protein Interaction Sites at Individual Nucleotide Resolution in Living Cells
SN Syed Nabeel-Shah
JG Jack F. Greenblatt
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4688 Views: 2781
Reviewed by: Gal HaimovichMarion Hogg Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Cell Sep 2022
Abstract
Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins’ (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins.
Key features
• Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution.
• iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells.
• iCLIP facilitates the identification of sequence motifs recognized by RBPs.
• Allows quantitative analysis of genome-wide changes in protein-RNA interactions.
• Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples.
Graphical overview
Keywords: iCLIP eCLIP Posttranscriptional regulation RNA-binding proteins UV crosslinking
Background
RNA-binding proteins (RBPs) are key players in posttranscriptional regulation, as they regulate essentially all aspects of mRNA metabolism, including 5′ cap formation, pre-mRNA splicing, 3′ end formation, mRNA export, and mRNA stability (Hentze et al., 2018). RBPs that decorate an RNA molecule during its life can dynamically change in space and time while they tightly co-ordinate posttranscriptional regulation (Hafner et al., 2021). Increasingly, links are being detected between defects in RBPs’ functions and human diseases, including neurological disorders and cancer (Pereira et al., 2017). Thus, understanding the role of RBPs in posttranscriptional regulation, and identifying their full repertoire of RNA targets in cells, is of fundamental importance for both better characterizing gene expression regulatory programs and drug development.
Although numerous methods exist to characterize RBPs’ target RNAs in cells (e.g., RIP, TRIBE, and APEX-seq), the cross-linking and immunoprecipitation (CLIP)-based methods are considered the gold standard for precisely identifying endogenous RNA-binding sites of RBPs (Hafner et al., 2021). Several variants of CLIP have been developed, including HITS-CLIP, individual-nucleotide resolution CLIP (iCLIP), ir-CLIP, enhanced CLIP (eCLIP), and iCLIP2, aiming to simplify the protocol and improve its efficiency (Buchbender et al., 2020;Konig et al., 2011;Lee et al., 2021;Van Nostrand et al., 2016; Zarnegar et al., 2016). These methods all rely on exposure of live cells to UV-C light, which covalently crosslinks RBPs to their target RNAs. The CLIP method can be carried out so as to provide precise positional and quantitative information on the crosslink sites. In particular, iCLIP aims to detect RNA fragments that truncate at the crosslink site during cDNA preparation, and thus reveals RBP binding sites on target RNA at individual nucleotide resolution. Since the original iCLIP method is laborious, and the library preparation efficiency can become limiting for low input samples (Huppertz et al., 2014), eCLIP was developed to omit the inefficient cDNA circularization step, improve the adapter ligation conditions, and implement a size-matched input (SMI) to control for background signal (Van Nostrand et al., 2016). Although eCLIP maintains the individual nucleotide resolution of iCLIP, it does not include direct visualization of protein-associated RNA. Hence, the quality of immunoprecipitated RNA, as well as the possibility of co-purifying background RBPs, cannot be directly monitored.
We have recently modified the original iCLIP method to interrogate the posttranscriptional regulatory functions of the ubiquitously expressed transcription factor SP1 (Song et al., 2022). By identifying its RNA-binding sites, we found that SP1 preferentially binds to A/G-rich sequence motifs on target RNAs. We further utilized our revised iCLIP procedure to examine the binding profiles of several cleavage and polyadenylation factors on target mRNAs (Song et al., 2022). Our data yielded high-resolution RNA-binding maps for SP1, as well as the cleavage and polyadenylation complex subunits, and unraveled a previously unknown function of SP1 in alternative cleavage and polyadenylation regulation. We have used this protocol with several cell lines (e.g., mouse neuroblastoma N2a cells, cgr8 embryonic stem cells, HeLa cells, glioblastoma, and HEK293 cells) to identify RNA targets of RBPs involved in diverse functions, including splicing regulation, mRNA stability, mRNA export, 5′ capping, and mRNA modifications (Nitoiu et al., 2021;Han et al., 2017and 2022;Nabeel-Shah et al., 2022 ). Our revised iCLIP protocol combines the improvements previously implemented in eCLIP (e.g., enhanced adapter ligation and SMI), with the optimization of several steps in the original iCLIP method, including improved circularization of the cDNA by increasing the incubation time and adding Betain in the reaction mixture, which helps the circularization of difficult-to-ligate substrates. Moreover, we utilize two-step dephosphorylation of RNA fragments to ensure the complete removal of the 3′-cyclic phosphate group left behind by RNase I cleavage. Since iCLIP2 is more similar to eCLIP (i.e., omission of circularization and linearization steps), whereas our protocol essentially follows the workflow of the original iCLIP procedure, we designate our revised protocol as iCLIP-1.5.
Materials and reagents
Protein G Dynabeads (Life Technologies, catalog number: 10004D)
Protein A Dynabeads (Life Technologies, catalog number: 10002D)
GFP recombinant rabbit monoclonal antibody (Thermo Fisher Scientific, catalog number: G10362)
Rabbit polyclonal anti-GFP (Abcam, catalog number: ab290)
SP1 antibody (Santa Cruz, catalog number: sc-17824)
Protease Inhibitor Cocktail Set III (Calbiochem/Merck, catalog number: 539134-1SET)
RNase I (Life Technologies, catalog number: AM2295)
Turbo DNase (Life Technologies, catalog number: AM2238)
T4 PNK plus 10× PNK buffer (NEB, catalog number: M0201L)
Murine RNase inhibitor 40 U/µL (NEB, catalog number: M0314L)
T4 RNA ligase 1 high conc 30 U/µL (NEB, catalog number: M0437M)
10× T4 RNA ligase reaction buffer (NEB, catalog number: B0216SVIAL)
FastAP 1 U/µL (Life Technologies, catalog number: EF0652)
Proteinase K 0.8 U/µL (NEB, catalog number: P8107S)
MyONE Silane beads (Life Technologies, catalog number: 37002D)
Pre-adenylated adapter L3-App IDT (rAppAGATCGGAAGAGCGGTTCAG/ddC/)
ATP [γ-32P] (PerkinElmer, catalog number: NEG502A250UC)
4%–12% NuPage gels (Life Technologies, catalog number: NP0322BOX)
LDS-4× sample buffer (NuPage loading buffer) (Life Technologies, catalog number: NP0007)
Pre-stained protein size marker (Fermentas, catalog number: SM1811)
Nitrocellulose membrane protran BA85 (VWR, catalog number: 732-4174)
20× transfer buffer (Life Technologies, catalog number: NP0006-1)
20× MOPS-SDS running buffer (Life Technologies, catalog number: NP0001)
Whatman filter paper (GE Healthcare, catalog number: 3030917)
Film (Fuji, catalog number: 4741019236)
Phenol/chloroform (Sigma, catalog number: P3803)
Phase lock gel heavy tube (VWR, catalog number: 713-2536)
GlycoBlue (Ambion, catalog number: 9510)
3 M sodium acetate pH 5.5 (Life Technologies, catalog number: AM9740)
2× TBE-urea loading buffer (Life Technologies, catalog number: LC6876)
6% TBE-urea pre-cast gels (Life Technologies, catalog number: EC68652B)
Low molecular weight marker (NEB, catalog number: N3233L)
TBE running buffer (Life Technologies, catalog number: LC6675)
SYBR green II (Life Technologies, catalog number: S-7564)
19 G syringe needle (BD, Microlance, catalog number: 300637)
Glass pre-filters (Whatman, catalog number: 1823010)
Costar SpinX column (Corning, catalog number: 8161)
CircLigaseTM (Lucigen, catalog number: CL4115K)
10× Circligase buffer (Lucigen, catalog number: CL4115K)
Betain solution 5 M (Sigma, catalog number: B0300-1VL)
Cut_oligo ‘GTTCAGGATCCACGACGCTCTTCaaaa’ (HPLC-purified, IDT)
BamHI (Fermentas, catalog number: FD0055)
Fast digest buffer (Fermentas, catalog number: FD0055)
2× Phusion High-Fidelity PCR Master Mix (NEB, catalog number: M0531L)
NaOH (Merck/Sigma, catalog number: S8045-500G)
RLT buffer (Qiagen, catalog number: 79216)
PBS (Sigma-Aldrich, catalog number: D8537)
UltraPureTM agarose (Invitrogen, catalog number: 16500-500)
QIAquick Gel Extraction kit (Qiagen, catalog number: 28706)
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)
Urea (Thermo Fisher Scientific, catalog number: 15505035)
Sodium deoxycholate (Thermo Fisher Scientific, catalog number: 89904)
Tween-20 (Thermo Fisher Scientific, catalog number: 28320)
Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: 28364)
Triton X-100 (Thermo Fisher Scientific, catalog number: 28314)
Primers (HPLC-purified from IDT):
P5_Solexa: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
P3_Solexa: CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Rt1clip/5Phos/NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt2clip/5Phos/NNACAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt3clip/5Phos/NNATTGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt4clip/5Phos/NNAGGTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt5clip/5Phos/NNCGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt6clip/5Phos/NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt7clip/5Phos/NNCTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt8clip/5Phos/NNCATTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt9clip/5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt10clip/5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt11clip/5Phos/NNGGTTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt12clip/5Phos/NNGTGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt13clip/5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt14clip/5Phos/NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt15clip/5Phos/NNTATTNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Rt16clip/5Phos/NNTTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
Solutions
Lysis buffer (see Recipes)
High-salt wash (see Recipes)
PNK buffer (see Recipes)
PK buffer (see Recipes)
PK buffer + 7 M urea (see Recipes)
1× RNA ligase buffer (see Recipes)
10× RNA ligase buffer (see Recipes)
1× FastAP buffer (see Recipes)
Recipes
Lysis buffer
50 mM Tris-HCl, pH 7.4
100 mM NaCl
1% Igepal CA-630
0.1% SDS
0.5% sodium deoxycholate
On the day: 1/100 volume of Protease Inhibitor Cocktail Set III, for tissues: 1/1,000 volume of ANTI-RNase
High-salt wash
50 mM Tris-HCl, pH 7.4
1 M NaCl
1 mM EDTA
1% Igepal CA-630
0.1% SDS
0.5% sodium deoxycholate
PNK buffer
20 mM Tris-HCl, pH 7.4
10 mM MgCl2
0.2% Tween-20
PK buffer
100 mM Tris-HCl, pH 7.4
50 mM NaCl
10 mM EDTA
PK buffer + 7 M urea
100 mM Tris-HCl, pH 7.4
50 mM NaCl
10 mM EDTA
7 M urea
1× RNA ligase buffer (no DTT)
50 mM Tris-HCl pH 7.5
10 mM MgCl2
10× RNA ligase buffer (no DTT)
500 mM Tris-HCl pH 7.5
100 mM MgCl2
1× FastAP buffer
10 mM Tris pH 7.5
5 mM MgCl2
100 mM KCl
0.02% Triton X-100
Equipment
Refrigerated microcentrifuge
Picofuge (Thermo Fisher Scientific, catalog number: 75004061)
Stratalinker UV crosslinker 2400 Stratagene
Denaturing agarose gel apparatus (Bio-Rad Laboratories)
Magnetic stand (Thermo Fisher Scientific, Invitrogen)
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)
Thermoblock (Thermo Fisher Scientific, Invitrogen)
Thermomixer (Boekel Scientific)
Sonicator (Branson Digital Sonifier)
Geiger counter sensitive to beta particles (Ludlum 3 survey meter Geiger counter radiometer 44-9 pancake probe)
3/8" or 1/2" Plexiglas benchtop shield (P32 protective shield)
Cell scraper
Liquid nitrogen (N2)
Saran wrap
Blue light transilluminator
UV transilluminator
X-ray film cassette
Software
iMAPs (Jernej Ule’s lab, https://imaps.goodwright.com/)
PureCLIP (Sabrina Krakau, https://pureclip.readthedocs.io/en/latest/index.html)
CTK package (Chaolin Zhang’s lab, https://zhanglab.c2b2.columbia.edu/index.php/CTK_Documentation)
Procedure
We describe below the step-by-step procedure for performing an iCLIP-seq experiment using GFP-tagged proteins with modifications/improvements implemented at various steps. This procedure (designated as iCLIP-1.5) has been applied to both epitope-tagged proteins (i.e., GFP- or FLAG-tagged proteins) and endogenous proteins (e.g., SP1) using validated antibodies. For doxycycline-inducible epitope-tagged cell lines, induce the expression of protein 24 h prior to harvesting. Since this protocol follows the workflow of the original iCLIP-seq method (Huppertz et al., 2014), the data generated using this protocol can be subjected to the standard iCLIP analysis pipeline.
Store all buffers at 4 °C 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 900µL unless stated differently.
UV crosslinking of cells
Grow HEK293 cells in 15 cm diameter tissue culture plates (~80% confluent) (Note 1).
Remove the media and wash cells once with ice-cold 1× PBS. Gently add 6 mL of ice-cold PBS and place the plate on a small ice tray that fits inside the Stratalinker (Note 2).
Remove the lid and irradiate the cells once with 150 mJ/cm2 at 254 nm in a Stratalinker 2400 (or equivalent UV-C crosslinker) (Notes 3, 4, 5).
Scrape off the cells and transfer to a 15 mL Falcon tube. Centrifuge at 1,000× g for 5 min at 4 °C, remove the supernatant, and snap freeze the cells for later use.
Lysis, partial RNA digestion, and immunoprecipitation
Prepare protein G Dynabeads for conjugation with anti-GFP antibody (Note 6). Wash 100 μL of protein G Dynabeads with 900 μL of lysis buffer (with protease inhibitors added) by gently pipetting up and down a few times. Place the tubes on a magnetic rack to separate the bead and remove the supernatant. Repeat the washing step three times and, after the last wash, resuspend beads in 100 μL of lysis buffer.
Add 10 μg of anti-GFP antibody. Rotate tubes for 1 h at room temperature (i.e., ~22 °C) (Note 7).
While beads are rotating, resuspend the cell pellets in 2.2 mL of iCLIP lysis buffer supplemented with protease inhibitors (1:100 Protease Inhibitor Cocktail Set III). Cells expressing free FLAG/GFP can be used as negative controls (Note 9).
Sonicate the samples with a probe sonicator: 15 cycles, 0.3 s on, 0.7 s off (three rounds) (Note 10).
Transfer 1 mL of cell lysate to a 1.5 mL Eppendorf tube.
Prepare at least two RNase I dilutions in PBS: 1:50 dilution for high RNase I and 1:250 dilution for low RNase I (Notes 11, 12, 13).
Add 20 µL of RNase I (from the dilution stocks) and 2 µL of Turbo DNase to 1 mL of lysate and place the tubes on a thermomixer. Shake the samples in a thermomixer (1,200 rpm) for exactly 5 min at 37 °C, and immediately put back on ice for 5 min.
Critical: Optimization of RNase I dilution is extremely important for the success of the experiment (see Note 11). Incubate the samples for exactly 5 min at 37 °C.
Centrifuge the lysate at 16,100× g in a microcentrifuge for 30 min at 4 °C. Transfer the clarified supernatant to a new 1.5 mL Eppendorf tube and keep on ice.
Transfer 20 µL of clarified lysate to an Eppendorf tube as input. Snap freeze in dry ice/liquid nitrogen and store at -80 °C until further use.
After 1 h, wash the antibody-conjugated beads (from Step B1) twice with 900 µL of ice-cold lysis buffer.
After the last wash, add the clarified cell lysate to the beads. Rotate in a cold room (4 °C) for 2 h or overnight (Note 14).
Place the tubes on a magnetic rack to separate the beads, remove the supernatant, and add 900 µL of high-salt buffer (Note 15).
Rotate for 5 min in the cold room. Repeat three times.
Wash three times with PNK buffer (no need to rotate in the cold room). Add 900 µL of PNK buffer and gently pipette up and down. Place the tubes back on a magnetic rack and discard the supernatant (Note 16).
Wash once with 1× FastAP buffer (no need to rotate in the cold room). Add 900 µL of 1× FastAP buffer and gently pipette up and down. Place the tubes back on a magnetic rack and discard the supernatant.
RNA 3′ end dephosphorylation, L3 adapter ligation, and 5′ end labeling
To prime RNA fragments for 3′ adapter ligation, RNA 3′ end dephosphorylation is carried out in two steps. This ensures the complete removal of the 3′-cyclic phosphate group left behind by RNase I cleavage.
Resuspend the beads in 100 µL of the following mix and shake at 37 °C for 15 min in a thermomixer (1,200 rpm).
H2O 79 μL
10× FastAP buffer 10 μL
RNase inhibitor 2 μL
Turbo DNase 1 μL
FastAP enzyme 8 μL
While tubes are shaking, prepare 300 µL of the following mix for each sample. Add the mix to each tube and incubate for another 20 min while shaking.
H2O 230 µL
5× PNK (pH 6.5) 60 µL
0.1 M DTT 3 µL
Turbo DNase 1 µL
RNase inhibitor (murine) 2 µL
T4 PNK enzyme 4 µL
Separate the beads and wash once with 900 µL PNK buffer (no need to rotate in the cold room).
Wash three times with high salt buffer. Rotate each time for 5 min in the cold room.
Wash three times with PNK buffer (no need to rotate in the cold room).
Wash once with 1× ligase buffer (no DTT) by gently pipetting up and down (Note 16).
Remove the buffer and resuspend the beads in 30 μL of the ligation mixture.
H2O 12 µL
10× ligase buffer (no DTT) 3 µL
100% DMSO 0.6 µL
50% PEG 8000 9 µL
RNase inhibitor (murine) 0.4 µL
T4 RNA ligase 1 (high conc 30 U/µL) 2.5 µL
Pre-adenylated adapter L3-App (20 μM) 2.5 µL
Incubate at room temperature for 90 min. Flick the tube every 10–15 min. Alternatively, the tubes can be shaken in a thermomixer at room temperature.
Add 500 μL of PNK buffer and place on the magnetic rack to separate the beads. Discard the supernatant.
Wash three times with high-salt buffer. Rotate each time in the cold room for 5 min.
Wash three times with PNK buffer without rotation in the cold room. Gently pipette up and down a few times. Place the tubes back on a magnetic rack and discard the supernatant.
Resuspend the beads in 1 mL of PNK buffer.
Transfer 200 μL of beads (20%) (hot beads) to a new tube. Keep the remaining 800 μL of beads in PNK buffer on ice (cold beads). Remove the supernatant from the hot beads and resuspend in the 5′ end labeling mixture.
T4 PNK 0.2 μL
32P-γ-ATP 0.4 μL
10× PNK buffer 0.4 μL
H2O 3.0 μL
Caution: All 32P-related work must be performed in a designated radioisotope area. Always work behind the protective shield when handling radioactive material.
Incubate for 5 min at 37 °C while shaking at 1,200 rpm in a thermomixer.
Separate the hot beads using the magnetic rack and discard the supernatant as radioactive waste.
Resuspend hot beads in 20 μL of 1× NuPage loading buffer (LDS sample buffer). Optional: add reducing agent (DTT) in the 1× NuPage loading buffer to break the antibody chains.
Remove supernatant from cold beads and mix them with hot beads.
Take out input tubes from -80 °C. Thaw on ice and add LDS sample loading buffer.
Heat the beads and input tubes at 70 °C for 5 min. Magnetically separate the beads and transfer the supernatant into a new tube.
Load the samples (20 μL) onto a 4%–12% NuPAGE Bis-Tris gel (Note 17). Use 1× MOPS-SDS running buffer (diluted from 20× MOPS-SDS running buffer). Also, load 5 μL of a pre-stained protein size marker.
1 2 3 4 5 6 7 8
Ladder Input Ladder Sample1 Empty Sample2 Empty Control
Run the gel for 1 h at 180V. For high molecular weight proteins, run the gel for 72 min.
Cut the ATP-containing dye front from the gel bottom and discard as solid radioactive waste.
Set up the transfer using a pure nitrocellulose membrane and Whatman filter papers according to the manufacturer's instructions. Use 1× transfer buffer (diluted from 20× transfer buffer) (Note 17).
Transfer in the cold room at 14 V overnight.
RNA isolation
Rinse the membrane in ice-cold PBS and wrap it with saran wrap. Expose to Fuji film at -80 °C for 30 min in a dark room (Notes 18, 19, 20).
Isolate the RNA-protein complexes using the autoradiograph as a mask for cutting the desired regions on the membrane (Figure 1). Cut the membrane into small pieces (~1–2 mm slices) using a new sterile razor blade for each sample and place them in Eppendorf tubes. Keep the tubes on ice until all samples have been excised.
Figure 1. Optimization of RNase 1 dilutions and CLIP-autoradiography. A. CLIP autoradiography of 32P-labeled RBP-RNA complexes was performed after RNase I treatment (1:25–1:250 dilution). Under high RNase 1 condition (1:25), the radiolabeled RNA signal diminished. Optimal RNase 1 concentration (1:250 here) results in radiolabeled RNA that runs as a smear above the predicted size of the RBP (~72 kDa here). Note: Antibody chains are often observed on autoradiographs. Bottom panel shows western blotting results to indicate the recovery of GFP-tagged bait protein. Black bar on the right indicates region on the membrane to be excised. B. Autoradiograph of input (INP) and 32P-labeled immunopurified (IP) RBP-RNA complexes after partial RNase I digestion (RNase I dilution, 1:250). Since the input sample was not radiolabeled, no radioactive signal was observed. Red boxes indicate the regions on the membrane that were excised to extract RNA. RBP (SP1) size is ~82 kDa here. GFP-only sample is shown as a negative control.
Note: Representative autoradiographs shown in A and B are for two unrelated RBPs.
For inputs, cut the membrane in parallel with the IP samples, matching the sizes of the excised areas (Figure 1B).
Add 20 μL of Proteinase K into 180 μL of PK buffer. Add this Proteinase K mixture to tubes containing the membrane pieces.
Shake the tubes in a thermomixer at 37 °C for 20 min.
Add 200 μL of PK-7M urea buffer and shake for another 20 min at 37 °C. For making PK-7M urea buffer, first dissolve 420 mg of urea in 500 µL of PK buffer, then bring to a final volume of 1 mL by slowly adding PK buffer.
Centrifuge the tubes briefly, collect the solution, and add it together with 400µL phenol/chloroform to a 2mL Phase Lock Gel Heavy tube.
Shake at 37 °C for 5 min in a thermomixer.
Separate the phases by centrifuging the tubes for 5 min at full speed in a tabletop centrifuge at room temperature.
Transfer the aqueous layer into a new tube (be careful not to touch the gel matrix). Centrifuge the supernatant again for 1min and transfer into a new tube.
Add 0.75µL of GlycoBlue and 40µL of 3M sodium acetate (pH 5.5), mix, and add 1mL of ice-cold 100% ethanol (EtOH). Mix by inverting the tubes several times and place overnight at -20 °C.
The next day, centrifuge the samples at 16,100× g for 20 min at 4 °C.
Remove the supernatant and wash the pellet with 900 µL of ice-cold 80% EtOH and centrifuge again for 10 min.
Resuspend the pellet in 5 μL of H2O and transfer to a PCR tube. Keep on ice (or -80 °C) until the input samples are ready for cDNA synthesis.
Input adapter ligation
Prior to converting RNA samples into cDNA, input RNA must undergo L3 adapter ligation. Of note, for certain spliceosome subunits (e.g., U2AF65 and PUF60), L3 adapter ligation to the immunoprecipitated protein-RNA complexes does not occur efficiently, presumably due to steric hinderance (Shao et al., 2014; Han et al., 2022). In such cases, we recommend that the IP samples should be processed along with inputs so that, after immunoprecipitation and three high-salt buffer washes (Step B12), you proceed directly to radiolabeling (Step C11) and omit the dephosphorylation and adapter ligation steps described above in section C. Isolate the RNA from the membrane as detailed above and continue with the steps noted below for input samples.
To 5 μL input samples, add:
H2O 15 μL
10× FastAP buffer 2.5 μL
RNase inhibitor 0.5 μL
FastAP enzyme 2.5 μL
Incubate at 37 °C for 15 min while shaking at 1,200 rpm. Then, add 75 μL of the following master mix:
H2O 48 µL
5× PNK (pH 6.5) 20 µL
0.1 M DTT 1 µL
RNase inhibitor (murine) 1 µL
Turbo DNase 1 µL
T4 PNK enzyme 4 µL
Incubate for another 20 min at 37 °C while shaking at 1,200 rpm.
Using the magnetic rack, separate 20 µL of MyONE Silane beads per sample.
Wash once with 900 µL RLT buffer. Pipette up and down a few times and then remove the buffer.
Resuspend beads in 300 µL of RLT buffer. Then, add these beads in RLT buffer to the samples.
Add 10 µL of 5 M NaCl and 615 µL of 100% EtOH.
Mix and rotate samples at room temperature for 15 min.
After 15 min, place tubes on the magnetic rack and remove supernatant.
Resuspend beads in 1 mL of 75% EtOH and move the suspension to a new tube.
Let it sit for 30 s and place back on the magnetic rack.
Remove the liquid and resuspend again in 1 mL of 75% EtOH. Let sit for 30 s. Remove the liquid and repeat the process once more.
After the final wash, spin down the tubes using a picofuge and place them back on the magnetic rack. Remove all residual liquid.
Let air dry for 3 min.
Resuspend in 10 µL of H2O. Let it sit for 5 min at room temperature.
Using the magnetic rack, separate the beads and collect RNA in 10 µL of H2O. Freeze 5 µL at -80 °C as the backup input sample (Note 21).
To the remaining 5 µL input samples, add:
H2O 1.2 µL
10× T4 RNA ligase reaction buffer 2 µL
100% DMSO 0.3 µL
50% PEG 8000 8 µL
RNase inhibitor (murine) 0.2 µL
T4 RNA ligase 1 (high conc 30 U/µL) 1.3 µL
Pre-adenylated adapter L3-App (20 μM) 2 µL
Incubate at room temperature for 90 min with shaking at 1,200 rpm.
Take 20 µL of MyONE Silane beads per sample.
Wash once with 900 µL of RLT buffer.
Resuspend beads in 61.6 µL of RLT buffer.
Add beads in 61.6 µL of RLT buffer to the sample.
Add 61.6 µL of 100% EtOH and incubate at room temperature for 15 min with the pipette tip left in the tube. Pipette up and down every 3–5 min to mix the samples.
After 15 min, pipette resuspend in 1 mL 75% EtOH and move to a new tube. Let it sit for 30 s at room temperature and then remove the liquid.
Add 1 mL of 75% EtOH, pipette resuspend, and after 30 s remove the liquid.
Wash once again with 1 mL of 75% EtOH.
After the third wash, briefly spin down the samples using a picofuge and remove residual liquid.
Air dry samples for 3 min at room temperature. Resuspend in 5 µL of H2O.
Reverse transcription and gel extraction of cDNA
Now that the Input and CLIP samples are synchronized, remove the CLIP samples from -80°C and perform reverse transcription (RT) for all samples (Figure 2).
Figure 2. Schematic representation of cDNA synthesis and iCLIP library preparation. Adapter-ligated RNA fragments are reverse transcribed using barcoded primers. Adapter and RT primer sequences are shown. cDNA is size selected, circularized, and linearized prior to performing PCR for library preparation.
Note: RT primer sequence corresponds to Rt1clip, as listed in the Materials and Reagents section.
Transfer RNA (5 µL) to PCR tubes and add the following reagents (Note 22):
Rt#CLIP primer (0.5 pmol/μL) 1 μL
dNTP mix (10 mM) 1 μL
Place tubes in PCR machine and use the following thermal program:
70 °C for 5 min.
Then, hold at 25 °C until RT mix is added.
In the meantime, prepare RT mix:
H2O 7 µL
5× first strand buffer 4 µL
0.1 M DTT 1 µL
RNase inhibitor (murine) 0.5 µL
Superscript III 0.5 µL
Add 13 µL of RT mix to each tube and use following thermal program:
25 °C 5 min
42 °C 20 min
50 °C 40 min
80 °C 5 min
4 °C hold
Add 1.65 μL of 1 M NaOH to each sample and incubate at 98 °C for 20 min.
Add 20 μL of 1 M HEPES–NaOH pH 7.3 to each sample. This will eliminate radioactivity from strongly labeled samples and prevent RNA from interfering with subsequent reactions (Note 23).
Add 350 μL of TE buffer, 0.75 μL of GlycoBlue, and 40 μL of 3 M sodium acetate (pH 5.5), mix, then add 1 mL of ice-cold 100% EtOH. Mix again and precipitate at -20 °C overnight.
The next day, centrifuge at 16,100× g for 15 min at 4 °C. Remove the supernatant and wash the pellet with 500 μL of ice-cold 80% EtOH. Centrifuge again, remove supernatant, and resuspend the pellet in 6 μL of H2O.
Add 6 μL of 2× TBE-urea loading buffer to the cDNA and heat samples to 80 °C for 5 min immediately before loading. Also, when loading the gel, leave one lane empty between each sample to avoid cross-contamination.
Set up the gel running apparatus with 6% TBE-urea gel in 1× TBE running buffer according to the manufacturer’s instructions. Make sure to flush out urea from the wells using a P1000 pipette tip prior to loading samples.
Run 6% TBE-urea gel for 40 min at 180 V until the lower (dark blue) dye is close to the bottom (Figure 3, Note 24).
Figure 3. Gel purification and size selection of cDNA. Cartoon illustration of 6% TBE-urea gel electrophoresis to guide the excision of iCLIP cDNA products. The pattern of migration for cDNA and two dyes (indicated) is highly reproducible when the gel is run for 40 min at 180 V (make sure to note the time required to obtain the indicated pattern of migration of the bands on the gel when using your own powerpack in initial experiments). Gel slices that are excised to purify cDNA corresponding to high (H), medium (M), and low (L) molecular size cDNA fractions are indicated. This figure was made after Konig et al. (2011).
Cut off the gel lane containing the size marker. Incubate it for 10 min in 20 mL of TBE buffer with 2 μL of SYBR green II added while gently shaking. Wash once with TBE and visualize by UV transillumination. Print the result using a 100% scale and use the printout as a mask to guide the excision of the cDNA bands from the rest of the gel (Note 25).
Together with the full L3-App sequence, the primer sequence accounts for 52 nt of the cDNA. Cut three bands, at 70–85 nt, 85–120 nt, and 120–200 nt with a sterile razor (Figure 3) (Note 26).
Use one of the following methods to crush the gel pieces:
Add 400 μL of TE to each gel piece and crush it with a 1 mL syringe plunger.
or
Prepare 0.5 mL tubes by piercing a hole in the bottom using a sterile 19G needle. Needle can be heated with a flame to assist in piercing a hole. Place a gel fragment inside and then place the tubes into 2 mL collection tubes. Centrifuge at room temperature for 2 min at full speed. Remove the 0.5 mL tubes and add 400 μL of TE to each collection tube.
Incubate samples for 1 h at 37 °C while shaking in a thermomixer. Then, place on dry ice for 5 min and put back for another hour at 37 °C while shaking.
Place two 1 cm glass pre-filters into a Costar SpinX column and transfer the liquid portion of the supernatant to these columns for each sample.
Centrifuge at full speed for 1 min in a tabletop centrifuge at room temperature. Collect the solution and add it together with 400 μL of RNA phenol/chloroform to a 2 mL phase lock gel heavy tube.
Shake at 37 °C for 5 min in a thermomixer.
Separate the phases by centrifuging the tubes for 5 min at full speed in a tabletop centrifuge at room temperature.
Transfer the aqueous layer into a new tube (be sure not to touch the gel matrix). Optionally, centrifuge the supernatant again for 1min and transfer the liquid into a new tube.
Add 1.5µL of GlycoBlue and 40µL of 3M sodium acetate (pH 5.5). Mix and add 1mL of ice-cold 100% EtOH. Mix by inverting the tubes several times and place overnight at -20°C.
Circularization of cDNA
The next day, centrifuge the samples at 16,100× g for 15 min at 4 °C. Remove the supernatants and wash once with 500 µL of ice-cold 80% EtOH. Centrifuge samples at 16,100× g for 10 min at 4 °C and remove supernatant.
Resuspend pellets in 8 μL of ligation mix:
H2O 4.9 μL
10× Circligase buffer 0.8 μL
50 mM MnCl2 0.4 μL
Circligase II 0.3 μL
Betain (5 M) 1.6 μL
Transfer to PCR tubes and incubate samples at 60 °C for 2 h (Note 27).
Then, add 30 μL of oligo annealing mix to each tube:
H2O 26 μL
FAST digest buffer 3 μL
10 μM Cut_oligo 1 μL
Anneal the oligonucleotide with the following program: 95 °C for 2 min, followed by successive incubations of 20 s, starting at 95 °C and decreasing the temperature by 1 °C each time until reaching 25 °C. Then, hold at 25 °C.
Add 2 μL of BamHI and incubate for 30 min at 37 °C; then, incubate for 5 min at 80 °C (Figure 2).
Transfer samples to 1.5 mL Eppendorf tubes. Add 350 μL of TE, 0.75 μL of GlycoBlue, and 40 μL of 3 M sodium acetate (pH 5.5) and mix. Then, add 1 mL of ice-cold 100% EtOH. Mix again and precipitate at -20 °C overnight.
Library preparation and gel extraction
The next day, centrifuge samples at 16,100× g for 15 min at 4 °C. Remove the supernatant and wash with 500 μL of ice-cold 80% EtOH as described above.
Resuspend the samples in 23 μL of H2O.
To determine the minimum number of PCR cycles required for library preparation, we recommend performing at least two initial PCR reactions using 17 and 20 cycles. Additional cycle numbers can also be tested. We usually do not sequence iCLIP libraries that require more than 22 cycles (Notes 28, 29).
Set up the following reaction mixture for each sample:
cDNA 1 μL
P3/P5 Solexa primer mix (10 μM each) 0.25 μL
2× Phusion HF PCR Master Mix 5 μL
H2O 3.75 μL
Run the PCR with the following conditions:
Mix 8 μL of PCR product with 2 μL of 5× TBE loading buffer, load on a 6% TBE gel, run at 180 V and 120 mA for 25–30 min, and stain the gel with SYBR Gold for 15 min.
Image the gel and estimate the minimum number of cycles needed for library preparation (Note 30). Overamplification during PCR often results in secondary bands that migrate at higher sizes, as detailed previously (Huppertz et al., 2014) (Figure 4). We amplify libraries in two halves using 10 μL of cDNA template in each half. Since 2.5-fold more concentrated cDNA is used for the first half (and second half), the number of cycles needed for library preparation is one less than the number of cycles used in the initial test PCR.
Figure 4. Representative PCR analysis of iCLIP libraries. Left: Preliminary PCR of iCLIP and input samples to determine the optimal cycle number. The iCLIP sample (labeled as IPs) was amplified using two different cycle numbers, i.e., 17 and 19, whereas the input (INP) was amplified using 17 cycles. While 19 and 17 cycles for IPs and input, respectively, lead to overamplification resulting in secondary products of higher sizes, 17 was chosen as the optimal cycle number to prepare the final libraries for iCLIP samples (labeled as IPs). Right: Gel image showing the final libraries for iCLIP and input samples prepared using the indicated cycle numbers. Note: Since the template cDNA was 2.5-fold more concentrated for the final PCR in comparison with the preliminary PCR, the iCLIP library (labeled as IPs) was prepared using 16 rather than 17 cycles. The final input library was prepared using 15 cycles. PCR products were resolved on 6% TBE gels and stained with SYBR gold.
Prepare the following reaction mix:
cDNA 10 μL
P3/P5 Solexa primer mix (10 μM each) 1 μL
2× Phusion HF PCR Master Mix 20 μL
H2O 9 μL
Run the same PCR program with the determined optimal cycle number.
Mix 8 μL of PCR product with 2 μL of 5× TBE loading buffer, load on a 6% TBE gel, run at 180 V and 120 mA for 25–30 min, stain, and visualize with SYBR Gold (Figure 4).
If the library appears under-amplified (i.e., the band is not clearly visible on the gel), immediately place back into the PCR machine for two or more cycles and re-run the gel.
Amplify the second half of the cDNA using the appropriate number of cycles, then combine both halves and proceed to agarose gel extraction steps.
Prepare 2% agarose gel in TBE. Let it cool down, add 1:10,000 SYBR Safe, mix, and pour gel.
Estimate the volume of each sample and accordingly add 6× loading dye into each.
Load samples on gel along with a low molecular weight DNA marker. Leave one well empty between samples.
Run at 90 V for 1 h (Note 31).
Use blue light illumination to visualize and then cut gel slices of appropriate sizes (see library amplification image for sizes) and place them into 15 mL Falcon tubes. Use a fresh razor blade for each sample.
Perform gel extraction using the Qiagen Gel Extraction kit according to the manufacturer’s instructions, with the following modifications: melt the gel pieces at room temperature. Elute in 20 μL of elution buffer. For improved yield, elute twice by taking the flowthrough from first elute and adding it back to the column.
Quantify the iCLIP libraries using Bioanalyzer and qPCR, as detailed previously (Huppertz et al., 2014). Mix High, Medium, and Low fractions into a 5:5:1 ratio for sequencing (Note 32).
iCLIP libraries can be sequenced using standard Illumina protocols. 50-nucleotide single-end runs are recommended.
Data analysis
Since this protocol essentially follows the workflow of the original iCLIP-seq method (Huppertz et al., 2014), the data can be subjected to the standard iCLIP analysis pipeline. Data analysis involves several steps, including de-multiplexing if multiple samples were run within one sequencing lane, mapping reads to the genome, collapsing of random barcodes and removing PCR duplicates, and identification of binding sites. Briefly, 51 nt raw reads that consist of three random positions, a 4 nt multiplexing barcode, and another two random positions, followed by the cDNA sequence, are initially de-duplicated based on the first 45 nt. Reads are de-multiplexed. The random positions, barcodes, and any 3′-bases matching Illumina adaptors are removed, and reads shorter than 25 nt are filtered out. The remaining reads are trimmed to 35 nt. These steps can be carried out using Trimmomatic (Bolger et al., 2014). The remaining reads are mapped to the human genome/transcriptome. To prevent false assignments of reads from repetitive regions, we generally remove any reads with a mapping quality <3 from further analysis. Several freely available pipelines can be utilized (e.g., PureCLIP, CTK, and iMAPs) (Krakau et al., 2017; Shah et al., 2017; Kuret et al., 2022). Moreover, the MEME package can be used to identify enriched sequence motifs (Bailey et al., 2015).
Validation of protocol
The number of PCR cycles required to amplify the cDNA during library preparation can directly impact the quality of the resulting data (i.e., libraries amplified with higher numbers of cycles contain higher amounts of PCR duplicates). The required number of cycles depends on several factors, including the protein abundance, quality of the antibody, IP efficiency, and technical limitations of the experimental procedure (Buchbender et al., 2020). In the original iCLIP method, 27–30 cycles were often needed to generate libraries (Huppertz et al., 2014). We have observed that, for our revised iCLIP-1.5 protocol, the number of cycles required for the amplification of cDNA libraries often lies between 16 and 20 cycles. Moreover, input material and abundant RBPs, such as PTBP1 and U2AF35, require even fewer PCR cycles, ranging from 14 to 16.
This revised protocol has been used to perform iCLIP-seq for a number of RBPs, including both well-characterized and relatively poorly studied RBPs, in several recent publications (Han et al., 2022; Nabeel-Shah et al., 2022; Song et al., 2022). In our most recent study, we performed iCLIP-seq for SP1 using our revised protocol (Song et al., 2022). SP1 iCLIP required 17 PCR cycles to obtain a DNA concentration suitable for high-throughput sequencing. After PCR duplicate removal, we obtained >18 million uniquely mapped reads per replicate (Figure 5A) (Song et al., 2022). In parallel to these experiments, we also performed one replicate of SP1 iCLIP-seq using the original protocol [data not published in Song et al. (2022)]. That iCLIP library required 28 cycles of PCR to obtain a DNA concentration sufficient for high-throughput sequencing. After discarding PCR duplicates, we obtained only ~1.6 million uniquely mapped reads (Figure 5A) (data unpublished), indicative of a low-complexity library and high PCR duplication rate. Thus, our revised iCLIP-1.5 protocol represents a substantial improvement to the original iCLIP-seq method, including enhanced adapter ligation, incorporation of SMI, and improved circularization of the cDNA.
In Figure 5B, we showcase the analysis of our published iCLIP-seq data generated using this revised protocol for a well-characterized RBP, CPSF5 (CFIm25) (Song et al., 2022). The analysis was performed using the CTK package to identify crosslink-induced truncation sites (Shah et al., 2017). We found that CPSF5 preferentially binds in the 3′ UTRs of target transcripts, consistent with its known function as a subunit of the cleavage and polyadenylation complex. Moreover, we identified the UGUA motif as the most enriched 4 mer around the cross-link sites of CPSF5, as reported in previous studies (Martin et al., 2012). These results show that our revised iCLIP-1.5-seq protocol faithfully captures known RNA-binding preferences of RBPs.
Figure 5. Sample results for iCLIP-seq data analysis. A. Comparison of number of uniquely mapping reads for SP1 iCLIP-seq data generated using either the revised protocol (reported here) or the original protocol. iCLIP-seq data for SP1 replicates 1–3 was reported as SP1.S2, SP1.S3, and SP1-E, respectively, in Song et al. (2022) (GSE165739). B. Left: Standardized metaplot profile showing the signal for CPSF5 along mRNA transcripts. iCLIP-seq was performed using GFP-tagged CPSF5 as reported in Song et al. (2022). Raw iCLIP data was acquired from the GEO database using the accession number GSE165739. Crosslink-induced truncation sites were identified using the CTK package (FDR ≤ 0.01). Right: Top RNA-binding motif identified in CPSF5 iCLIP-seq. MEME software was used for de novo motif discovery. U in the target RNA sequences was replaced by T for MEME analysis. Statistical significance was calculated against randomly assorted sequences.
Notes
For a standard experiment, ∼5–7 million HeLa cells or ∼10 million HEK293 cells are sufficient. We usually grow cells in two independent batches to represent biological replicates.
Make sure that tray/plate does not touch the UV bulbs or cover the energy detector. This can lead to errors in UV exposure readings.
The length of UV exposure can be optimized for new RBPs (range 150–400 mJ/cm2). However, increasing the UV dose could distort library preparation due to damaged RNA and could trigger DNA damage response pathways in the cells. Therefore, care should be taken when increasing the UV dose.
Cells grown in a monolayer are equally exposed to the UV light and hence only require a single round of irradiation to crosslink equally.
For suspension cells, centrifuge the cells, wash three times with 1× PBS (6 mL), leave cells in 1× PBS, and transfer to the 100 mm dishes. Irradiate and harvest cells as described.
Although Proteins A and G are structurally similar, they have different affinities for IgG subclasses across different species. For example, Protein A has greater affinity for rabbit, pig, dog, and cat IgG, whereas protein G has greater affinity for mouse and human IgG. Therefore, host species of the RBP-specific primary antibody should match the target species of the Dynabeads used. Protein A Dynabeads should be used for rabbit antibodies.
The quantity of required antibody and time of incubation depend on antibody quality and purity. This should be optimized in preliminary experiments. For GFP-tagged cell lines, we routinely use GFP recombinant rabbit monoclonal antibody from Thermo Fisher Scientific (G10362) or rabbit polyclonal anti-GFP from Abcam (ab 290). For SP1 iCLIP-seq reported in Song et al. (2022), we used 10 μg of SP1 antibody (Santa Cruz sc-17824).
RNA or protein concentration should be determined using Nanodrop or the Bradford assay, respectively. Normalize all the samples to the lowest concentration. 2 mg/mL is recommended as the optimal protein concentration. We take 1 mL of cell lysate per purification for each sample. Therefore, 2.2 mL of cell lysate is usually sufficient for two iCLIP experiments.
We employ several kinds of controls as needed, including samples where the RBP is absent from the original material (such as knockout or knockdown cells), a control where no crosslinking is done, and a control where no antibody is used during IP. In the case of epitope-tagged proteins (e.g., GFP and FLAG), use of cells expressing free FLAG/GFP should be used. It should be noted that FLAG/GFP-only cells usually yield iCLIP libraries with extremely low complexity, which are dominated by PCR duplicates, and hence not ideal for downstream computational analysis.
Sonication is strongly recommended for nuclear proteins. It shears the genomic DNA and helps to release proteins from the chromatin. Keep the probe ~0.5 cm above the bottom and avoid touching the tube sides. Repeat sonication (15 cycles, 0.3 s on, 0.7 s off) three times on ice. Leave the tubes on ice for ~3 min between rounds. Wash the probe with 70% EtOH and then with dH2O before sonication and in between samples.
Optimization of RNA fragmentation is extremely important for the success of the experiment. Enzymatic RNA digestion depends on the cell type and quantity of RNA present in the sample. Each new batch of RNase I should be tested for the optimal dilution. Treatment with high RNase I concentration (1:5 or 1:50) results in sharp bands on SDS-PAGE, corresponding to ~5 kDa above the predicted size of the protein. The optimal RNA fragments should range between 50 nt and 200 nt. The optimal RNase I dilution should yield RBP-RNA complexes that run in a diffused fashion ~20–80 kDa above the predicted size of RBP. Optimal RNase I dilution must be determined in the initial experiments prior to performing full iCLIP-seq experiment for a given RBP. For initial experiments, dephosphorylation and L3 adapter ligation steps should be omitted (see also Note 16 below). The presence of multiple sharp bands under high RNase I conditions generally indicates the co-purification of strongly interacting non-specific RBPs. To prevent the co-purification of a non-specific RBP, we recommend performing dual immunoprecipitation with urea denaturation, as detailed in Huppertz et al. (2014). For a detailed description of the RNase I optimization step, see Huppertz et al. (2014).
Although the optimal RNase I dilution should be determined for each new RBP, in our experience 1:250 dilution works for most RBPs in HEK293. For GFP-only, no-UV, and/or no-antibody control samples, RNase I dilution of 1:250 should suffice, and there is no need to test several dilutions since these controls do not yield any appreciable radioactive signal.
iCLIP lysis buffer contains SDS, which disables RNase I after prolonged incubation. Additionally, SUPERase•In RNase Inhibitor can be used to inhibit RNase I.
In our experience, overnight IP at 4 °C does not cause any appreciable RNA degradation. Optional: Cell lysate can be pre-incubated with 25 µL of protein A/G Dynabeads for 2 h at 4 °C to reduce background binding. Take 25 µL of protein A/G Dynabeads, wash three times with iCLIP lysis buffer, and incubate with the clarified lysate (before taking input samples) for 2 h at 4 °C with end-to-end rotation. After 2 h, place tubes on a magnetic rack and transfer the supernatant to a new tube. Continue with the procedure from Step 9.
If antibody depletion efficiency needs to be monitored, collect 20 μL at this step (flowthrough) for western blot comparison of the lysate before and after IPs. Additionally, it is possible to take input from the flowthrough rather than whole-cell lysates. Input from the clarified whole-cell lysate (recommended) retains the target RBP-RNA complexes, whereas flowthrough is specifically depleted of the bait.
For experiments aimed only at optimization of the RNase I concentration, and not for library preparation, steps involving washing with FastAP buffer, RNA 3′ end dephosphorylation, and L3 adapter ligation should be omitted. Proceed straight to radiolabeling after washing with high-salt and PNK buffers. Moreover, no-UV and no-antibody controls do not need to be subjected to RNA 3′ end dephosphorylation and L3 adapter ligation steps, since these controls often fail at the library preparation steps. In rare instances when we were able to successfully prepare iCLIP libraries for controls, sequencing data were almost always dominated by PCR duplicates. Since DTT can decrease the IP efficiency, it is removed from the ligation buffer used for L3 adapter ligation steps for IPs.
The Novex NuPAGE gels are important for the success of iCLIP. A pour-your-own SDS-PAGE gel (Laemmli) changes its pH during the run, which can get to pH ∼9.5 and lead to alkaline hydrolysis of the RNA. The Novex NuPAGE buffer system maintains the pH around 7 throughout the whole run. MOPS NuPAGE running buffer is recommended. Do not forget to add methanol when diluting 20× transfer buffer.
If the experiment is aimed only at optimizing RNase I conditions, then also perform western blotting to assess the recovery of the bait.
Different exposure times might be needed for autoradiography depending upon the RBP and IP efficiency. Expose for 30 min, 1 h, or even overnight at -80 °C. Although iCLIP buffer conditions eliminate physical protein–protein interactions, it is possible that undesired interacting proteins might co-purify with your protein of interest. If contaminating bands of other protein–RNA complexes are observed above your protein–RNA complex, only cut up to, but not including, these bands. Alternatively, IPs can be performed under denaturing urea conditions, as detailed in Huppertz et al. (2014). We do not use 32[P] that is older than two weeks. If radioactive smearing in +UV samples is not observed even after prolonged exposure, it might be due to failed IP, your protein binding very weakly to RNA, or cells not being properly exposed to UV during the crosslinking step. We do not recommend continuing the experiment for library preparations in such cases.
When comparing different RNase I conditions, the size of the radiolabeled band must change. Under high-RNase conditions, the radioactive smearing will diminish in comparison to the low-RNase, confirming that the band corresponds to a protein–RNA complex. Furthermore, the high-RNase condition helps to determine the size of the immunoprecipitated RBP, as the RBP will be bound to short RNAs and thus will migrate as a less diffuse band ∼5 kDa above the expected molecular weight of the protein. The radioactive band should become more diffuse in the low-RNase condition. On this basis, proceed to RNA isolation using the following guidelines: the average molecular weight of 70 nt RNA is ∼20 kDa. To isolate a broad range of RNAs between 40 and 300 nt in size (including the adapter), we recommend cutting a wide band of ∼15–80 kDa above the expected molecular weight of the protein. For further details refer to Huppertz et al. (2014). Note: At extremely low-RNase condition (e.g., 1:1,200), RBP–RNA complexes might not enter the wells. Control samples should not show any bands.
The remaining 5 µL can be used later in case the input cDNA libraries fail to amplify. When using antibody against endogenous proteins, the same input can be used for RBPs of similar molecular weights.
iCLIP primers are barcoded, as reported in Huppertz et al. (2014). Use distinct primers (Rt1clip–Rt16clip) for the control and the experimental replicates. Since primers contain distinct 4 nt barcode sequences, samples can be multiplexed. Distinct barcodes also allow us to control for cross-contamination between samples during library preparation. Moreover, RT polymerases other than Superscript III can also be used. For example, irCLIP utilized f TIGRT-III reverse transcriptase (Ingex, #TGIRT50).
It is possible to multiplex up to three samples with different barcodes at this stage. Alternatively, cDNA libraries can be made separately for each sample and mixed after the PCR for sequencing.
We have observed that the gel running time can vary depending on the power pack that is used. For initial experiments, it is important to note the time it takes for the lower (dark blue) dye to nearly reach the bottom. At least in the initial experiments, it is recommended to include DNA size markers.
This step is not needed if you are not using DNA size markers.
We recommend avoiding cutting gel slices higher than 200 nt or lower than 70 nt. cDNAs fragments below 70 are often too short to accurately map to the genome. cDNA fragments that are too long are not suitable to obtain desired resolution. If binding to miRNAs or other short RNAs is not of interest, it is not necessary to isolate low band.
We have increased the incubation time and included betain in the reaction mixture. Increasing the incubation time and the addition of betain to a final concentration of 1M in the reaction mixture have been found to improve the circularization of difficult-to-ligate substrates.
cDNA libraries must never be brought to the area where iCLIP RNA or cDNA work is done, in order to avoid cross-contaminating samples. Library preparation should be carried out in a designated area, ideally far away from general lab workplaces.
Additional cycle numbers can also be tested. It is important to optimize PCR cycle numbers in order to avoid overamplification. In our experience, 17–20 cycles produce a sufficient cDNA concentration for sequencing for most RBPs. Strong RBPs and input libraries often require fewer cycle numbers, usually ranging from 14 to 16.
The size of the cDNA will be the size of the product minus the combined length of the P3/P5Solexa primers and the barcode (128 nt). Therefore, a band cut at 70–85 nt on the cDNA gel (with 20–30 nt cDNA + 52 nt primer) is expected to generate 145–160 nt PCR products. For a detailed description, see Huppertz et al. (2014).
Longer runs may give better resolution but larger cut sizes. We incubate the gel running apparatus in 20% bleach in between different runs. Rinse thoroughly with water to clean the apparatus before using it for the next run.
The ratio of the bands can be adjusted. For example, if miRNAs are important, use more of the lower bands. Also, take the quality of each PCR sample into account; if one of the ½ PCR reactions was overamplified, use more of the reaction that is in the right range of amplification.
Acknowledgments
This research was funded by the Canadian Institutes of Health Research Foundation Grant FDN-154338 to JFG. We thank Dr. Benjamin J Blencowe and his laboratory members including Drs. Thomas Gonatopoulos-Pournatzis, Ulrich Braunschweig, Andrew Best, and Esha Sharma for helpful discussions and technical advice. We also thank all members of the Greenblatt laboratory, particularly Nujhat Ahmed for helpful discussions and her critical reading of the manuscript, and Shuye Pu for his help with iCLIP data analysis. This protocol was used in Song et al. (2022).
Competing interests
The authors declare no competing interests.
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4,689 | https://bio-protocol.org/en/bpdetail?id=4689&type=0 | # Bio-Protocol Content
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Automated 384-well SYBR Green Expression Array for Optimization of Human Induced Pluripotent Stem Cell Differentiation
MC Max Y. Chen
LH Laurin Heinrich
FZ Faria Zafar
KS Kamilla Sedov
BS Birgitt Schuele
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4689 Views: 889
Reviewed by: Xi FengSubhra Prakash HuiLei Gao
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Original Research Article:
The authors used this protocol in Cell Stem Cell Dec 2022
Abstract
Cell populations and tissues exhibit unique gene expression profiles, which allow for characterizing and distinguishing cellular subtypes. Monitoring gene expression of cell type–specific markers can indicate cell status such as proliferation, stress, quiescence, or maturation. Quantitative reverse transcriptase PCR (qRT-PCR) allows quantifying RNA expression of cell type–specific markers and distinguishing one cell type from another. However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive. Processing RNA sequencing data can take several weeks, which is not optimal for quality control and monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type.
A more cost-effective assay is based on SYBR Green technology. SYBR Green is a nucleic acid dye that binds to double-stranded DNA, absorbs blue light at 497 nm, and emits green light at 520 nm up to 1,000-fold upon intercalation with double-stranded DNA. Amplification of a region of interest can be quantified based on the level of fluorescence intensity when normalized to a housekeeping gene and compared to control conditions. Previously, we established a SYBR Green qRT-PCR protocol to characterize samples using a limited set of markers plated on a 96-well plate.
Here, we optimize the process and increase throughput to a 384-well format and compare mRNA expression to distinguish iPSC-derived neuronal subtypes from each other by increasing the number of genes, cell types, and differentiation time points. In this protocol, we develop the following: i) using the command-line version of Primer3 software, we design primers more easily and quickly for the gene of interest; ii) using a 384-well plate format, electronic multichannel pipettes, and pipetting robots, we analyze four times more genes on a single plate while using the same volume of reagents as in a 96-well plate. The advantages of this protocol are the increased throughput of this SYBR Green assay while limiting pipetting errors/inconsistencies, reagent use, cost, and time.
Graphical overview
Figure 1. Overall optimized SYBR Green qRT-PCR workflow. (A) Primers are designed through the command-line version of Primer3. The program takes a couple of files as arguments: 1) an input file containing a sequence of the region of interest and a target, and 2) settings file with custom settings and primer picking conditions. The results are saved to a text file, checked for secondary and tertiary structures, then synthesized. (B) Primers are then plated using either multichannel pipettes with a pipetting aid or an automated pipetting robot. Plates are left to dry at room temperature and can be stored for an indefinite time. (C) Meanwhile, RNA is extracted from cell samples, reverse-transcribed into cDNA, then plated onto pre-coated 384-well plates. SYBR Green qRT-PCR is run and analyzed with QuantStudio software and Microsoft Excel.
Keywords: SYBR Green Quantitative polymerase chain reaction (qRT-PCR) Gene expression RNA cDNA Automation 384-well format
Background
Quantitative reverse transcriptase PCR (qRT-PCR) is a fundamental laboratory technique to quantify the RNA gene expression of a biological sample. Previously, we developed a protocol that quantifies PCR products using an SYBR Green array in a 96-well format. SYBR Green is a dye that fluoresces when intercalated into double-stranded DNA (Srinivasaraghavan et al., 2022). As a region of interest is PCR-amplified, the amount of fluorescence is directly proportional to the amount of DNA content in the sample. This assay provides an accurate, cost-effective, and fast way to quantify the mRNA gene expression of a cell or tissue sample. Here, we optimized the throughput with a 384-well format, increasing the number of markers, simplifying and standardizing primer design, and automating pipetting of the plate to reduce manual pipetting errors and ensure volume accuracy. Each well uses 5 μL of reaction mix instead of the 20 μL in a 96-well plate, but the whole plate contains four times the number of wells. Thus, for the same volume of reagents used, one plate can output four times the data.
Scaling up the assay from 96- to 384-well plates requires optimization
However, qRT-PCR methods such as TaqMan technology require fluorescent reporters to characterize target genes and are challenging to scale up as they need different probes for each reaction. Bulk or single-cell RNA transcriptomics is time-consuming and expensive (Table 1). Processing RNA sequencing data can take several weeks, which is not optimal for monitoring gene expression, e.g., during a differentiation paradigm of induced pluripotent stem cells (iPSCs) into a specialized cell type. We improved primer design by using the command line version of Primer3 software. This avoids using the web interface, where user input errors can occur. Instead, the program uses 1) a text file containing all settings and primer conditions; 2) an input file containing the gene name, full gene sequence, and target region to amplify; and 3) an output file/folder containing compatible primers. Coating 384-well plates with primers is labor-intensive and yields potential user error. To mitigate this, we use one of three methods for plating primers: 1) use of a PlatR pipetting aid, which uses a tablet that lights up wells from underneath, 2) an E1-ClipTip adjustable tip-spacing electronic pipette, or 3) Integra ASSIST PLUS pipetting robot to pipette larger arrays with more plates. All three methods increase the throughput of generating coated plates and the consistency of volumes across all assays.
With the optimized format we describe in this protocol, we can assess 31 markers in triplicates on four samples on a single 384-well plate. Alternatively, 40 markers can be plated in triplicates on a plate with three samples. The number of wells needed for each will vary depending on the number of genes, replicates, and samples.
Human iPSC modeling
Patient-specific iPSC lines can be generated and differentiated to study cell processes, developmental defects, or disease mechanisms on a human genomic background. However, since differentiating these cells requires careful design of differentiation protocols, a method is needed to validate and confirm that this differentiation resembles that of in vivo tissue.
The use of many markers allows SYBR Green to be used to characterize many cell types. In contrast, the customizable plate allows flexible comparison of samples at different differentiation time points or between different cell types. Because most of the workflow for this SYBR Green protocol can be done within 1–2 days, the low turnaround time is ideal for characterizing cells at various time points during the differentiation process.
Optimization and validation of differentiation protocols using cell type–specific markers
The data analysis for RNA-seq methods can take some time and requires advanced bioinformatics tools. Our SYBR Green qRT-PCR gene expression can be completed in ~2 days, in which cells are harvested, cDNA synthesis is performed, and cDNA samples are quantified (Figure 1).
To show that the optimized primer design process can generate more markers and establish markers for optimizing current and future differentiation protocols, we have developed a library of markers to characterize multiple neuronal types at different developmental time points (Supplemental Table 1). This primer inventory includes cortical, striatal, and dopaminergic neurons and progenitors, pluripotency genes, astrocytes, hematopoietic precursors, microglia, retinal genes, synaptic markers, and germ layer markers.
Please note that we have saved Supplemental Table 1 and Supplemental Table 2 as individual files.
Table 1. Comparison between different RNA analysis tools
Assay type # of genes Multiplexing Approx. time Cost
TaqMan ~96 (4 samples) 1–6 ~1 day $498/"scorecard"
Bulk RNA-seq 300,000 1 Weeks $200–300
scRNA-Seq ~7–8,000 Up to 8 Weeks ~$1,000
SYBR Green ~30 (4 samples) 1–8 ~1 day ~$85/384-well plate
Materials and reagents
MicroAmpTM Optical 384-well reaction plate (Applied Biosystems, catalog number: 4309849)
8-channel VOYAGER adjustable tip electronic pipette (Integra Biosciences, catalog number: 4721)
1.5 and 0.6 mL RNase-free microcentrifuge tubes
RNase-free filter pipette tips (P1000, P200, P20, and P2)
PowerUpTM SYBRTM Green Master Mix (Thermo Fisher, catalog number: A25742)
High-Capacity cDNA rev Transcription kit (Thermo Fisher, catalog number: 4368814)
DNase I, amplification grade (Invitrogen, Thermo Fisher Scientific, catalog number: 18068015)
PurelinkTM RNA Mini kit (Invitrogen, catalog number: 12183018A)
Homogenizer (Invitrogen, catalog number: 12183026)
2-mercaptoethanol (Aldrich Chemistry, Sigma-Aldrich, catalog number: M2650)
Nuclease-free water
Phosphate buffered saline without calcium and magnesium (PBS) (Thermo Fisher, catalog number: 70-011-044)
Custom-designed forward and reverse primers diluted to 100 μM in nuclease-free water
Integra GRIPTIP 6000 for ASSIST PLUS (Integra Biosystems, catalog number: 6403)
Bucket with wet ice
Personal protective equipment (gloves, lab coat, goggles)
10% bleach for decontamination
Equipment
Microcentrifuge 5415C (Eppendorf, catalog number: M7282)
Refrigerated centrifuge (Beckman Coulter, catalog number: GS6 Allegra)
Mini centrifuge (Fisher Scientific, catalog number: 05-090-100)
MiniAmpTM thermal cycler (Applied Biosystems, Thermo Fisher, catalog number: A37834)
NanoDrop spectrophotometer (Thermo Fisher, catalog number: 13-400-525)
PlateR visual pipetting aid tablet (Biosistemika, catalog number: P-10)
QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, catalog number: 4485691)
Integra ASSIST PLUS pipetting robot (Integra Biosciences, catalog number: 4505)
E1-ClipTip 200 Bluetooth electronic single channel pipette (Thermo Fisher, catalog number: 4670020BT
E1-ClipTip 384 12.5 electronic adjustable tip-spacing multichannel pipette (Thermo Fisher, catalog number: 4672010BT)
Software
QuantStudio 6 and 7 Flex Real-Time PCR software v1.7.2 (Thermo Fisher, https://www.thermofisher.com/us/en/home/global/forms/life-science/quantstudio-6-7-flex-software.html)
Primer3 (Steve Rozen, Helen Skaletsky, Triinu Koressaar, Maido Remm, and Andreas Untergasser, https://github.com/primer3-org/primer3)
Beacon Designer (Premier Biosoft, http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1)
In silico PCR prediction (UCSC Genome Browser, https://genome.ucsc.edu/cgi-bin/hgPcr)
UNAFold (Integrated DNA Technology, https://www.idtdna.com/UNAFold). Create an account to use the software.
Integra VIALAB automation software (Integra, https://www.integra-biosciences.com/united-states/en/pipetting-robots/assist-plus/download-vialab)
RStudio Desktop (RStudio, https://www.rstudio.com/products/rstudio/download/)
Procedure
RNA extraction with PureLink RNA Mini kit
We optimized RNA extraction for neuronal cultures. Previously, cells were dissociated from the culture well and then pelleted before lysis for RNA extraction (Srinivasaraghavan et al., 2022). However, RNA in axons and dendrites is lost as cells detach and round up during dissociation, resulting in only capturing RNA from the cell soma. Alternatively, TRIzol can be used to extract RNA but risks phenol contamination due to its manual pipetting of the separated supernatant layer. Use the protocol according to the manufacturer's instructions [section titled "Purifying RNA from Animal and Plant Cells" (LifeTechnologies, 2012)] with the following modifications:
Instead of performing “Lysis and Homogenization” steps, perform the following:
Aspirate culture media from adherent cells.
Wash twice with PBS. Bring culture plate to RNA extraction lab bench.
Note: RNA extraction should be performed on a separate bench from cell culture and other assays to avoid RNAse contamination from surfaces. Clean and wipe down all surfaces, have a dedicated set of pipettes for RNA work, and use RNAse/DNAse-free plastic consumables, especially if working close to DNA extraction kits with RNAse.
Aspirate PBS and add 300 μL of lysis buffer (containing 2-mercaptoethanol) for each 6-well plate (9.6 cm2). Discard PBS into the liquid waste container containing 10% bleach.
Ensure lysis buffer has lysed all cells on the plate (liquid is slimy/viscous and has no precipitate or cell pellet) and then transfer to homogenization tubes.
Note: Do not wait more than ~5 min to process the sample through the homogenization tubes, as this might reduce RNA yield.
Spin homogenization tubes in the centrifuge at 12,000× g for 2 min.
Proceed with "Binding, Washing, and Elution steps" with the cell lysate.
Elution of RNA and aliquoting
Add 30 μL of nuclease-free water to the cartridge.
Incubate at room temperature for 1 min.
Centrifuge at 12,000× g for 2 min.
Repeat steps 1–3 with eluted RNA.
Measure RNA content
Measure RNA concentration with a Nanodrop or comparable spectrophotometer.
Clean Nanodrop surface with nuclease-free water.
Add 1 μL of a blank (nuclease-free water).
Wipe off the water drop.
Add 1 μL of purified RNA to the Nanodrop surface. Close the lid and press Analyze.
Repeat steps c–d for each sample. High-quality RNA has an A260/A280 absorbance ratio of ~2.0 and an A260/A230 ratio of ~2.2.
Note: Low-yield RNA samples can be eluted a second time through the spin column, if samples were not incubated for 1 min prior to centrifugation. Low-quality RNA is usually a result of either RNA degradation or other compounds contaminating the sample from previous steps. For more troubleshooting, see the Troubleshooting section of the PureLink manual (LifeTechnologies, 2012).
Important: Aliquot RNA into 1 μg aliquots to avoid freeze-thaw cycles.
Eliminate gDNA contamination with DNase I treatment
Use protocol as outlined in the previous protocol (Srinivasaraghavan et al., 2022). Perform on each 1 μg aliquot.
Generate cDNA with a High-Capacity cDNA rev Transcription kit
Use protocol as outlined in the previous protocol (Srinivasaraghavan et al., 2022).
It is recommended to dilute cDNA to a 10 ng/μL working concentration.
Primer design and automated primer plating
Primer design
All primer design conditions are the same as previously described (Srinivasaraghavan et al., 2022), except for using the command-line version of the Primer3 software. Once set up, the command-line interface of Primer3 eliminates user error, generates primer pairs for different genes much more quickly, and automates the primer selection process. The use of the command-line interface is beyond this protocol's scope, but a few commands are needed to get started (Table 2).
Table 2. Basic command line functions to help with installing/using Primer3
Action Command
Show current working directory pwd
Enter a folder/directory cd *name of folder*
Move up a folder/directory (exit a folder) cd ..
List folder contents ls
Make a new folder mkdir *name of folder
Delete file rm *name of file*
Tab Autocomplete file/folder name if it exists
Use the command-line version of Primer3 software to design primer pairs.
Install the Primer3 software:
Method 1 (manual install)
i. Navigate to the Primer3 GitHub page (https://github.com/primer3-org/primer3).
ii. Under "Code," select "Download ZIP."
iii. Extract the folder to the local drive, not a cloud drive.
iv. Navigate to the extracted folder in the command line and run the following commands:
make
make test
Method 2 (MacOS command-line install)
i. Open Terminal.
ii. Install XCode command line tools (if not already installed) by entering the following command:
xcode-select –install
iii. Enter the following commands line by line into Terminal:
git clone https://github.com/primer3-org/primer3.git primer3
cd primer3/src
make
make test
iv. Primer3 is now installed.
Method 3 (Linux command-line install)
i. Follow Method 2, but instead of xcode-select --install, run the following command:
sudo apt-get install -y build-essential g++ cmake git-all
ii. Proceed with the rest of the steps from Method 3.
Navigate to the primer3 folder and create an "input.txt" file and "settings.txt" file by entering the following commands into Terminal:
touch input.txt
touch settings.txt
Run ./primer3_core ../example to ensure the program runs correctly.
Edit the settings.txt file by following the format listed under "20. Primer3 settings file format" on the Primer3 website and with the settings values listed in SupplementalTable 2.
Edit the input.txt file described under "13. Input and output conventions" on the Primer3 website. This will serve as the template sequence for which Primer3 will pick primers (Table 3).
Table 3. Sample input file containing gene name, template sequence to pick primers from, and the target region
Note: Setting the sequence target around an exon-exon junction at position 54 of the sequence will require the program to design the primers surrounding this location for the listed number of nucleotides. In the above case, the program will be required to design primers that surround the four base pairs around sequence number 54 (highlighted in yellow).
Go to the NCBI website and choose the gene of interest, taking note of the exon junction locations and coding sequence locations. For additional detail, see Section E. Primer Design (Srinivasaraghavan et al., 2022).
Copy and paste the sequence into the "SEQUENCE_TEMPLATE" section of the input.txt file and choose a target location.
Note: The sequence must be formatted in a continuous string without newline characters. To remove newline characters from the sequence:
i. Download the FASTA file.
ii Navigate to the file in the terminal and run the following command, where sequence.txt is the FASTA file:
tr -d ‘\n’ < sequence.txt
In the terminal or command line interface, navigate to the primer3 folder (if not already in there) and run the following command:
./src/primer3_core --p3_settings_file settings.txt input.txt --output output.txt
i. This runs "primer3_core" in the /src/ folder, using the input file "input.txt," the settings file "settings.txt," and output file in the /output/ folder called "output.txt".
Note: More arguments are described in the section "Command line arguments" on the Primer3 website. The input file does not need a flag.
ii. Resulting primer results are printed onto the output.txt file that is designated above.
Run quality control on primer results to find optimal primer pairs as stated in the original protocol (Srinivasaraghavan et al., 2022):
Beacon designer
Checks for primer dimers and self-dimers. Ensure ΔG values are >-3.5 (more positive).
In-silico PCRChecks if primers amplify the correct regions in the genome.
IDT UNAfold
Checks if the amplified product produces any secondary structures, such as stem loops. Ensure ΔG >3.5.
Order primers at 40 nmol without purifying.Reconstitute primers in nuclease-free water to 100 μM.
Make separate working concentrations of mixed forward/reverse primers at 1.5 μM for 384-well plating.
For each gene, combine the forward and reverse primers and nuclease-free water (Table 4). For a 5 μL SYBR Green reaction mix, 2 μL of 1.5 μM primer solution will be needed to make a 300 nM primer concentration. Adjust volumes.
Table 4. Diluting forward and reverse primer solutions
Reagent Volume (μL)
Fwd primer (100 μM) 22.5
Rev primer (100 μM) 22.5
Nuclease-free water 1,455
Total volume 1,500
Store stock 100 μM primer solutions at -20 °C.
Primer plating
We developed three different approaches to plating primers:
1. Manual pipetting
2. Multichannel pipetting
3. Automated
While primers can be plated in any position and order, we find it most efficient and straightforward to plate them in an order consistent across all samples (Figure 2). It also helps in reducing pipetting errors.
Figure 2. Example plate template for SYBR Green. Each filled colored circle is a unique gene; colored squares represent a different cDNA sample. Leave the bottom row for controls.
Manual pipetting with PlatR pipetting aid
This system is ideal for small-scale pipetting with single-channel pipettes on 384-well plates. It consists of three components (Figure 3):
i. Tablet and tablet stand.
ii. Plate holder.
iii. Foot pedal.
Figure 3. PlatR pipetting aid setup to reduce manual pipetting errors in 96 and 384-well plates. Front (A) and side (B) setup of the PlatR pipetting aid. Plates are held in place by the adhesive-backed and removable plate holder (C). A foot pedal (D) is used to advance to the next well, which frees up hands. All these together increase ergonomics, pipetting consistency, and reduce user error.
Place tablet on tablet stand.
Open the app and create a new 384-well plate.
Select the Reagents tab and create/assign primers to each well (Figure 4); then, select the Samples tab to assign cDNA/SYBR Green samples to each well (Figure 5) (How to set up your pipetting protocol | Pipetting Aid PlatR, 2021).
Attach the plate holder to the tablet and place the plate in the holder.
Connect the foot switch via Bluetooth and place it on the ground.
Proceed with pipetting primers or master mix.
Figure 4. PlatR Reagents setup screen for designating primers
Figure 5. PlatR Samples setup screen for designating cDNA/SYBR Green master mix
Electronic multichannel pipetting with E1-ClipTip adjustable tip-spacing multichannel
This system can be supplemented with the PlatR pipetting aid.
On the pipette, choose "Presets" -> "Stepper" and adjust the volume per step to 2 μL.
Proceed with pipetting as usual.
Note: Ensure tips touch the bottom of the well when pipetting, especially for such low volumes.
Integra ASSIST PLUS pipetting robot
Plate design: the pipetting robot picks up/pipettes eight wells at once and cannot rotate, so choose a plate design/pipetting scheme that allows pipetting 8 wells in one direction. The 384-well plates in slots B and C can be rotated, but not the working solution of primers in slot A (Figure 6). See an example plate design below. For a 4-sample plate, columns 1–6 contain one sample, 7–12 contain another sample, etc. Eight different primers will be plated in rows A–H or I–P in triplicates.
Download, install, and open Integra VIALAB to design automated pipetting instructions for the ASSIST PLUS (“Download VIALAB | INTEGRA,” 2018).
i. Create a new plate and select "3 Position Universal Deck."
ii. “Choose Pipette” -> Voyager 8-channel 12.5 μL, part number 4721.
iii. “Choose Tips” -> 12.5 μL GripTip LONG, part number 6403.
iv. Under the gray square labeled "A", select "Choose labware." This will be used as a reservoir containing primers. Choose a 96-well plate from the list, as dimensions will be different between different 96-well plates.
v. In the gray boxes labeled "B" and "C," select "Choose labware” -> "384-microampTM Optical plate." Click the two circular arrows button to rotate the plate as needed (Figure 6).
Figure 6. Materials tab on VIALAB to specify materials (pipette, tips, plates, etc.)
vi. Choose the Method tab at the top.
vii. With the "Initial Volumes" block selected, select all wells of the stock primer solutions to be used. Enter the initial volume.
viii. Click the "+" button and select the "Repeat Dispense" step.
1) In the Pipetting location tab (Figure 7):
(a) "Edit Source" -> select the first eight wells of the 96-well plate from which to choose primers.
(b) "Edit Target" -> click or drag the wells on the right to pipette to the 384-well plate. Select tip spacing and pipetting direction.
(c) Select the "close" tip spacing option. Select "Pipetting direction" to the right.
Figure 7. Initial Volumes block under the Methods tab
2) In the Volumes tab (Figure 8):
(a) If the stock primers are 1.5 μM forward/reverse mixed primers, select "Volume" -> 2.00 μL.
(b) Select all "Post Dispense," "Volume," and "Pre-dispense" settings as in Figure 8:
Figure 8. Volumes tab to designate the volume to dispense, as well as pre- and post-dispense to ensure accurate pipetting
3) In the Source Details tab:
(a) Pipetting Height -> 3.0 mm
(b) Safety Bottom Offset -> 2.0 mm
4) In the Target Details tab:
(a) “Pipetting Height” -> 1.8 mm
(b) “Safety Bottom Offset” -> 0.7 mm
(c) "Tip Touch" tab:
“Tip Touch Height” -> 1.6 mm
5) Keep all other settings as default.
6) Copy and repeat these settings for every set of eight primers to pipette.
ix. Select the "Simulation" tab to check if the pipette moves as intended.
x. Connect the pipette to the ASSIST PLUS.
xi. On the pipette: Toolbox -> Communications.
xii. In the "Transfer" tab, select the connected device.
xiii. Begin the pipetting program.
After plating primers, centrifuge plates at 200× g for ~2 min. Then, dry and store covered at room temperature.
Run qRT-PCR
Use qRT-PCR settings and protocol as indicated in Srinivasaraghavan et al. (2022), except for the following:
Change the plate type to 384-well.
Combine nuclease-free water, cDNA, and SYBR Green master mix into a 5 μL reaction mix for each well (Table 5).
Table 5. SYBR Green master mix reaction volumes per well
Reagent Volume (μL)
PowerUp SYBR Green Master Mix 2.5
cDNA (10 ng/μL) 1.0
Nuclease-free water 1.5
Total per reaction 5.0
After adding the Master Mix and cDNA combination to each well, spin down the plate at ~2,000 rpm for ~3 min to eliminate air bubbles in the reaction mix.
Important: Air bubbles at the bottom of the well will result in inaccurate results.
Cover the plate with an optical adhesive cover and place it in the QuantStudio instrument.
Data analysis
We use the QuantStudio software to analyze the data, such as calculating mean Ct, Ct SE, ΔΔCt, and fold-change if target and housekeeping genes for both treated and untreated samples are present on the same plate. We run each gene and sample combination in triplicate wells. The fold change of each gene of each sample is calculated with the following equation:
where each Ct is the mean Ct. In other words,
Since fold change is calculated using mean Ct values from differentiated neurons (treated) and iPSC samples (untreated), some genes such as Neurofilament or MAP2 are not expressed in iPSCs. This results in Ct values of "undefined," since undetected mRNA might take more than 40 cycles to amplify, and thus make it difficult to calculate the fold change. To circumvent this, Ct values for undefined results are arbitrarily set to 35 or higher, since Ct values above 35 likely represent low or undetectable mRNA expression levels in the sample.
Alternatively, the data can be analyzed using R. By using R packages such as gdata, tidyr, and dplyr, .xls files can be imported into RStudio and analyzed. This method has not been implemented here yet.
To demonstrate the high throughput capabilities of this assay, iPSCs from the KOLF2.1J cell line were differentiated into three neuronal subtypes: developing cortical, developing striatal, and developing mesencephalic floorplate progenitors for 20 days in vitro (Calatayud et al., 2022). Developing mesencephalic floorplate progenitors were differentiated with two different concentrations of Sonic Hedgehog (SHH), as well as an SHH alternative, Smoothened agonist (SAG) (Figure 9). All five samples were differentiated simultaneously. Each gene for each sample type was amplified as technical triplicates. The advantage of using electronic pipettes and pipetting aids is reducing pipetting volume variability, which is crucial to ensuring consistency among low-volume technical replicates.
Each plate can be customized to contain unique layouts with different genes. The number of genes included on a given plate varies depending on the number of samples. As shown in Figure 3, a consistent plate design helps to reduce pipetting errors and maintain consistent results. For example, the plate in Figure 2 can fit four samples, each with 30 genes in triplicates.
Results
While throughput has increased, reagent use has not. Whereas the SYBR Green assay on 96-well plates previously used 20 μL per reaction, this assay only used 5 μL of cDNA/SYBR Green master mix, thus increasing the maximum number of reactions per plate from 96 to 384.
Previously, forward and reverse primers were designed using the graphical user interface version of the Primer3 website. The command-line interface of the Primer3 software is identical in parameters and results to the website, while being more suitable for designing multiple primers concurrently. Several files are uploaded into the program to simplify the primer design process: a settings file, an input file, and an output file. Since primer conditions and settings are not changing for SYBR Green, a settings file containing all those primer conditions is created and fed into the program, as opposed to manually entering conditions (max/min melting temperature, ion concentrations, etc.). An input file is also provided to the system, containing the gene name, the RNA sequence or template to choose primers from, and a target region. The gene sequence can be imported as a FASTA file from the NCBI website, and the target is preferably chosen based on exon-spanning regions. Finally, an output file can be specified to save results for later review. This serves to view past results and is an easy way to update results if the selected target region does not have suitable primer pairs. This program creates a digital trail that can be easily referenced if the primer conditions (e.g., secondary/tertiary structures form) are not optimal.
The use of various electronic pipetting aids increases pipetting ease, throughput, and ergonomics. Using a single-channel pipette on such small 384-size wells introduces high variability of volumes, user error, and inefficient use of time, and places strain on hand/arm ergonomics. The PlatR was introduced initially as a quick, cheap, and easy alternative to easily keep track of which well is being pipetted, since all reagents are a similar color and wells are extremely small and tight. The use of adjustable tip-spacing electronic multichannel pipettes further improves ergonomics and pipetting consistency by decreasing the variability of pipetted volumes across multiple wells. Adjustable tip spacing allows versatile pipetting from Eppendorf tubes or 96-well plates into 384-well plates.
This assay was also designed to emphasize rigor and reproducibility. We aimed to eliminate as many opportunities for human error and variability as possible, including implementing the command line interface for the primer design and using multichannel electronic pipetting for primer plating. Differences in manual pipetting, especially at low volumes in a 384-well plate, can contribute to variability between technical replicates. While other commercial arrays include more genes per plate, our plate design contains fewer markers to maintain multiple replicates. Every gene and sample combination is always performed in triplicate. In addition, both differentiated and undifferentiated samples and housekeeping genes are included on each plate to reduce plate-to-plate variability and normalize target gene expression.
Application to iPSC-derived neuronal subtypes
KOLF2.1J iPSCs were differentiated into three neuronal subtypes: developing cortical, striatal, and developing floorplate progenitors for 20 days. As shown in Figure 9, there seems to be an upregulation of cortical developmental markers among cortical and striatum progenitors. We also detect the upregulation of ganglionic eminence developmental markers among cortical and striatal progenitors. These results allow us to quickly modify the differentiation protocols or terminate the experiment when used as a quality control measure of an existing protocol.
Figure 9. Fold-change mRNA gene expression of cortical, striatal, and floorplate neuronal progenitors normalized to KOLF2.1J iPSCs (Pantazis et al., 2022)
Pitfalls and future directions
As with any non-specific dsDNA binding molecules, a disadvantage of SYBR Green is the potential for SYBR Green to bind to primer dimers or other non-specific dsDNA products. However, this is mainly prevented by the several in silico quality control steps, Beacon Designer, In-Silico PCR, and IDT's UNAFold, which limit primer dimer formation, ensure the correct region is amplified, and reduce amplicon nucleic acid folding (Srinivasaraghavan et al., 2022). Using SYBR Green without these quality control methods can result in overestimating the target or even a false positive, as more fluorescence is detected from the non-specific binding than that bound to the target of interest. In addition, there can be a reduction in dynamic range as non-specific binding increases the noise, which leads to a more minor perceived difference between the controls and sample/genes of interest.
As throughput increases, so will the amount of data to analyze. While the QuantStudio software and Microsoft Excel are sufficient for analyzing data with one reference sample or data from one or two plates, increasing the number of samples, genes, and plates makes data analysis more sophisticated beyond what an Excel spreadsheet can handle. Thus, as mentioned before, utilizing R, a programming language commonly used by bioinformaticians, will be the subsequent development to make data easier to organize and analyze. Passing the Excel spreadsheet through an R script that manages exported QuantStudio data would make analyzing statistical data, generating graphs, and pooling multiple runs together more effortless.
In the case of low conversion or differentiation rate, the small percentage of differentiated cells would still result in elevated lineage-specific marker expression compared to iPSCs, especially when iPSCs do not express those markers. Therefore, the qPCR array needs to be combined with additional quality control steps, such as visual check and/or FACS analysis for differentiation efficiency.
Optimizing the SYBR Green assay to 384-well plates increases throughput considerably by increasing the number of genes and samples that can be analyzed in a single experimental run, while using the same quantity of reagents as a standard 96-well SYBR Green array. In addition, because this can be performed in a relatively short time, this assay is ideal for time-sensitive scenarios, such as analyzing gene expression during a differentiation protocol.
Acknowledgments
This was supported by the California Institute for Regenerative Medicine Bridges program grant #EDUC2-08394.
Figure 1 was created with BioRender.com.
This protocol is an advancement for automation and throughput from Srinivasaraghavan et al. (2022).
Competing interests
Nothing to disclose.
Ethics
The human KOLF2.1 iPSC line was used in accordance with the Stem Cell Research Oversight protocol 754 (Stanford University).
References
Calatayud, C., Muñoz-Pedrazo, E., Fernández-Gallego, S. and Verstreken, P. (2022). Modular generation of cortical, striatal and ventral midbrain progenitor cells [WWW Document]. protocols.io. URL https://www.protocols.io/view/modular-generation-of-cortical-striatal-and-ventra-b54cq8sw (accessed 8.19.22).
Download VIALAB | INTEGRA [WWW Document], 2018. URL https://www.integra-biosciences.com/united-states/en/pipetting-robots/assist-plus/download-vialab (accessed 8.30.22).
How to setup your pipetting protocol | Pipetting Aid PlatR, 2021.
Life Technologies (2012). PureLink® RNA Mini Kit. Life Technologies, Carlsbad.
Srinivasaraghavan, V., Zafar, F. and Schüle, B. (2022). Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays.Bio-protocol 12(14): e4283.
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Cycloheximide (CHX) Chase Assay to Examine Protein Half-life
YM Ying Miao
QD Qian Du
HZ Hong-Guang Zhang
YY Yukang Yuan
YZ Yibo Zuo
HZ Hui Zheng
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4690 Views: 9164
Reviewed by: Ralph Thomas BoettcherDeepali Bhandari Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Cell Research Oct 2022
Abstract
Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay.
Graphical overview
Keywords: Cycloheximide (CHX) chase assay Protein half-life Protein degradation Ubiquitin Ubiquitination
Background
Regulation of protein abundance is crucial for the maintenance of normal cell function; all proteins in the cell are constantly being degraded and replaced. Specifically, rapid protein degradation plays an important role in regulating protein abundance in a variety of cellular processes, including cell cycle, signal transduction, and differentiation (Goldberg, 2003). Studies on protein degradation rate and half-life are important for understanding protein function, developing drugs to regulate the degradation of disease-specific proteins, and researching small molecule compounds to regulate protein levels (Paiva and Crews, 2019). There are two main protein degradation pathways in eukaryotic cells—the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. The former is the main pathway of protein degradation in cells and is involved in the degradation of various proteins (Glickman and Ciechanover, 2002). It degrades proteins in cells in a specific, highly complex mechanism, and can be reversed by deubiquitinating enzymes (Sun et al., 2020). Autophagy-lysosome pathway is a self-degradation process, which degrades proteins through lysosomal mediation, by coating them and forming an autophagic lysosome, to fulfill the metabolic needs of the cells. Both pathways play vital roles in maintaining the normal protein metabolism of cells, and abnormal protein degradation may lead to many diseases, especially neurodegenerative disorders and cancer (Huang et al., 2018; Park et al., 2020).
The half-life of proteins varies widely, from a few minutes to days, and different rates of protein degradation are important for protein function. The classical method for protein degradation detection is the pulse-chase assay (Fritzsche and Springer, 2014); this assay is based on the addition of radionuclide-labeled amino acids, such as 35S-labeled methionine, to cell culture medium. The change process of a specific protein in the cells can be detected by immunoblotting or other methods. However, this method uses isotopes, which have some negative effects on the health and safety of researchers, and the operation process is complicated. Another common approach to study protein degradation is the protein translation blocker cycloheximide (CHX), which we will describe in detail later. In recent years, mass spectrometry technology has developed to detect the degradation of the primary structure of proteins. Therefore, protein degradation detection by mass spectrometry can be used to study protein structure, function, and quality control (Spradlin et al., 2021). In addition, it has been reported that CuAAC (copper-catalyzed azide–alkyne cycloadditions) can be used to label newly synthesized proteins in cells and to study protein degradation (Lu et al., 2013). Besides, protein degradation can be studied through the addition of pharmacological inhibitors of the degradation pathway to cells (Hanzl and Winter, 2020). For example, proteasome inhibitors such as MG-132, epoxomicin, and bortezomib block protein degradation via the ubiquitin-proteasome pathway. Inhibitors such as chloroquine and bafilomycin A1 work by neutralizing the acidic pH in the lysosome, which is necessary for the function of lysosomal protease.
Here, we describe a method that inhibits protein synthesis by adding CHX to assess the degradation of the target protein over time. CHX is a global translational inhibitor that can block the eukaryotic ribosome translocation process (Schneider-Poetsch et al., 2010). CHX is a product of Streptomyces griseus that can be used to determine the half-life of proteins or enzymes in molecular biology. It is noteworthy that CHX can inhibit eukaryotic protein synthesis but not prokaryotic protein synthesis. The advantages of CHX are that it is easy to operate, does not require considering the effect of ongoing protein synthesis, and allows for high-throughput screening. On the other hand, its main disadvantages are i) blocking all protein translation without specificity, and ii) having high cytotoxicity, able to cause large-scale cell death after prolonged treatment, so it is not suitable for proteins with a slow degradation rate. Therefore, CHX needs to be selected according to the appropriate conditions.
CHX chase assay visualizes protein degradation kinetics through CHX treatment over different times and western blotting analysis. Briefly, cells are treated with CHX at a suitable concentration, and then harvested at different timepoints. Next, western blot is employed to analyze the expression levels of a given protein. A short half-life protein will decrease in abundance over time, while a long half-life protein could exhibit little change in abundance. Thus, the CHX chase assay visualizes protein degradation efficiently without the use of radioactive isotopes, which provides an easy and reliable means to observe the change of intracellular protein levels and therefore could contribute to the study of therapeutic targets for a range of diseases.
Materials and reagents
Materials
Cell culture dish, 100 mm diameter (NEST, catalog number: 704001)
12-well plate (NEST, catalog number: 712011)
1,000 μL blue tip (Biosharp, catalog number: BS-1000-T)
200 μL yellow tip (Biosharp, catalog number: BS-200-T)
10 μL clear tip (Biosharp, catalog number: BS-10-T)
1.5 mL tube (AXYGEN, catalog number: AXYMCT150CS)
0.6 mL tube (AXYGEN, catalog number: AXYMCT060C)
Medical X-ray film (FUJIFILM, catalog number: SUPER RX-N)
Cell scraper (BIOLGIX, catalog number: 70-1180)
Cells
A549 cells (American Type Culture Collection)
Antibodies and plasmids
Anti-Tyk2 antibody (Cell Signaling Technology, catalog number: 14193; 1:1,000)
Anti-AHI1 antibody (Santa Cruz, catalog number: sc-515382; 1:500)
Anti-tubulin antibody (Proteintech, catalog number: 66031-1-Ig; 1:3,000)
Goat anti-rabbit IgG (H+L) HRP (Bioworld, catalog number: BS13278; 1:10,000)
Goat anti-mouse IgG (H+L) HRP (Bioworld, catalog number: BS12478; 1:10,000)
shCtrl plasmid (RNAi-Ready pSIREN-RetroQ-ZsGreen vector)
shAHI1 plasmid
shAHI1(#1)-forward: 5'-CACCGCGGAGACATTATCCGAGTGTTCGAAAACACTCGGATAATGTCTCCG-3'
shAHI1(#1)-reverse: 5'-AAAACGGAGACATTATCCGAGTGTTTTCGAACACTCGGATAATGTCTCCGC-3'
shAHI1(#2)-forward: 5'-CACCGCCATATTGGTCCGACAGTTTCGAAAAACTGTCGGACCAATATGGC-3'
shAHI1(#2)-reverse: 5'-AAAAGCCATATTGGTCCGACAGTTTTTCGAAACTGTCGGACCAATATGGC-3'
Reagents
Dulbecco’s modified Eagle medium (DMEM basic medium) (Gibco, catalog number: C11995500BT)
Fetal bovine serum (FBS) (PAN, catalog number: P30-3302)
Penicillin-streptomycin (Biosharp, catalog number: BL505A)
0.25% trypsin-EDTA (Biosharp, catalog number: BL512A)
Cycloheximide (CHX) (Sigma, catalog number: C7698-1G)
SuperSignal West Dura Extended kit (Thermo Scientific)
LongTrans (UCallM, catalog number: TF07)
Dimethyl sulfoxide for cell culture (DMSO) (panreac applichem, catalog number: 67-68-5)
Bradford (Sangon Biotech, catalog number: C900164-0200)
Non-fat milk (Biosharp, catalog number: BS102-500g)
BSA (Solarbio, catalog number: A8020)
Serum-free cell freezing medium (NCM Biotech, catalog number: C40100)
KCl (Sangon Biotech, catalog number: 7447-40-7)
NaCl (Sangon Biotech, catalog number: 7647-14-5)
EDTA (Solarbio, catalog number: 9002-07-7)
NP40 (Solarbio, catalog number: 9016-45-9)
Inhibitor cocktail (Sigma, catalog number: 58914000)
NaF (Sangon Biotech, catalog number: 7681-49-4)
PMSF (Solarbio, catalog number: 329-98-6)
Acr-Bis (30%) (Biosharp, catalog number: BL513B)
TEMED (Beyotime, catalog number: SY728)
APS (Sangon Biotech, catalog number: 7727-54-0)
SDS (Solarbio, catalog number: 151-21-3)
Glycine (Solarbio, catalog number: 56-40-6)
Tris base (Solarbio, catalog number: 77-86-1)
Tween 20 (Sangon Biotech, catalog number: 9005-64-5)
β-mercaptoethanol (Sigma, catalog number: 60-24-2)
Isopropyl alcohol (Sangon, catalog number: A507048-0500)
PageRuler prestained protein ladder (Thermo Fisher Scientific, catalog number: 26616)
Na3VO4 (Sangon Biotech, catalog number: 13721-39-6)
Na2HPO4 (Solarbio, catalog number: 7558-79-4)
KH2PO4 (Solarbio, catalog number: 7778-77-0)
Bromophenol blue (Solarbio, catalog number: 34725-61-6)
Glycerol (Solarbio, catalog number: G8190-500 ml)
HCl (Yonghua, catalog number: 210101204)
10× PBS Buffer (see Recipes)
Lysis buffer (1% NP40) (see Recipes)
8% separation gel (see Recipes)
5% stacking gel (see Recipes)
10× running buffer (see Recipes)
10× transfer buffer (see Recipes)
100 mM PMSF (see Recipes)
10% APS (see Recipes)
Stacking mix (see Recipes)
3× loading buffer (see Recipes)
SDS-sample buffer (see Recipes)
1× PBST buffer (see Recipes)
1% NP40 washing buffer (see Recipes)
1 M Tris-HCl (see Recipes)
Equipment
37 °C, 5% CO2 forced-air incubator (Thermo Scientific, catalog number: 3111)
Electric thermostatic water tank (Shanghai Jing Hong, catalog number: DK-8D)
Dry thermostat (AOSHENG, catalog number: MK-10)
SDS-PAGE gel casting apparatus (Tanon, catalog number: VE-180)
Protein electrophoresis and blotting instruments (Tanon, catalog number: EPS 300)
Centrifuge (Eppendorf, catalog number: 5425R)
Software
Microsoft PowerPoint
ImageJ
GraphPad Prism
Procedure
CHX can be used to determine changes in protein stability/half-live between different conditions, such as knockout vs. wild-type, overexpressed or in a knock-down situation. This protocol uses the example of Tyk2 stability in AHI1 knock-down cells.
Cell culture and transfection
Cells were cultured at 37 °C under 5% CO2 in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (complete medium). Cells were cultured with 0.25% trypsin-EDTA enzyme digesting technique and passaged in vitro.
Take A549 cells from -80 °C or liquid nitrogen and quickly place them in a 37 °C constant temperature water bath for approximately 2–3 min until the cells are completely thawed.
Place the cells into a centrifuge at 157× g for 3 min.
Remove the serum-free cell freezing medium, add 1 mL of complete medium to resuscitate cells, and transfer cells to a 100 mm culture dish.
Culture A549 cells in a 100 mm culture dish until 90% confluent.
Seed the cells at the ratio of 1:24 (5 × 105 cells) into six wells of a 12-well plate.
After 18–24 h, observe the cell density (approximately 80%–90%), and then perform the transfection of plasmids (such as shCtrl or shAHI1).
Remove the medium and add 1 mL of fresh complete medium before transfection.
Label 1.5 mL tubes as required and add 50 μL of DMEM basic medium to the tubes.
Prepare transfection solution A: add 1 μg of plasmid into DMEM medium and gently mix in the 1.5 mL tubes.
Prepare transfection solution B: add the LongTrans (DMEM:plasmid:longtrans = 50 μL:1 μg:2 μL) into DMEM medium and gently mix in the 1.5 mL tubes.
Gently mix transfection solutions A and B and leave at room temperature (RT) for 10–15 min.
Gently add the mixture to the corresponding medium and shake gently.
Place the plates in the incubator for approximately 36–48 h (for overexpressed plasmid) or 48–72 h (for knockdown plasmid).
Treat cells with CHX (50 μg/mL) for the appropriate timepoints (Section B).
CHX treatment
CAUTION: CHX is a highly toxic chemical and very toxic if swallowed; please avoid direct contact and release to the environment. In case of accident or if you feel unwell, seek medical advice immediately.
Remove the cell medium and replace with 1 mL of complete medium.
Distribute 500 μg of CHX into 0.6 mL tubes.
Add 10 μL of DMSO to the tube and mix well (the final concentration of CHX should be 50 mg/mL). CHX is better dissolved fresh to ensure maximal activity.
Slowly add 1 μL of CHX into the 1 mL cell culture medium and shake the plate gently to mix well. Similarly, add 1 μL of DMSO into the corresponding well as a control.
Harvest cells and extract cellular proteins (Section C).
Extracting cellular proteins
Prepare cell lysis buffer: add PMSF (1:100) to 1× NP-40 lysis buffer before use.
Place the cells on ice and aspirate the medium.
Gently add 1× PBS to wash and discard the PBS thoroughly. Repeat twice.
Add lysis buffer to the cells (100 μL per well).
Place the cells on ice for 30 min.
Scrap down the fully lysed cells with a cell scraper.
Transfer cell lysates to a 1.5 mL tube.
Place the 1.5 mL tubes into a 4 °C centrifuge at 12,000× g for 15 min.
Transfer the supernatant to a new 1.5 mL tube to get the cleared lysate (save at -80 °C); discard the cell debris sediment.
Use Bradford to determine protein concentration.
Denature 30–50 μg of proteins and add SDS-sample buffer to the samples.
Boil the samples in a metal bath at 96 °C for 10 min.
Centrifuge at 12,000× g for 1 min.
Use the samples for western blot or save them at -20 °C for future use.
Protein degradation analysis by western blot
Prepare the 8% SDS-PAGE gels (the gel concentration can be adjusted to 8%–12% depending on the molecular weight of the detected protein) with 8% separation gel and 5% stacking gel.
Dilute the 10× running buffer to 1× with double-distilled water (ddH2O).
Add 1× running buffer to the vertical electrophoresis chamber.
Gently pull out the comb of SDS-PAGE gel under running water and assemble it into vertical electrophoresis chamber.
Fill the chamber with running buffer.
Load the denatured protein samples into the lane of stacking gel and place 2 μL of PageRuler prestained protein ladder on both sides to indicate the molecular weight of the proteins.
Run for approximately 30 min using constant 80 V when the samples are in the stacking gel, and for approximately 90 min in the resolving gel at 110 V for a better separation of proteins.
Soak the PVDF membrane in absolute methanol for approximately 1 min to activate.
Prepare 1× transfer buffer (10× transfer buffer:absolute methanol:double-distilled water = 1:1:8).
Put the gel onto the activated PVDF membrane in 1× transfer buffer in the sequence of sponge, filter paper, gel, filter paper, and sponge.
Transfer the proteins to the gel using constant 360 mA for 90–120 min and immerse the electrophoresis tank in ice water.
Block the PVDF membrane with 5% non-fat milk with gentle shaking for 30 min at RT.
Dilute primary antibodies in 1% BSA at the indicated dilution.
Incubate the membrane with diluted primary antibodies with gentle shaking at RT for 2 h or 4 °C overnight.
Wash the membrane in 1× PBST buffer at RT for 10 min. Repeat three times.
Dilute the anti-mouse or anti-rabbit secondary antibodies in 5% non-fat milk at 1:10,000.
Incubate the membrane with the secondary antibodies with gentle shaking at RT for 1 h.
Wash the membrane in 1× PBST buffer at RT for 10 min. Repeat three times.
Visualize immunoreactive bands with a SuperSignal West Dura Extended kit using X-ray film in the dark room.
Edit gel images to the right size using Microsoft PowerPoint.
Use ImageJ to count pixels of the bands and calculate protein half-life.
Use GraphPad Prism to make the line chart.
Data analysis
Figure 1 displays a representative result showing the protein stability of Tyk2 in A549 cells transfected with shCtrl or shAHI1.
Figure 1. AHI1 knockdown accelerated Tyk2 protein degradation. (A) A549 cells were transfected with either control shRNAs (shCtrl) or shRNAs against AHI1 (shAHI1) plasmids. Seventy-two hours after transfection, cells were treated with either DMSO (for 0 h timepoint) or CHX (50 μg/mL, for 6 h and 12 h timepoints). Whole-cell extracts were analyzed by western blot using the antibodies against Tyk2, AHI1, or Tubulin. Tubulin serves as a loading control. (B) The striped images of Tyk2 and Tubulin were imported into Image J. The pixel count of all the strips was measured, and then the percentage of Tyk2 that remained was calculated in GraphPad Prism software. From Zhang et al. (2022).
Notes
The degradation rates of proteins can vary considerably from one another. Thus, it is suggested to determine the time course of protein degradation using the CHX chase assay in advance. Some endogenous proteins degrade very rapidly, and therefore the treatment time of CHX can be adjusted to 30–60 min. Sometimes it is necessary to set as few as five timepoints to detect the half-life of the protein. You can also find other experimental data about CHX chase assay in other papers of our research group (Feng et al., 2018; Yuan et al., 2020).
Recipes
10× PBS buffer
Reagent Amount (g)
NaCl 80.00
KCl 2.00
Na2HPO4 14.20
KH2PO4 2.70
Dilute the 10× PBS buffer to 1× PBS with ddH2O.
Lysis buffer (1% NP40)
Reagent Volume (mL)
1% NP40 washing buffer (Recipe 13) 9.85
EDTA (0.5 M) 0.05
Na3VO4 (0.2 M) 0.05
NaF (0.5 M) 0.02
Inhibitor cocktail 0.02
8% separation gel
Reagent Volume (mL)
ddH2O 3.80
Tris-HCl (pH 8.0) (Recipe 14) 2.00
Acr-Bis (30%) 2.13
10% APS (Recipe 8) 0.08
TEMED 8.00 × 10-3
5% stacking bel
Reagent Volume (mL)
Stacking mix (see Recipe 9) 3.00
10% APS (Recipe 8) 0.02
TEMED 5.00 × 10-3
10× running buffer
Reagent Amount (g)
Tris base 30.00
Glycine 144.00
SDS 40.00
Dilute the 10× running buffer to 1× running buffer with ddH2O
10× transfer buffer
Reagent Amount (g)
Tris base 24.00
Glycine 142.00
Dilute the 10× transfer buffer to 1× transfer buffer with ddH2O
100 mM PMSF
Reagent Amount
PMSF 0.696 g
Isopropyl alcohol 40.00 mL
10% APS
Reagent Amount
APS 1.00 g
ddH2O 10.00 mL
Stacking mix
Reagent Volume (mL)
Acr-Bis (30%) 16.70
1 M Tris-HCl (pH 6.8) (Recipe 14) 12.50
20% SDS 0.50
ddH2O 70.30
3× loading buffer
Reagent Amount
1 M Tris-HCl (pH 6.8) (Recipe 14) 1.88 mL
20% SDS 3.00 mL
Glycerol 3.00 mL
Bromophenol blue 6.00 × 10-3 g
ddH2O 2.20 mL
SDS-sample buffer
Reagent Ratio (%)
3× loading buffer (Recipe 10) 85
β-mercaptoethanol 15
1× PBST buffer
Reagent Volume (mL)
1× PBS 500
Tween 20 5
1% NP40 washing buffer
Reagent Volume (mL)
5 M NaCl 1.50
1 M Tris-HCl (pH 7.4) 2.50
NP40 0.50
ddH2O 45.50
1 M Tris-HCl
Reagent Amount
Tris 121.10 g
HCl to pH = 6.8, 7.4, or 8.0
ddH2O to 1 L
Acknowledgments
This work was supported by grants from the Project of Natural Science Research in Colleges and Universities of Jiangsu Province (20KJB310008), the Foundation for Young Scholars of Jiangsu Province (BK20200864), and the National Key R&D Program of China (2018YFC1705500): 2018YFC1705505. This protocol was adapted from previous work published in Cell Research (Zhang et al., 2022).
Competing interests
The authors declare no conflict of interest.
Ethics
The study did not involve human subjects or animal work.
References
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Lu, B., Al-Ramahi, I., Valencia, A., Wang, Q., Berenshteyn, F., Yang, H., Gallego-Flores, T., Ichcho, S., Lacoste, A., Hild, M., et al. (2013). Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance. Nat Neurosci 16(5): 562-570.
Paiva, S. L. and Crews, C. M. (2019). Targeted protein degradation: elements of PROTAC design. Curr Opin Chem Biol 50: 111-119.
Park, H., Kang, J. H. and Lee, S. (2020). Autophagy in Neurodegenerative Diseases: A Hunter for Aggregates. Int J Mol Sci 21(9).
Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., Green, R., Shen, B. and Liu, J. O. (2010). Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Cem Biol 6(3): 209-217.
Spradlin, J. N., Zhang, E. and Nomura, D. K. (2021). Reimagining Druggability Using Chemoproteomic Platforms. Acc Chem Res 54(7): 1801-1813.
Sun, T., Liu, Z. and Yang, Q. (2020). The role of ubiquitination and deubiquitination in cancer metabolism. Mol Cancer 19(1): 146.
Yuan, Y., Miao, Y., Qian, L., Zhang, Y., Liu, C., Liu, J., Zuo, Y., Feng, Q., Guo, T., Zhang, L., et al. (2020). Targeting UBE4A Revives Viperin Protein in Epithelium to Enhance Host Antiviral Defense. Mol Cell 77(4): 734-747 e737.
Zhang, H. G., Wang, B., Yang, Y., Liu, X., Wang, J., Xin, N., Li, S., Miao, Y., Wu, Q., Guo, T., et al. (2022). Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis. Cell Res 32(10): 897-913.
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Biochemistry > Protein > Degradation
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Transformation and Detection of Soybean Hairy Roots
XX Xing Xu *
TY Tai-Fei Yu *
JM Jian Ma
JC Jun Chen
YZ Yong-Bin Zhou
MC Ming Chen
ZC Zhan-Yu Chen
YM You-Zhi Ma
ZX Zhao-Shi Xu
ZZ Zhi-An Zhang
(*contributed equally to this work)
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4691 Views: 1091
Reviewed by: Zhibing LaiAli ParsaeimehrRaviraj Mahadeo Kalunke
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Original Research Article:
The authors used this protocol in Plant Biotechnology Journal Dec 2021
Abstract
Agrobacterium rhizogenes is a soil bacteria with extensive infectivity, which can infect almost all dicotyledonous plants and a few monocotyledonous plants to induce root nodules. This is caused by the root-inducing plasmid, which contains genes responsible for the autonomous growth of root nodules and crown gall base synthesis. Structurally, it is similar to the tumor-inducing plasmid in that it mainly contains the Vir region, the T-DNA region, and the functional region of crown gall base synthesis. Its T-DNA is integrated into the nuclear genome of the plant with the assistance of Vir genes, causing hairy root disease in the host plant and the formation of hairy roots. The roots produced by Agrobacterium rhizogenes–infested plants are characterized by a fast growth rate, high degree of differentiation, physiological, biochemical, and genetic stability, and ease of manipulation and control. In particular, the hairy root system is an efficient and rapid research tool for plants that have no affinity for transformation by Agrobacterium rhizogenes and low transformation efficiency. The establishment of germinating root culture system for the production of secondary metabolites in the original plants through the genetic transformation of natural plants mediated by root-inducing plasmid in Agrobacterium rhizogenes has become a new technology combining plant genetic engineering and cell engineering. It has been widely used in a variety of plants for different molecular purposes, such as pathological analysis, gene function verification, and secondary metabolite research. Chimeric plants obtained by induction of Agrobacterium rhizogenes that can be expressed instantaneously and contemporarily are more rapidly obtained, compared to tissue culture and stably inheritable transgenic strains. In general, transgenic plants can be obtained in approximately one month.
Keywords: Agrobacterium rhizogenes Soybean transformation Detection hairy roots Transient expression Molecular biology research
Background
The methods for soybean genetic transformation can be roughly divided into two types, with different applications: (1) Agrobacterium-mediated transformation, and (2) foreign target genes being introduced into the recipient soybean cells by auxiliary means, such as instruments (gene guns) or reagents (cell wall enzymes). This second type includes the microinjection, pollen tube channel, electroporation (Zhou and Qiu, 2010), polyethylene glycol (Liu and Friesen, 2012), and gene gun methods.
Agrobacterium rhizogenes–mediated genetic transformation is a new plant tissue culture technology that emerged in the late 20th century. As A. rhizogenes can induce hairy roots without hormone promotion and resistance screening, it has been widely used in the research of many plants for multiple molecular purposes, such as pathological analysis, gene function verification, secondary metabolite research, etc. (Fathi et al., 2019; Jiang et al., 2019; Meng et al., 2019). Reported plants that can be induced into hairy roots by A. rhizogenes include soybean, mesembryanthemum crystalline, white campion, and Salix (Gomes et al., 2019; Hudzieczek et al., 2019; Hwang et al., 2019; Yu et al., 2021; Zhang et al., 2022; Zhao et al., 2022).
In this protocol, we improved the method of genetic transformation of soybean mediated by A. rhizogenes, to establish various genetic transformation systems for inducing hairy roots in soybean and to provide technical support for soybean molecular biology research.
Materials and reagents
Soybean seeds (William 82) (Glycine max) or other soybean varieties suitable for transformation
Agrobacterium rhizogenes K599 chemically competent cell (Beijing Zoman Biotechnology Co., Ltd. catalog number: ZC1506)
pCAMBIA3301 or other plasmid (CAMBIA)
NcoI and BstEII (Thermo Fisher Scientific, Waltham, MA, USA)
Glufosinate-ammonium (Basta®) (Shanghai Acmec Biochemical Co., Ltd, catalog number: A607805g)
Kanamycin solution (100 mg/L) (Inalco Pharmaceuticals. catalog number: 1758-9316)
Streptomycin solution (100 mg/L) (Inalco Pharmaceuticals. catalog number: 1758-9319)
GUS stain solution (Biorigin Inc, catalog number: BN20175)
Tryptone (OXOID LIMITED, catalog number: LP0042B)
Yeast extract (OXOID LIMITED, catalog number: LP0021T)
NaCl (Beijing Biomed Gene Technology Co., Ltd. catalog number: SH5001-01)
CaCl2·2H2O (Sigma-Aldrich catalog number: C7902-500G)
90% acetone (Sigma-Aldrich catalog number: 270725-500mL)
Glycerol
Liquid and solid LB medium (for 1 L) (see Recipes)
0.1 M CaCl2 solution (see Recipes)
Equipment
Disposable plastic Petri dishes (Beijing Ruiaizhengte Biotechnology Co., Ltd)
Medical disposable sterile syringe
Gauze
Disposable transparent cup
1 L culture flasks
Procedure
Preparation of soybean seeds
Select full and uniform seeds, removing any that are diseased, small, or damaged. Prepare a suitable number of clean Petri dishes with water-moistened gauze, place the selected seeds in the clean Petri dishes, spread the moistened gauze on top of the soybean seeds, cover the Petri dishes, and incubate in the dark for 12 h.
Before transferring the seeds to the nutrient soil, pre-moisten the soil with water, fill each pot with an equal amount of pre-moistened wet soil, and press the pots gently to flatten the soil. Next, place the absorbed seeds evenly on top of the pressed soil. Then, lightly cover each pot with a layer of remaining soil as needed. Spray each pot with an appropriate volume of water using a spray bottle.
Incubate in a greenhouse at 28 °C for 5–7 days until 10 cm seedlings have grown for the next transformation step.
Generation of Agrobacterium rhizogenes competent cells
Take frozen K599 chemically competent cells stored at -80 °C and dip them gently into a sterile inoculation loop on a UV-irradiated ultra-clean table. Draw a line on an antibacterial-free plate and then incubate for 16–20 h at 37 °C.
Take a single colony and inoculate it in 5 mL of LB liquid medium. Oscillate at 220 rpm at 28 °C for 12–16 h.
Transfer 2 mL of bacterial solution into 100 mL of LB liquid medium and incubate at 28 °C at 220 rpm until OD600 = 0.5.
Transfer to a sterile centrifuge tube and centrifuge at 3,000× g for 5 min. Remove the supernatant.
Add 10 mL of pre-cooled 0.1 M CaCl2 solution, gently suspend the cells, and place on ice for 20 min. Centrifuge at 3,000× g for 5 min at 4 °C and remove the supernatant.
Add 4 mL of pre-chilled 0.1 M CaCl2 solution containing 15% glycerol and gently suspend.
Dispense Agrobacterium rhizogenes competent cells in sterile Eppendorf tubes, each tube with 200 μL. Freeze at -80 °C.
Preparation of A. rhizogenes
Insert target gene into the pCAMBIA3301 vector with kanamycin resistance with a phosphinothricin selection marker from both NcoI and BstEII enzymatic cut sites. In this vector, the lac promoter drives the expression of the inserted gene. To screen transformed roots, the vector contains a promoter encoding an enhanced glufosinate resistance gene derived from the cauliflower mosaic virus gene CaMV control, which provides strong root expression. The gene encoding kanamycin resistance provides antibiotic selection. Transfer the fusion expression vector plasmid with the target gene into A. rhizogenes K599 by the freeze-thaw method.
Place the K599 competent cells stored at -80 °C at room temperature or in an ice water bath for a moment and wait for its partial melting. Just-thawed cells have the highest conversion efficiency.
Add 1 μg of plasmid DNA per 100 μL of competent cells and mix well by gently toggling the bottom of the tube with your hands. Then, let it stand on ice for 5 min, liquid nitrogen for 5 min, 37 °C water bath for 5 min, and ice bath for 5 min, in this order.
Add 800 μL of LB liquid medium without antibiotics and incubate at 28 °C at 200 rpm for 2–3 h.
Centrifuge at 3,000× g for 1 min at room temperature. Leave 100 µL of the supernatant to resuspend the remaining bacteria onto LB plates containing the appropriate antibiotics and incubate upside down in an incubator at 28 °C for 2–3 days.
Induction of hairy roots
Prepare sterile disposable syringes. Puncture (5 mm) the cotyledons of young soybean seedlings with a syringe to infect the (approximately 7-days-old) soybean seedlings with A. rhizogenes containing plant expression vectors (Figure 1). Prepare a sterile disposable syringe, scrape the colonies on the plate transformed with the target gene and collect them with a needle tip. Then, pass the needle through the cotyledon hypocotyl (Figures 2 and 3) to stab the plant, ensuring that the needle passes through the central part of the hypocotyl. Place a clear plastic cup (with approximately 20 cm in height) upside down above the seedlings to facilitate moisture retention and thus improve the efficiency of infection, while incubating for 24 h under light-proof conditions.
Figure 1. Vector contains kanamycin resistance gene for bacterial selection and bialaphos resistance (BlpR) gene for plant selection
Figure 2. Inoculation with bacterial paste
Figure 3. Stabbing of the hypocotyl close to the cotyledonary node
Then, grow under normal conditions (25 °C; 16:8 h light/dark cycle). Cut off the bottom of the cup, put it upside down on the plant to fix the soybean plant, cover the cup with soil to completely cover the infected part, and pour sufficient water.
After approximately two weeks, observe whether new roots have grown at the infected location (Figure 4). During this period, apply sufficient water and fertilizer to ensure normal plant growth and promptly remove any damaged and dead plants.
Figure 4. New roots growing at the infected location
At the two-leaf stage, directly spray glufosinate-ammonium to the plants to be selected. (Different plants have different sensitivities to Basta®, and the same plant has different sensitivities at different growth stages. Criteria for determining the critical screening concentration: Basta® concentration at which wildtype plants can hardly germinate, or germinate but then cannot survive. The optimal screening concentration of Basta® is determined according to the yellowing degree and mortality rate of soybean seeds.)
Note: The general working concentration of spraying selected plants is 0.001%–0.002%.
Alternatively, the expression of the reporter gene can be detected by GUS staining. The specific procedures are as follows:
Pretreatment: cut the roots into small pieces, put them in a 1.5 mL centrifuge tube, add pre-cooled 90% acetone to completely cover the material, and treat at room temperature for 20–30 min. This step can pre-fix the tissue and remove some chlorophyll.
Staining: rinse the material with distilled water and place in a 1.5 mL centrifuge tube; add an appropriate volume of prepared GUS stain solution until the material is completely covered, wrap in aluminum foil, and leave overnight at room temperature.
Elution: Gradient elution with 25%, 50%, 70%, and 95% ethanol with gentle shaking on a shaker for 20 min each time.
Observation: The blue dots on the white background are the GUS expression sites when observed with the naked eye or under a microscope.
Seven days later, when the hairy root is approximately 5–10 cm (long enough to support the normal growth of the plant), select plants with consistent growth and cut the hypocotyl approximately 1 cm below the wound formed by the hairy root. Remove the primary root, retain the new root, and transplant it to a new basin.
Recipes
Liquid and solid LB medium (for 1 L)
Tryptone 10 g
Yeast extract 5 g
NaCl 10 g
Dissolve in 1 L of double-distilled water; then, sterilize by autoclaving at 120 °C for 20 min
0.1 M CaCl2 solution
Weigh 0.28 g of CaCl2·2H2O, dissolve in 50 mL of double-distilled water, fix the volume to 100 mL, and autoclave.
Acknowledgments
This protocol is adapted from our previous work (Zhang et al., 2022). This research was financially supported by the National Key R&D Program of China (2022YFD1201700), Hainan Yazhou Bay Seed Lab (B21HJ0215) and Nanfan special project, CAAS (YBXM04).
Competing interests
There are no conflicts of interest or competing interests.
References
Fathi, R., Mohebodini, M. and Chamani, E. (2019). High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Cichorium intybus L. via removing macronutrients. Ind Crops Prod 128: 572-580.
Gomes, C., Dupas, A., Pagano, A., Grima-Pettenati, J. and Paiva, J. A. P. (2019). Hairy Root Transformation: A Useful Tool to Explore Gene Function and Expression in Salix spp. Recalcitrant to Transformation. Front Plant Sci 10: 1427.
Hudzieczek, V., Cegan, R., Cermak, T., Bacovska, N., Machalkova, Z., Dolezal, K., Plihalova, L., Voytas, D., Hobza, R. and Vyskot, B. (2019). Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia. N Biotechnol 48: 20-28.
Hwang, H. H., Wang, C. H., Chen, H. H., Ho, J. F., Chi, S. F., Huang, F. C. and Yen, H. E. (2019). Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum). Bot Stud 60(1): 1.
Jiang, H., Li, K. and Gai, J. (2019). Agrobacterium rhizogenes-induced soybean hairy roots versus Soybean mosaic virus (ARISHR-SMV) is an efficient pathosystem for studying soybean-virus interactions. Plant Methods 15: 56.
Liu, Z. and Friesen, T. L. (2012). Polyethylene glycol (PEG)-mediated transformation in filamentous fungal pathogens. Methods Mol Biol 835: 365-375.
Meng, D., Yang, Q., Dong, B., Song, Z., Niu, L., Wang, L., Cao, H., Li, H. and Fu, Y. (2019). Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants. Plant Biotechnol J 17(9): 1804-1813.
Yu, T. F., Liu, Y., Fu, J. D., Ma, J., Fang, Z. W., Chen, J., Zheng, L., Lu, Z. W., Zhou, Y. B., Chen, M., Xu, Z. S. and Ma, Y. Z. (2021). The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stress tolerance. Plant Biotechnol J 19(12): 2589-2605.
Zhang, H. Y., Hou, Z. H., Zhang, Y., Li, Z. Y., Chen, J., Zhou, Y. B., Chen, M., Fu, J. D., Ma, Y. Z., Zhang, H. and Xu, Z. S. (2022). A soybean EF-Tu family protein GmEF8, an interactor of GmCBL1, enhances drought and heat tolerance in transgenic Arabidopsis and soybean. Int J Biol Macromol 205: 462-472.
Zhao, J., Zheng, L., Wei, J., Wang, Y., Chen, J., Zhou, Y., Chen, M., Wang, F., Ma, Y. and Xu, Z. S. (2022). The soybean PLATZ transcription factor GmPLATZ17 suppresses drought tolerance by interfering with stress-associated gene regulation of GmDREB5. Crop J 10(4): 1014-1025.
Zhou, G. A. and Qiu, L. J. (2010). Identification and Functional Analysis on Abiotic Stress Response of Soybean Cl− Channel Gene GmCLCnt. Agricultural Sciences in China 9(2): 199-206.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Plant Science > Plant transformation > Agrobacterium
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4,692 | https://bio-protocol.org/en/bpdetail?id=4692&type=0 | # Bio-Protocol Content
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Measurement of Total Phosphorus and Polyphosphate in Chlamydomonas reinhardtii
YY Yujie Yang *
SR Suna Ren *
XJ Xianqing Jia
HZ Houqing Zeng
LW Long Wang
YZ Yiyong Zhu
KY Keke Yi
(*contributed equally to this work)
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4692 Views: 511
Reviewed by: Ansul Lokdarshi Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Plant May 2021
Abstract
Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.
Keywords: Green algae Chlamydomonas Polyphosphate Total phosphorus Vacuolar phosphate Plant nutrient
Background
As an essential nutrient, phosphorus is required for the constitution of cellular components such as nucleic acids, phospholipids, and ATP. P is also required for the regulation of protein phosphorylation in plants. P is stored as inorganic phosphate (Pi) in the vacuoles of land plants and as inorganic polyphosphate (polyP) in chlorophyte algae. PolyP is a linear chain composed of several phosphate residues that are linked together by phosphoanhydride bonds. PolyP is an important metabolite and a signaling molecule acting as an energy source, a regulator of gene expression, a channel-forming component, and a storage form of Pi found in green plants (Wang et al., 2021).
Since the polyP is a mixture with different chain lengths, few analytical methods are currently available for polyP quantitation. In order to determine the content of polyP, it usually has to be degraded into Pi first, and then its content can be estimated by measuring the amount of Pi released. Two experimental methods are widely used for quantitatively estimating the amount of polyP in vitro (Aschar-Sobbi et al., 2008). The first one is based on the hydrolysis of polyP by hydrochloric acid and subsequent measurement of the Pi released. The second one is based on the activity of two enzymes, exopolyphosphatase and polyphosphate kinase, which can digest and convert the polyP into Pi for measurement. However, the expression and purification of these enzymes are complicated and time consuming (Christ et al., 2020).
Here, we describe an updated protocol for quantitative measurement of the polyP content in algae. We use silica gel columns to purify polyP and then convert it into Pi through digestion by hydrochloric acid. Although this method is not very accurate, because some short-chain-length polyP could not be completely extracted by the silica gel columns, it is economical and convenient. This protocol is relatively simple and time saving, leading to a fast determination of the polyP in Chlamydomonas, and should be considered suitable for polyP determination in other microalgae.
Materials and reagents
Centrifuge tubes (1.5, 2, 15, and 50 mL) (Sangon Biotech)
Sterile culture flasks (50 and 250 mL) (Sangon Biotech)
Pipettes (10, 20 and 200 μL, 1 and 5 mL) (Eppendorf)
96-well ELISA plate (Sangon Biotech, catalog number: F605031)
Petri dishes (Sangon Biotech, catalog number: F611003)
47 mm filter paper sheets (Whatman, catalog number: 1822047)
Spin Columns CA2 from TIANgel Midi Purification kit (TIANGEN, catalog number: DP209)
Hydrochloric acid (HCl)
Nitric acid (HNO3)
98% sulfuric acid (H2SO4)
Tris (C4H11NO3) (Sangon Biotech, catalog number: A100826)
Glacial acetic acid (CH3COOH) (Sangon Biotech, catalog number: A501931)
Ammonium chloride (NH4Cl) (Sangon Biotech, catalog number: A501569)
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sangon Biotech, catalog number: A610329)
Calcium chloride dihydrate (CaCl2·2H2O) (Sangon Biotech, catalog number: A610050)
Dipotassium hydrogen phosphate (K2HPO4) (Sangon Biotech, catalog number: A610447)
Potassium phosphate monobasic (KH2PO4) (Sangon Biotech, catalog number: A600445)
EDTA disodium salt (C10H14N2O8Na2·2H2O) (Sangon Biotech, catalog number: A100105)
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sangon Biotech, catalog number: A602906)
Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sangon Biotech, catalog number: A600461)
Boric acid (H3BO3) (Sangon Biotech, catalog number: A100588)
Manganese (II) chloride tetrahydrate (MnCl2·4H2O) (Sangon Biotech, catalog number: A500331)
Cobalt chloride hexahydrate (CoCl2·6H2O) (Sigma-Aldrich: catalog number: 255599)
Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sangon Biotech, catalog number: A600063)
Ethanol absolute (Sangon Biotech, catalog number: A500737)
Neutral red (Sangon Biotech, catalog number: A600895)
1 M Tris-HCl (pH 7.5) (Sangon Biotech, catalog number: A610283)
Sodium iodide (NaI) (Sangon Biotech, catalog number: A610283)
Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sangon Biotech, catalog number: A600067)
Malachite green oxalate salt (Sangon Biotech, catalog number: A620330)
TAP medium (see Recipes)
40× TAP salt mixture (see Recipes)
Phosphate solution (50 μg/mL) (see Recipes)
Hutner's trace elements (see Recipes)
TAP solid medium (see Recipes)
1 M sulfuric acid (H2SO4) (see Recipes)
2 M NaOH (see Recipes)
70% ethanol (see Recipes)
0.1% neutral red solution (w/v) (see Recipes)
1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v) (see Recipes)
6 M NaI (see Recipes)
Wash buffer (see Recipes)
2 M hydrochloric acid (HCl) (see Recipes)
Phosphate (Pi) solution (10 μg/mL) (see Recipes)
28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes)
0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes)
Equipment
Spectrophotometer (Shimadzu, model: UVmini-1240)
Centrifuge (Thermo Scientific, catalog number: 75002411)
Hitachi Himac CT15RE tabletop high-speed microcentrifuge
Hitachi CR21N high-speed refrigerated centrifuge
Vortex (IKA, model: IKA MS 3 basic)
Thermal cycler (Thermo Scientific, catalog number: 4484073）
Microplate reader (Tecan, model: Infinite® F50 microplate reader)
Vacuum pump (JinTeng, model: GM-0.5B)
Analytical balance (Sangon Biotech, catalog number: G001142)
Air dry oven (Sangon Biotech, catalog number: G003409)
Clean bench (AIRTECH, catalog number: SW-CJ-2F)
Fume hood (ALPHA, VAV controller)
Nikon Digital Camera D7200
Software
Microsoft Excel (Microsoft, Inc.)
Microsoft PowerPoint (Microsoft, Inc.)
Prism GraphPad (Dotmatic, Inc.)
Procedure
In this section, we describe the procedure to measure polyP and total P in C. reinhardtii. The first step is to culture algae cells. Next, collect an appropriate number of algae cells to extract P (polyP or total P) and then digest polyP or total P into Pi by hydrochloric acid or nitric acid from dried cells. To quantify the Pi released, the malachite green colorimetric method is used.
Figure 1. Basic workflow of cell culture and P measurement. A. Procedure for algae cell culture. B and C show the procedures for polyP and total P measurement, respectively. Drawn and composed using Microsoft PowerPoint.
Culture methods
Protocols adapted from Wang et al. (2021).
Cell culture is grown using 20 mL of TAP medium in a 50 mL flask. Stationary-phase cell culture with a volume of 200 μL is transferred into 20 mL of fresh medium every week. The culture growth conditions are adjusted to a speed of 150 rpm shaking at 25 °C using a rocking incubator, and the culture itself is illuminated continuously with white fluorescent light (150 μmol photons m-2 s-1). A cell density of approximately 1 × 107 cells (3–5 days after transfer, late linear phase) is used for the experiment.
The following provides specific steps:
Pick wildtype (CC-4533) and mutant (Crptc1) or other desired strain(s) from a fresh plate with a sterile toothpick or pipette tip and inoculate with 20 mL of liquid medium (TAP medium or appropriate selection medium) using a 50 mL flask. Rotate at 150 rpm and 25 °C for 5–7 days under continuous white light. This primary culture is grown to a stationary phase.
Inoculate cultures into a 50 mL flask with 20 mL of TAP medium and adjust inoculum size appropriately to make the OD750 of inoculated solution approximately 0.2–0.3. Cultures will reach the stable period (~1 × 107 cells) again after 5–7 days. The OD750 should be measured every 12 h to count the number of algae cells.
Transfer 1 mL of the secondary algae cells into a 250 mL flask containing 100 mL of TAP medium and incubate for 3–5 days.
After three times of uniformization, different algae strains reach the same or similar growth status.
Optional:
Determine the OD750 every 12 h, calculate the regression equation, and draw the growth curve.
Note: The growth phase can be determined by measuring cell density and drawing the growth curve.
Extraction of P
This section contains two parts. The first one is the purification and degradation of polyP and the second one is the digestion of total P in algae with nitric acid. After purification and degradation, polyP and total phosphorus content is converted into Pi released and can be measured using the malachite green colorimetric method.
Purification and degradation of polyP
Protocol adapted from Werner et al. (2005), Canadell et al. (2016), and Wang et al. (2021).
Determine the OD750 of the algae using a spectrophotometer and record it.
Add 500 μL of algae (record the exact weight) into a 1.5 mL centrifuge tube. After centrifugation at 8,000× g for 5 min at room temperature, remove all the supernatant by pipetting and harvest the algae cells.
Note: The sample can be directly purified or stored at -20 °C. For more accurate measurements of dry weight, 5 mL of algae is filtered on 47 mm filter paper sheets using a vacuum pump, and then put in a drying oven for several hours (Figure 1). The weight of the 47 mm filter paper sheet should be measured using an analytical balance before and after sample loading.
Add 50 μL of 1 M H2SO4 (see Recipes) into the algae, mix by pipetting or vortex to homogenate, and then place the tube at room temperature for 5 min.
Neutralize the suspension with 50 μL of 2 M NaOH (see Recipes) and 100 μL of 1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (see Recipes). Gently mix the tubes upside down four times.
Note: The pH indicator helps to assure that the pH is around 7 (the sample color becomes orange red), which is a critical step for the reproducibility of polyP extraction. Samples too acidic (pink) or too basic (yellow) have to be corrected to the appropriate pH (orange red) by the addition of 2 M NaOH or 1 M H2SO4, respectively.
Centrifuge at 1,600× g for 10 min at 4 °C.
Drain 150 μL of supernatant with a pipette into a new 1.5 mL centrifuge tube containing 450 μL of 6 M NaI (freshly prepared; see Recipes).
Mix by pipetting 3–5 times and then transfer to a silica gel column (Spin Columns CA2 from TIANgel Midi Purification kit). Centrifuge the column at 12,000× g for 1 min at room temperature.
Discard the flowthrough and place the column back into the collection tube. Add 400 μL of wash buffer (see Recipes) into the column and centrifuge the column at 12,000× g for 1 min at room temperature.
Repeat step B1h.
Discard the flowthrough, place the column back into the collection tube, and centrifuge at 12,000× g for 2 min at room temperature to remove residual wash buffer.
Transfer the column into a clean 1.5 mL centrifuge tube and place the column with the cap open for 2–5 min to dry the membrane.
Add 50 μL of ddH2O to the center of the membrane, incubate at room temperature for 2 min, and then centrifuge at 12,000× g for 2 min at room temperature.
Note: The eluted polyP can be directly degraded; however, leaving it at room temperature for a longer time should be avoided. For long-term storage, it can be kept at -20 °C.
Pipette 10 μL of eluted polyP and add 10 μL of 2 M HCl (see Recipes), mix thoroughly, and centrifuge for several seconds.
Incubate at 95 °C for 30 min in the thermal cycler and then transfer onto the ice.
The degraded polyP sample can be directly measured with the malachite green colorimetric method or stored at -20 °C.
Determination of total P
Mark a 2 mL centrifuge tube and weigh it.
Collect 10–15 mL of cells into a 15 mL centrifuge tube and centrifuge at 2,070× g for 5 min at room temperature.
Discard the supernatant, wash the algae cells with ddH2O, and centrifuge at 2,070× g for 5 min at room temperature.
Repeat step B2c.
Discard the supernatant, suspend the algae cells with 1 mL of ddH2O, and transfer the entire suspension to the 2 mL centrifuge tube.
Centrifuge at 12,000× g for 5 min at room temperature and pipette all the supernatant carefully in case of loss of algae samples.
Dry and weigh the tube with algae samples.
Add 0.4–1.0 mL of concentrated HNO3 and incubate overnight at 4 °C until the solution is clean.
Transfer the solution into a 50 mL centrifuge tube, wash the inside of the 2 mL centrifuge tube with ddH2O, transfer all the wash water into the 50 mL centrifuge tube, and dilute with ddH2O to 50 mL.
This sample can then be analyzed for P concentration determination using the malachite green colorimetric method.
Quantification of P with the malachite green colorimetric method
Drawing of the standard curve (Table 1)
Table 1. Standard curve
1 2 3 4 5 6 7
Concentration μg/mL 0 0.25 0.5 0.75 1 1.5 2
Pi solution (10 μg/mL) μL 0 2.5 5 7.5 10 15 20
ddH2O μL 100 97.5 95 92.5 90 85 80
According to the Pi concentration gradient in the table, add these substances in order:
Add Pi solution (10 μg/mL) (see Recipes) and the corresponding volume of ddH2O.
Add 86 μL of 28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes).
Add 64 μL of 0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes).
Leave the 96-well ELISA plate at room temperature for 30 min and measure the OD595 with a microplate reader.
Note: Do not let the reaction proceed for more than 1 h as it can cause the appearance of small precipitates in highly concentrated Pi samples and polyP degradation in background samples, thus interfering with the correct Pi measure.
Draw the standard curve by taking the volume of Pi solution (10 μg/mL) as the x-axis and the OD595 as the y-axis. Then, the P concentration of the samples can be calculated according to the standard curve.
Figure 2. Standard curve. Pi concentration as x-axis and the OD595 as the y-axis. Drawn using Microsoft Excel and Prism GraphPad.
Determination of P in samples
Use a 96-well plate for the reaction and measurement of polyP. To quantify released Pi, add to each well 10 μL of polyP after degradation, 90 μL of ddH2O, 86 μL of 28 mM ammonium heptamolybdate in 2.1 M H2SO4 (see Recipes), and 64 μL of 0.76 mM malachite green in 0.35% polyvinyl alcohol (see Recipes), in this order. Mix the well by pipetting gently.
The blank control consists of 100 μL of ddH2O, 86 μL of 28 mM ammonium heptamolybdate in 2.1 M H2SO4, and 64 μL of 0.76 mM malachite green in 0.35% polyvinyl alcohol.
Leave the 96-well plate at room temperature for 30 min and measure the OD595 with a microplate reader.
Results
Figure 3. The content of PolyP and total P in wildtype strain CC-4533 and the Crptc1 mutant. (A) and (B) show filter paper sheets of 5 mL cells through a vacuum pump. The weight of algae cells was calculated using the dry weight difference of filter paper before and after filtration. PolyP content (C) and total P content (D) of the wildtype strain CC-4533 and the Crptc1 mutant. CrPTC1 is a vacuole efflux phosphate transporter in algae and catalyzes Pi transport out of acidocalcisomes. The Crptc1 mutant over-accumulates total P and polyP (Wang et al., 2021). Data is presented as the mean ± SD from five independent experiments. Pictures were taken using a Nikon Digital Camera D7200. C and D were drawn using Microsoft Excel and Prism GraphPad.
Recipes
TAP medium
Tris 2.42 g
40× TAP salt mixture 25 mL (Recipe 2)
Phosphate solution (50 μg/mL) 0.4 mL (Recipe 3)
Hutner’s trace elements 1 mL (Recipe 4)
Glacial acetic acid 1 mL
Adjust to pH 7.0 with HCl
Add ddH2O to 1 L and autoclave at 121 °C for 20 min
40× TAP salt mixture
NH4Cl 15 g
MgSO4·7H2O 4 g
CaCl2·2H2O 2 g
Add ddH2O to 1 L
Phosphate solution (50 μg/mL)
K2HPO4 28.8 g
KH2PO4 14.4 g
Add ddH2O to 100 mL
Hutner’s trace elements
EDTA disodium salt 50 g
ZnSO4·7H2O 22 g
FeSO4·7H2O 4.99 g
H3BO3 11.4 g
MnCl2·4H2O 5.06 g
CoCl2·6H2O 1.61 g
CuSO4·5H2O 1.57 g
(NH4)6Mo7O24·4H2O 1.10 g
Add ddH2O to 1 L
TAP solid medium
TAP medium (Recipe 1) with 1% (w/v) agar
Autoclave at 121 °C for 20 min
1 M sulfuric acid (H2SO4)
Mix 5.45 mL of 98% sulfuric acid with 94.55 mL of ddH2O and store at room temperature
2 M NaOH
Mix 8 g of NaOH with 80 mL of ddH2O
Add ddH2O to 100 mL and store at room temperature
70% ethanol
Mix 70 mL of ethanol absolute with 30 mL of ddH2O
0.1% neutral red solution (w/v)
Mix 0.1 g of neutral red with 100 mL of 70% ethanol (Recipe 8) and store at room temperature
1 M Tris-HCl (pH 7.5) supplemented to 6% (v/v) with 0.1% neutral red solution (w/v)
Mix 12.1 g of Tris with 70 mL of ddH2O
Add 6 mL of 0.1% neutral red solution (w/v) (Recipe 9)
Adjust pH to 7.5 with HCl
Add ddH2O to 100 mL and store at room temperature
6 M NaI
Mix 8.9934 g of NaI with ddH2O up to 10 mL
The solution has to be freshly prepared each time and kept in darkness
Wash buffer
10 mM Tris-HCl buffer (pH 7.5), 50% ethanol, 1 mM EDTA, and 100 mM NaCl
Mix 5 mL of 1 M Tris-HCl (pH 7.5), 250 mL of 100% ethanol, 1 mL of 0.5 M EDTA, 10 mL of 5 M NaCl, and 200 mL of ddH2O
Adjust pH to 7.5 with HCl
Add ddH2O to 500 mL and store at room temperature
2 M HCl
Mix 16.67 mL of 37% HCl with 83.33 mL of ddH2O and store at room temperature
Phosphate (Pi) solution (10 μg/mL)
Mix 50 μL of Phosphate solution (50 μg/mL; Recipe 3) and 200 μL of ddH2O
28 mM ammonium heptamolybdate in 2.1 M H2SO4
Mix 3.46 g of ammonium heptamolybdate tetrahydrate with 80 mL of ddH2O
Add 11.2 mL of 98% H2SO4
Add ddH2O to 100 mL and store at room temperature
0.76 mM malachite green in 0.35% polyvinyl alcohol
Mix 350 mg of polyvinyl alcohol in 100 mL of ddH2O at 80 °C and stir vigorously on a magnetic stirrer until all polyvinyl alcohol dissolves completely
Add 35 mg of malachite green oxalate salt
Add ddH2O to 100 mL and store at room temperature
Acknowledgments
The protocol for algae culture was adapted from Wang et al. (2021). The protocol for polyP purification was modified from Werner et al. (2005) and Canadell et al. (2016). This work was supported by the National Natural Science Foundation of China (32130096, 32202593, and 32102478) and the National Key Research and Development Program of China (2021YFF1000404). K.Y. was supported by the Innovation Program of the Chinese Academy of Agricultural Sciences. X.J. and L.W. were supported by the China Postdoctoral Science Foundation (2021M693447 to X.J.; 2021M693449 and 2022T150707 to L.W.).
Competing interests
No conflict of interest or competing interests declared.
References
Wang, L., Jia, X., Zhang, Y., Xu, L., Menand, B., Zhao, H., Zeng, H., Dolan, L., Zhu, Y. and Yi, K. (2021). Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants. Mol Plant 14(5): 838-846.
Aschar-Sobbi, R., Abramov, A. Y., Diao, C., Kargacin, M. E., Kargacin, G. J., French, R. J. and Pavlov, E. (2008). High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach. J Fluoresc 18(5): 859-866.
Christ, J. J., Willbold, S. and Blank, L. M. (2020). Methods for the analysis of polyphosphate in the life sciences. Anal Chem 92(6): 4167-4176.
Werner, T. P., Amrhein, N. and Freimoser, F. M. (2005). Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase. Arch Microbiol 184(2): 129-136.
Canadell, D., Bru, S., Clotet, J. and Ariño, J. (2016). Extraction and quantification of polyphosphate in the budding yeast Saccharomyces cerevisiae. Bio-protocol 6(14): e1874.
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4,693 | https://bio-protocol.org/en/bpdetail?id=4693&type=0 | # Bio-Protocol Content
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Large-scale Isolation of Exosomes Derived from NK Cells for Anti-tumor Therapy
HL Heyong Luo
JZ Jing Zhang
AY Anqing Yang
WO Weiwei Ouyang
SL Shiqi Long
XL Xiaojin Lin
NY Na Yang
ZY Zhiru Yang
YZ Yingchun Zhang
WY Wei Yang
QC Qiyuan Che
YY Yuxin Yang
TG Ting Guo
XZ Xing Zhao
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4693 Views: 2408
Reviewed by: Kazem NouriIvan ShapovalovDanielle Harper
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Abstract
Exosomes are lipid bilayer–enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell–derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.
Keywords: Exosome Natural killer cells Tangential flow filtration Ultracentrifugation Immunotherapy
Background
Adoptive NK cell therapy is a promising new approach for the treatment of hematological and solid malignancies, whose safety and efficacy have been tested in multiple studies mainly by utilizing autologous or allogeneic NK cells (van Vliet et al., 2021; Laskowski et al., 2022). Although the efficacy of adoptive NK cell therapy in the treatment of hematologic tumors has been verified, there are limitations to their application in solid tumors (van Vliet et al., 2021). Due to the existence of various biological barriers in the body, the migration and infiltration of NK cells to tumors are insufficient (Kang et al., 2021; Russo et al., 2021). The tumor microenvironment (TME) has immunosuppressive effects on NK cell function (Terrén et al., 2019; Di Pace et al., 2020); in addition, storing and transportation of NK cells is difficult, which increases the cost of treatment.
Exosomes are vesicles with a phospholipid bilayer membrane structure that are actively secreted by a variety of cells and carry the characteristic substances of their source cells (Shoae-Hassani et al., 2017; Dai et al., 2022; Rezaie et al., 2022). NK cell–derived exosomes (NK-Exo) have some significant advantages over NK cell therapy. Due to their nanoscale size and good tissue permeability, NK-Exo can cross some biological barriers such as the blood-tumor and blood-brain barriers (Yang et al., 2021). These are difficult for NK cells to cross (Nayyar et al., 2019; Yong et al., 2019; Kang et al., 2021), leading to the possibility of NK-Exo having a better tumor-targeting effect. It has been demonstrated that NK-Exo express perforin, granzyme B, Fas/FasL, and other anti-tumor substances (Zhu et al., 2017; Di Pace et al., 2020; Federici et al., 2020; Han et al., 2020). NK-Exo is not restricted to the TME and retains its original anti-tumor activity (Federici et al., 2020). In addition, NK-Exo has simpler preservation conditions than NK cells. The potential of implementing exosomes as an anti-tumor therapy has been demonstrated in several clinical trials (Chen et al., 2020; Nikfarjam et al., 2020; Lee et al., 2021; Rezaie et al., 2022). Therefore, we speculate that NK-Exo could also be used as a cell-free therapy to provide alternative anti-tumor immunotherapy.
However, the dose-demand for NK-Exo when used in anti-tumor therapy is enormous (Kibria et al., 2018; Kimiz-Gebologlu and Oncel, 2022). At present, laboratory methods for exosome isolation include ultracentrifugation, size exclusion chromatography, and exosome acquisition kits. However, it is challenging to isolate enough exosomes in supernatants in laboratory studies, such as exosomes derived from NK cells. Therefore, the large-scale preparation of exosomes is a key issue to be investigated urgently for further application.
Our protocol focuses on the following components: first, NK cell supernatants are collected and concentrated by tangential flow filtration (TFF) system, allowing us to obtain NK-Exo by ultracentrifugation. The interception efficiency of TFF system is evaluated by nanoflow assay. The obtained NK-Exo is then characterized to evaluate their phenotype, as well as morphological and structural integrity. Finally, the anti-tumor functionality of NK-Exo is evaluated in vitro. This protocol provides an efficient and convenient method to concentrate and isolate NK-Exo from large-scale NK cell culture supernatants, which can provide enough NK-Exo for solid tumor therapy.
Materials and reagents
0.22 μm filter (PALL, catalog number: 4612)
50 mL centrifuge tube (NEST, catalog number: 602001)
0.45 μm PVDF (Millipore)
RPMI 1640 medium (Gibco)
Fetal bovine serum (FBS) (Gibco)
1% penicillin/streptomycin (Gibco)
Centrifuge ware (Hitachi, catalog number: 3 29607A)
Annexin -FITC/PI Apoptosis Detection Kit (Absin, catalog number: abs50001)
Cell Counting kit 8 (Dojindo, catalog number: CK04)
FITC-conjugated mouse anti-human CD107a (BioLegend, catalog number: 328606)
PBS (meilunbio, catalog number: MA0015)
PE-conjugated mouse anti-human CD56 (BioLegend, catalog number: 304605)
PKH67 green fluorescent cell linker Midi kit (Sigma-Aldrich, catalog number: MIDI67)
Silica nanosphere cocktail (NanoFCM, catalog number: S16M-Exo)
DAPI (Sigma, catalog number: D9592)
4% paraformaldehyde (ChemCruz, catalog number: sc281692)
12% SDS-PAGE (TGX Stain-Free FastCast Acrylamide Kit, 12%) (Bio-Rad, catalog number: 161-0185)
SDS-PAGE sample loading buffer (5×) (Beyotime, catalog number: P0015L)
SDS-PAGE running buffer powder (Servicebio, catalog number: G2081-1L)
SDS-PAGE transfer buffer powder (Servicebio, catalog number: G2017)
Western blocking buffer (Beyotime, catalog number: P0023B-100ml)
Uranyl acetate (HSA Biotech)
RIPA (Solarbio)
PMSF (Sigma)
TEMED (Bio-Rad)
TBST (20×) (Solarbio)
Loading buffer (5×) (Beyotime)
10–180 kD marker (Absin)
Western chemiluminescent HRP substrate (Millipore, catalog number: WBKIS0100)
SKOV3 cells (China Center for Type Culture Collection, CCTCC)
OV-90 cells (China Center for Type Culture Collection, CCTCC)
Primary and secondary antibodies (Table 1)
Table 1. Primary and secondary antibodies
Antibody Species Host Company Catalog number Dilution ratio
CD63 Human Rabbit Abcam ab134045 1:1,000
CD81 Human Rabbit CST 56039s 1:1,000
Calnexin Human Rabbit Biodragon BD-PT0613 1:1,000
CD56 Human Rabbit CST 99746s 1:1,000
Perforin Human Rabbit Biodragon BD-PT5792 1:1,000
Rabbit-HRP Rabbit Goat Absin Abs20040 1:5,000
Equipment
Transmission electron microscope (Hitachi, catalog number: HT-7700)
Ultracentrifuge (Hitachi, catalog number: CPN100N)
ChemiDoc XRS high sensitivity chemiluminescence instrument (Bio-Rad Laboratories)
Filter cassettes (100 kD) (Sartorius, catalog number: VF05H4)
Flow cytometer (Beckman Coulter Inc., model: FC500)
Fluorescence microscope (Olympus BX51)
Nanoflow cytometry (NanoFCM)
P28S rotor (Hitachi)
Peristaltic pump (Millipore, model: XX80EL005）
Autoclave (MEDNIF, model: LS-B50L)
Ultraviolet irradiation (SJMAEA)
Centrifuge (Eppendorf)
Biosafety cabinet (Thermo Scientific)
PowerPacTM (Bio-Rad)
Software
Fiji: ImageJ 1.53a
GraphPad Prism 8
NanoFCM software (NanoFCM Profession V1.0)
FlowJo 10
Procedure
NK cell culture supernatants preparation
Maintain the NK cells expanded in vitro in RPMI 1640 medium supplemented with 5% FBS and 1% penicillin/streptomycin.
Change the FBS to exosome-free FBS on the 14th day of culture and continue the culture for two days.
Note: Exosome-free FBS is obtained by centrifugation of FBS at 100,000× g for 16 h followed by the collection of supernatants.
Adjust cell density to 1 × 109 cells/500 mL and collect the cell culture supernatant for pretreatment.
Centrifuge the cell culture supernatant at 300× g for 10 min to remove cells and then transfer the supernatant to a new centrifuge tube.
Note: The centrifugal force of the first step should be controlled below 350× g to avoid damage to NK cells.
Centrifuge the supernatant obtained in the previous step again at 2,000× g for 10 min to remove cell debris, and then transfer the supernatant to the sample cup of the TFF system for subsequent treatment.
Note: The volume of supernatant can be increased by increasing the number of sample cups or by adding liquid multiple times to a single sample cup.
Concentration of NK cell culture supernatants by TFF system
Note: All the following steps are performed at 4 °C; for reference, we performed all steps in cold storage.
Connect the sample cup, the liquid inlet pipe, the peristaltic pump, the filter cassettes (100 kD), the liquid outlet pipe, the waste liquid pipe, and the waste liquid cup successively, and install the pressure indicator at the connection of the liquid outlet pipe to complete assembly of the TFF system (Kim et al., 2021).
Note: The sample cup should be autoclaved in advance, and the entire TFF system should be sterilized by ultraviolet irradiation 30 min before operation.
Add the pretreated cell culture supernatants from steps A4–A5 into the sample cup, start the peristaltic pump, and concentrate at a pressure of 2.5 bar until the concentrated volume is approximately 13 mL.
Transfer the concentrate to a 50 mL centrifuge tube and add 10 mL of PBS into the sample cup.
Reduce the pressure of the peristaltic pump to 1 bar.
After recirculating the TFF system approximately five times, shut down the peristaltic pump and transfer PBS to the above 50 mL centrifuge tube.
Note: All residual fluid in the pipeline should be drained as far as possible to improve exosome recovery.
Preparation of NK-Exo using ultracentrifugation
Note: All following steps should be performed at 4 °C as much as possible; steps in the biosafety cabinet should be performed on ice.
Transfer the concentrated solution from step B5 to an ultra-speed centrifuge–fitted centrifuge tube and centrifuge at 10,000× g for 30 min at 4 °C; then, filter through a 0.22 μm filter.
Note: The centrifuge tube should be irradiated by UV 30 min in advance, and all following steps should be performed aseptically in the biosafety cabinet.
Centrifuge the liquid from the previous step at 100,000× g for 70 min at 4 °C.
Remove the supernatants, re-suspend the exosome pellet with 20 mL of precooled PBS, and centrifuge again at 100,000× g for 70 min at 4 °C.
Note: The suspended liquid should account for at least 1/2 of the centrifugal tube volume, so as not to damage the centrifugal tube due to high centrifugal force.
Remove and re-suspend the supernatants with 500 μL of PBS. Then, immediately use or store at -80 °C.
Note: NK-Exo should be used as soon as possible or stored at -80 °C for up to three months.
Detection of particle concentration, particle size, and phenotypes by nanoflow cytometry
Calibrate the particle concentration of the instrument with 200 nm PE and AF488 fluorescent-conjugated polystyrene beads and calibrate the particle size of the instrument with silica nanosphere cocktail.
Dilute NK-Exo serially to an optimal recording range of 2,000–12,000 particles/min during the final detection and record all particles passing through the 1 min interval during each detection.
Notes:
NK-Exo should be diluted at a ratio of 1:10, 1:100, and 1:1,000 until its detection rate reaches 2,000–12,000 particles/min.
Incubation should be done in dark conditions.
Then, wash the dyeing mixture with PBS and centrifuge at 100,000× g for 70 min at 4 °C.
Note: PBS should account for at least 1/2 of the centrifugal tube volume, so as not to damage the centrifugal tube due to high centrifugal force.
Re-suspend NK-Exo with 50 μL of PBS and test by nanoflow cytometry.
Using a standard curve, convert the flow rate and lateral scattering intensity to the corresponding concentrations and size in NanoFCM software.
NK-Exo detection by transmission electron microscopy
Re-suspend exosomes (> 109/mL) purified from NK cell supernatants in 50 μL of PBS.
Obtain a 10 μL sample and drop it on a copper grid. After 1 min of precipitation, use a filter paper to remove the excess liquid.
Obtain 10 μL of uranyl acetate and add it onto the copper grid to precipitate for 1 min; use a filter paper to remove the excess liquid.
After drying at room temperature for several minutes, the image results are obtained using a transmission electron microscope (100 kV).
NK-Exo detection by western blot analysis
Add 150 μL of RIPA containing 1.5 μL protease inhibitor (PMSF) to NK-Exo. After leaving to crack on ice for 5 min, collect the supernatants after centrifugation at 13,000× g for 20 min.
Note: These steps are performed at 4 °C.
Add 5× loading buffer to the supernatants obtained in the previous step and heat at 95 °C for 10 min.
Separate the products from the previous step by 12% SDS-PAGE and use 10–180 kD marker as an indicator ruler.
Note: Choose the gel percentage according to the molecular weight of the protein of interest.
Run the gel in running buffer at 80 V for approximately 30 min, then adjust the voltage to 120 V and run for 50 min until the loading dye reaches the bottom of the gel.
Note: The run time may vary according to the equipment used or type and percentage of gel.
Transfer the gel using a PVDF membrane at 300 mA for 80 min in transfer buffer.
Block the membrane with Western blocking buffer at room temperature for 2 h.
Incubate the membranes with primary antibodies (Table 1) overnight and shake at 4 °C.
Wash the membranes in 1× TBST three times for 10 min each time.
Incubate the membrane with the appropriate secondary antibody (Table 1) and shake at room temperature for 2 h.
Wash the membrane three times with 1× TBST for 10 min each time
Images are displayed using Western chemiluminescent HRP substrate under chemiluminescence instrument.
For phenotype analysis, NK-Exo is first suspended in 50 μL of PBS according to the concentration of 1 × 109 particles/mL, mixed with 5 μL of PE-conjugated mouse anti-human CD56 (a marker of NK cells) and FITC-conjugated mouse anti-human CD107a (a marker for NK cell degranulation), and incubated at 37 °C for 30 min.
NK-Exo uptake
Stain NK-Exo with PKH67 green fluorescent cell linker Midi kit and mix NK-Exo with diluent buffer (Diluent C).
Note: Do not use a vortex to mix vigorously; use a pipette to gently mix.
Add the NK-Exo mixture from the previous step into the pre-prepared 2× PKH67 dye solution, mix gently, and incubate for 4 min.
Note: NK-Exo must be added to the dye solution—the order must not be changed—and the dye must be mixed from time to time during incubation.
Add an equal volume of FBS to stop staining for 1 min.
Centrifuge at 100,000× g for 70 min to wash the residual dye.
Note: If the dyed exosomes are not used immediately, they can be stored at -80 in the dark for one week.
Culture SKOV3 cells in 24-well plates with 5 × 104 cells per well and add 20 μg of NK-Exo to each well for a 6 h co-culture at 37 .
After incubation, wash the cells with PBS and fix in 4% paraformaldehyde, and stain the nuclei with DAPI.
Note: Washing with PBS should be sufficient to avoid residual dye affecting the experimental results.
Image using a fluorescence microscope and quantify the uptake of NK-Exo by SKOV3 cells using ImageJ 1.53a software.
Functional characterization of NK-Exo
To evaluate the cytotoxicity of NK-Exo on tumor cells, add SKOV3 and OV-90 ovarian cells into 96-well plates at the concentration of 1 × 104 cells per well.
Add NK-Exo with a concentration of 80 μg/mL to each well and incubate at 37 °C for 24 h.
Add 10 μL of Cell Counting kit 8 (CCK8) reagent to each well and incubate at 37 °C for 2 h.
Note: For this suspension volume, 10 μL of CCK8 reagent was added to each well. For other volumes, a corresponding volume of CCK8 is added.
Measure cell viability at 450 nm. Repeat each experiment in triplicate and analyze statistically using GraphPad Prism 8 software.
Calculate cell survival rate using the following formula:
Survival rate = (ODexperiment - ODblank)/(ODcontrol - ODblank) × 100%
Use an Annexin -FITC/PI Apoptosis Detection kit to detect the proportion of tumor cell apoptosis induced by NK-Exo.
Pre-treat SKOV3 and A2780 ovarian cancer cells with 80 μg/mL of NK-Exo for 24 h.
Wash cells twice with 1× PBS and re-suspend in 1× binding buffer.
Stain with 5 μL of Annexin -FITC for 10 min in the dark, followed by 5 μL of PI for 5 min before flow cytometry analysis.
Use a flow cytometer for quantification of apoptotic cells and analyze with FlowJo 10.
Data analysis
Quantification of interception efficiency of TFF system
The particle size and concentration of the filtrate from the TFF system and the prepared NK-Exo were detected by nanoflow to evaluate the interception efficiency of exosomes in the TFF system.
Record all particles passing through the 1 min interval during each detection (Table 2).
Table 2. Comparison of particle distribution in ultrafiltered solution and concentrated solution after TFF system concentration. The particles in the filtrate are below 70 nm, while the particle size of NK-Exo ranges from 60 to 90 nm. By comparing the total particle numbers of the two, it can be concluded that the retention efficiency is 94.40%.
Concentrate Ultrafiltrate
Size (nm) Events Size (nm) Events
40–50 0 40–50 3
50–60 577 50–60 177
60–70 1474 60–70 75
70–80 1205 70–80 17
80–90 661 80–90 5
90–100 397 90–100 1
100–150 592 100–150 3
150–250 86 150–250 1
Total 4992 Total 282
Convert and analyze the collected data by NanoFCM software to obtain the particle concentration and particle size (Figure 1A–1B).
Figure 1. Isolation and nanoflow detection of exosomes derived from NK cells (NK-Exo). The particle concentration and size of ultrafiltrate (A) and NK-Exo (B) were detected by nanoflow.
Characterization of NK-Exo
Morphological images of exosomes were collected by transmission electron microscopy (Figure 2A). They show a typical saucer shape with a particle size of approximately 80 nm, which was consistent with nanoflow results. The expression of exosome surface markers CD63, CD81, and TSG101 were identified by western blot (Figure 2B).
Figure 2. Characterization of NK cell–derived exosomes (NK-Exo). (A) Transmission electron microscopy images of NK-Exo. Scale bar: 100 nm. (B) Western blot analysis of CD81 (22 kDa), CD63 (46 kDa), TSG101 (44 kDa), and calnexin (90 kDa) expression on NK-Exo (A: cell lysate, B: NK-Exo).
Internalization of NK-Exo
Fix SKOV3 cells treated with PKH67-labeled NK-Exo for 6 h with 4% paraformaldehyde for 30 min and wash three times with PBS. Stain with DAPI at 0.1 μg/mL for 20 min and wash three times with PBS.
Take immunofluorescent images of those labeled cells (Figure 3A).
Quantify fluorescence intensity and area using ImageJ software to calculate uptake rate of PKH67-labeled NK-Exo (Figure 3B).
Figure 3. Cellular uptake of NK cell–derived exosomes (NK-Exo) by ovarian cancer cells. (A) NK-Exo uptake by SKOV3 cells. SKOV3 cells were stained blue by DAPI and NK-Exo were stained green by PKH67. (B) NK-Exo uptake quantified by green fluorescence intensity (10×, scale bar: 200 μm; 100×, scale bar: 40 μm, n = 3, mean ± SEM, t-test, **p < 0.01). Quantification of fluorescence intensity demonstrated significant uptake of PKH67-labeled NK-Exo by SKOV3 cells and the uptake rate reached 50% in 6 h.
Functional verification of NK-Exo
Detect the expression of typical NK cell marker CD56 and degranulation marker CD107a on NK-Exo by nanoflow analysis (Figure 4A).
Detect the expressions of CD56 and perforin in NK-Exo by western blot (Figure 4B). NK cells are known to exert their cytolytic effect through the release of effector molecules such as perforin, which may contribute to the cytotoxicity of NK-Exo against tumor cells.
Detect the cytotoxicity of NK-Exo to SKOV3 and OV-90 ovarian cancer cells by CCK8 assay (Figure 4C).
Use flow cytometry to detect NK-Exo–mediated apoptosis of SKOV3 and A2780 ovarian cancer cells (Figure 4D). Apoptosis analyzed by flow cytometry confirmed that NK-Exo can induce apoptosis in SKOV3 and A2780 cells; the proportion of apoptotic cells was comprised of both late apoptotic (upper right quadrant) and early apoptotic cells (bottom right quadrant).
Figure 4. Functional characterization of NK cell–derived exosomes (NK-Exo). (A) Expression of CD56 and CD107a on the surface of NK-Exo was detected by nanoflow. Red dots represent positive signals. (B) Western blot analysis of CD56 (122 kD) and perforin (61 kD) expression (A = cell lysates, B = NK-Exo). (C) CCK-8 assay of NK-Exo against SKOV3 cells and OV-90 cells (n = 3, mean ± SEM, t-test, *p < 0.05, ***p < 0.001). (D) Apoptosis analysis of SKOV3 and A2780 cells after NK-Exo treatment by flow cytometry.
Discussion
The traditional exosome separation method is ultracentrifugation. However, this method has limitations on the volume for application, and can only achieve a large number of exosomes by using a small volume and multiple centrifuges, which is very costly and therefore poses a great challenge to the application of exosomes in clinical treatment (Liang et al., 2018).
To overcome these issues, we used a TFF system combined with a conventional ultra-high speed centrifugal method to concentrate a large volume of cell culture supernatants. Concentration ensures that exosomes are retained while smaller impurities and excess water are removed. This step can achieve 50–160-fold concentration of cell culture supernatants and adequately trap NK-Exo to ensure no exosome loss.
Before concentration, we performed a two-step low-speed centrifugation on cell culture supernatants to remove large cell fragments. This step reduces the wastage of the TFF system and speeds up the concentration. The inlet pressure was maintained at 2.5 bar for concentration to avoid damaging the structural integrity of NK-Exo. The pressure is then reduced at a slow rate for final concentration before completing enrichment to improve the recovery rate of NK-Exo. NK-Exo were then separated from the concentrated solution by ultracentrifugation. The particle concentration and size of tangential flow filtrate and NK-Exo were detected by nanoflow, and it was determined that the interception efficiency of TFF system for NK-Exo was higher than 90%.
As confirmed by the characterization results of NK-Exo, this method does not damage the structural integrity of NK-Exo, which still maintained their characteristic morphology and size, with removal of most of the cell debris contamination.
In this study, NK-Exo were further functionally validated. First, NK-Exo were confirmed to be able to enter ovarian cancer cells and further exert their effect. We detected some cytotoxic substance in NK-Exo and demonstrated NK-Exo were able to exert cytotoxic effects on OC cells and induce apoptosis in vitro. Therefore, NK-Exo isolated from large-scale cell culture supernatants by our protocol can maintain their complete morphological structure, phenotype, and anti-tumor activity, all of which provide a possibility for the application of NK-Exo in clinical tumor therapy.
Acknowledgments
X.Z. was supported by the Science and Technology Program of Guizhou Province, China [grant number: (2019)1263] and Key Program for Science and Technology of Guizhou Province [grant number: ZK (2021)012]. J.Z. was supported by Guizhou Provincial Natural Science Foundation [grant number: J (2015)2017]. This work was supported by Foundation of Development and Related Diseases of Women and Children Key Laboratory of Sichuan Province (Grant No. 2022002).
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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Synthesis and Purification of Lipid-conjugated Fluorescent pH Sensors
WW Wiebke Wiesner
RK Ronja Marie Kühnel
TP Thomas Guenther Pomorski
Published: Vol 13, Iss 11, Jun 5, 2023
DOI: 10.21769/BioProtoc.4694 Views: 712
Reviewed by: Gal HaimovichPhilipp A.M. Schmidpeter Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Analyst May 2019
Abstract
Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.
Graphical overview
Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE)
Keywords: Electrochemical gradients Fluorescence Labelled lipids Liposome Membrane transporter pH sensor Reconstitution
Background
Cells and organelles depend on electrochemical gradients across their membranes, which are maintained through ATP-driven, membrane-embedded transporter proteins. Transport of ions across cellular membranes can be primary or coupled to drive the uptake or export of other solutes. Perturbations of the highly regulated transport systems are the cause to many diseases and can be the reason for failure of pharmacotherapy (Padan and Landau, 2016; Clausen et al., 2017; Picci et al., 2022). Given the pivotal role of these transporters in cell homeostasis and health, a thorough understanding of their organization, functioning, and dynamics is highly desirable. A powerful tool to visualize the activity of ion transporters in living cells and reconstituted membrane systems are fluorescent compounds based on synthetic molecules. Although a variety of ion-targeting probes are commercially available, many have limitations in studies with membrane transporters. For example, water-soluble dyes such as pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid) and fluorescamine (4-phenylspiro-[furan-2(3H),1-phthalan]-3,3′-dion) have been used to monitor the intravesicular pH values in studies of H+-translocating proteins (S. Li et al., 2011; Ohlsson et al., 2012; M. Li et al., 2015; Berg et al., 2017). However, their modest brightness (pyranine) and high bleaching rates (fluorescamine) make them difficult to use for pH measurements in vivo and in vitro. Rhodamine-based dyes provide an alternative due to their improved brightness and photostability; however, for reconstituted systems, problems with insufficient encapsulation during protein reconstitution and leakage out of vesicles were reported (Schubert, 2003; Leiding et al., 2009). More recently, membrane-bound pH sensors based on lipid-conjugated fluorophores have been utilized (Kemmer et al., 2015; Veshaguri et al., 2016; Schwamborn et al., 2017; Gerdes et al., 2018; Kühnel et al., 2019). These lipid-conjugated pH sensors efficiently co-reconstitute with membrane proteins into large liposomes, thereby avoiding loss during reconstitution and allowing studies in reconstituted systems down to the single vesicle level (Kemmer et al., 2015; Veshaguri et al., 2016; Kühnel et al., 2019). Lipid-conjugated pH sensors were also instrumental to study the activity of lipases on native substrate systems (Bohr et al., 2020). Furthermore, short-chain lipid-conjugated pH sensors enable monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells (Kühnel et al., 2019).
Here, we describe the synthesis of lipid-conjugated pH sensors from amine-reactive pHrodo esters and phosphatidylethanolamines. The method described here enables fast preparation of the sensor and can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.
Things to consider before starting
Choice of the fluorophore
The fluorophore must contain an amine-reactive functional group such as NHS- or STP-ester, to allow for coupling with the ethanolamine head group of the lipid. Furthermore, the fluorophore characteristics must be suitable for the later use of labelled lipids, e.g., pH range to induce a change in fluorescence emission. In Table 1, a number of reactive fluorophores are listed that we coupled effectively to phosphatidylethanolamines by using the protocol described here.
Choice of lipid
The lipid headgroup must contain an ethanolamine group to allow for the coupling reaction with amine-reactive dyes. The lipid chains can vary. Herein, dioleoyl- and dipalmitoylphosphatidylethanolamine (DOPE and DPPE, respectively) were employed. The usage of other lipids might lead to decreased yields, as the reaction conditions are optimized for DOPE and DPPE.
Choice of analysis method
A method to verify the successful formation of the labelled lipid is e.g., high resolution mass spectroscopy (MS), since small amounts of substance can be reliably detected. Two potential methods are MALDI-TOF MS (matrix-assisted laser desorption/ionization with time-of-flight analysis MS) and ESI MS (electrospray ionization MS).
Table 1. Fluorophores that have been coupled effectively to phosphatidylethanolamine (PE)
Fluorophore Excitation (nm) Emission (nm) pH range# pKa#
pHrodo Red NHS-ester 566 588 2.7–7.0 4.6
pHrodo Green STP-ester 505 543 2.7–7.0 4.6
SNARF-1 NHS ester 488–530 585/650† 6–11 9.3
Alexa FluorTM 488 NHS-ester 494 517 insensitive ---
TAMRA NHS-ester 541 567 insensitive ---
#Values upon coupling to DOPE (Kemmer et al., 2015; Kühnel et al., 2019).
†Values upon coupling to DOPE. Note that the emission spectrum of SNARF-1 undergoes a pH-dependent wavelength shift, thus allowing the ratio of the fluorescence intensities from the dye at two emission wavelengths to be used for more accurate determinations of pH.
All catalog numbers provided below shall serve as guide; alternative sources can be used as well.
Materials and reagents
All catalog numbers provided below shall serve as guide; alternative sources can be used as well.
Materials
Aluminium foil
Chromatography column, 200 × 10 mm, 15 mL (VWR, catalog number: 552-5410)
Disposable glass Pasteur pipettes (150 mm; VWR, catalog number: 612-1701)
Glass beads, 3 mm (Supelco, catalog number: 1040150500)
Glass cutter (Bohle, catalog number: BO 400.1)
Glass marbles (Fisher Scientific, catalog number: S04581)
Glass pipettes (e.g., graduated pipettes BLAUBRAND® Type 3 Class AS, 10 mL, graduation: 10 mL; Carl Roth, catalog number: HXT8.1)
Glass wool (VWR, catalog number: 519-3101)
Hamilton 700 Series Syringes of 25, 100, and 500 μL (Peter Oehmen GmbH, catalog numbers: 9221013, 9221015, 6055335)
Long necked glass tubes (Carl Roth, catalog number: 0486.2)
Magnetic bars ROTILABO® Micro, diameter 2 mm, length 5 mm (Carl Roth, catalog number: 0955.2)
Pipette tips 200 μL and 1,000 μL (SARSTEDT AG & Co. KG, catalog numbers: 70.760.002 and 70.3050.020)
Pointed-bottom glass tube, 10 mL (Merck, catalog number: CLS9950210)
Round-bottom glass tube, 10 mL, with joint and plug (Carl Roth, catalog number: NY90.1)
Spray bottle for primuline staining (Carl Roth, catalog number: YC44.1)
TLC plates, silica gel 60 (Merck, catalog number: 105626)
TLC chamber (Carl Roth, catalog number: 1N64.1)
Chemicals
Acetone p.a. (VWR, catalog number: 32201)
Ammonium molybdate tetrahydrate (Carl Roth, catalog number: 3666.1)
L-ascorbic acid (Carl Roth, catalog number: 3525.2)
Chloroform, ethanol-stabilized and certified for absence of phosgene and HCl (CHCl3) (Roth, catalog number: 7331.2)
CM SepharoseTM Fast Flowing (Sigma-Aldrich, catalog number: 17 0719-01)
Deionized water
Dichloromethane (DCM) (VWR, catalog number: 32222)
N’,N’-Diisopropylethylamine (DIPEA) (Sigma-Aldrich, catalog number: D125806)
N’,N’-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: D4551)
Ethanol p.a. (VWR, catalog number: 20821.321)
Methanol (MeOH) (VWR, catalog number: 20847.307)
Nitrogen gas, 99.999% (ALPHAGAZ, Air Liquide, Düsseldorf, Germany)
Perchloric acid (VWR, catalog number: 20589)
Primuline (Sigma-Aldrich, catalog number: 206865)
Di-sodium hydrogen phosphate dihydrate (VWR, catalog number: 28029)
Triethylamine (VWR, catalog number: 8.08352)
Ascorbic acid solution (see Recipes)
Extraction solution (see Recipes)
Molybdate solution (see Recipes)
Phosphate standard solution (see Recipes)
Primuline staining solution (see Recipes)
Reaction solvent (see Recipes)
TLC eluent (see Recipes)
Lipids
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipids, Alabaster, AL, USA, catalog number: 850725)
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE, Avanti Polar Lipids, Alabaster, AL, USA, catalog number: 850705)
Note: This procedure has also been successfully performed using short-chain lipids including 1,2-dihexanoyl-sn-glycero-phosphoethanolamine (diC6-PE, catalog number: 850697), 1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine (diC8-PE, catalog number: 850699), and 1-hexadecanoyl-2-hexanoyl-sn-glycero-3-phosphoethanolamine (C16C6-PE, custom made), all purchased from Avanti Polar Lipids (Alabaster, AL, USA).
Fluorescent dyes
pHrodo Red NHS-ester (Invitrogen, catalog number: P36600)
pHrodo Green STP-ester (Invitrogen, catalog number: P35369)
Alexa FluorTM 488 NHS-ester (Invitrogen, catalog number: A20000)
Note: This procedure has also been successfully performed using TAMRA NHS-ester (Lumiprobe, catalog number: 18120) and SNARF-1 NHS ester (Invitrogen, catalog number: S22801).
Equipment
Pipettes P200 and P1000 (GILSON®, catalog numbers: FD10005 and FD10006)
Analytical balance (Sartorius Entris-i II, 220 g/0.1 mg; Buch Holm, catalog number: 4669128)
Chemidoc MP imaging system (Bio-Rad) with illumination at 460–490 nm and 520–545 nm with 530/28 nm and 695/55 nm emission filters, respectively
CLARIOstar plate reader (BMG LABTECH)
Freezer -20 °C
Glass desiccator Boro 3.3 with socket in lid, 20 cm, including stopcock (BRAND GmbH, catalog number: 65238)
Heating block (Rotilabo® Block Heater H 250; Carl Roth, catalog number: Y264.1)
Magnetic stirrer (e.g., IKAMAG®, DREHZAHL ELECTRONIC, IKA, Staufen im Breisgau, Germany)
Refrigerated centrifuge (e.g., Multifuge® 3-R, Kendro Laboratory Products, catalog number: 75004371)
Vacuum Pump V-100 with interface I-100 and rotary evaporator Rotavapor® R-100, SJ29/32, V, 220–240V (Buchi, catalog numbers: 11593636, 11593655D, and 11100V111, 11061895)
Vortex mixer (Vortex Genie 2 Scientific Industries Inc., catalog number: SI-0236)
Water bath (Julabo CORIO C-BT5, catalog number: 9011305)
Software
ImageLab software version 5.2.1 (Bio-Rad)
Procedure
Coupling reaction of DOPE with pHrodo Red NHS-ester (Figure 1)
Glassware should be used throughout the procedure, since lipids stick to plasticware, and chloroform can extract components from plasticware. Lipid stocks are handled on ice to reduce evaporation of chloroform during pipetting. In this section, the coupling of DOPE with pHrodo Red NHS-ester will be described. The procedure was also successfully performed using pHrodo Green STP-ester and Alexa Fluor 488 NHS-ester with DPPE as lipid. The labelling of short-chain lipids such as C16C6PE is also possible using this procedure.
Note: The volumes given below serve just as an orientation. All volumes need to be calculated properly for each reaction depending on the used fluorophore and lipid.
Figure 1. Synthesis scheme of pHrodo red–labelled DOPE
Lipids are received in chloroform and packaged in sealed glass ampoules; store at -20 °C until use.
Note: For long-term storage, evaporate the solvent in a vacuum desiccator and store the lipids at -80 °C to avoid oxidation of unsaturated lipids. To evaporate the solvent, we use a glass desiccator connected to a chemically resistant vacuum pump, reaching a final vacuum of 10 mbar (± 2 mbar) and a suction capacity of 1.5 m3/h.
To prepare lipid stocks, transfer 10 mg aliquots from the Avanti glass ampoule into glass screw neck vials. Evaporate the solvent from the glass vials in a desiccator at 250 mbar for 3 h with an additional incubation for 1 h at 30 mbar. Close the vials with screw caps and store at -20 °C until further use.
Note: Chloroform is a hazardous solvent. Conduct all work in a fume hood while wearing proper protective clothing.
Remove desired lipid stocks from freezer, place on ice, and dissolve in chloroform to a final lipid concentration of 10 mg/mL.
Note: Lipids other than DOPE may have limited solubility in chloroform and require a mixture of chloroform/methanol/water. Further directions and guidance can be found on the Avanti Polar Lipids’ web site (https://avantilipids.com/products).
Transfer the desired quantity of lipid (usually between 1 and 2 mg for 500 μg of fluorophore NHS-ester) from the CHCl3 stock solution to a round-bottom glass tube with a joint (NS 14). To maximize the reaction yield, we use a 2-fold molar excess of the lipid over fluorophore NHS- or STP-ester.
Evaporate the solvent carefully under a N2 stream at room temperature. After solvent removal, add a small magnetic stirring bar.
Note: We slide the large end of a disposable glass Pasteur pipette into laboratory tubing attached to the N2 gas cylinder. The flow is adjusted for a gentle stream of N2 gas through the pipette. The pipette can be held with a ring stand clamp to hold it in a fixed position.
Solubilize the fluorophore (500 μg) in 100 μL of the reaction solvent (see Recipes) and transfer it to the lipid-containing glass tube. Use approximately 100 μL for 500 μg of fluorophore.
Add DIPEA to a final concentration of 130 mM (e.g., 2.3 μL of DIPEA for 100 μL of reaction solvent).
Note: DIPEA is toxic and can cause severe skin burns.
Seal the vessel with a glass plug (NS 14) and wrap it in aluminium foil. Place the vessel on top of a magnetic stirrer and let the reaction stir overnight at room temperature.
Extraction of the labelled lipid
For extraction, add 0.5 mL of CHCl3, 1 mL of MeOH, and 0.5 mL of H2O followed by another 0.5 mL of CHCl3 and 0.5 mL of H2O. The reaction mixture should be monophasic at first and then turn biphasic (Figure 2).
Figure 2. Result of the extraction process of fluorescent-labelled lipids. Coloured organic phase at the bottom and aqueous phase on top.
Transfer the organic phase (coloured bottom phase) to a new glass tube.
Note: Short-chain lipids may require several subsequent washes with CHCl3 to yield a decolorized aqueous phase.
Dry down the organic phase either by N2 stream or using rotary evaporator, depending on the volume of solvent.
After complete evaporation of the solvent, either continue with purification (see Section C1 and C2) or store the sample at -20 °C.
Purification of the labelled lipid
C1. Purification via preparative thin layer chromatography (TLC)
Before starting the preparative TLC, the retention factor (Rf) of the product needs to be determined. For this, a test TLC can be performed (Figure 3).
Prepare an approximately 5 cm wide TLC plate using a glass cutter.
Carefully draw a line approximately 1 cm above the bottom of the plate with a pencil.
Apply small volumes of the dissolved lipid stock and dissolved crude reaction product (e.g., with a glass Pasteur pipette) to the thin line at the bottom and let them dry completely.
Fill a TLC chamber with TLC eluent (see Recipes) up to a filling level of approximately 0.8 cm.
Close the lid and let the chamber saturate for approximately 5 min.
Place the TLC plate carefully and upright into the chamber and close the lid. Remove the TLC plate from the chamber when the solvent front is approximately 1 cm from the top.
Mark the solvent front line with a pencil.
Let the TLC plate dry under the fume hood for approximately 1 h before taking an image under white light and long-wave UV, to make fluorophore-coupled lipids and unreacted fluorophore visible using e.g., the Chemidoc MP imaging system.
Spray the plate with a primuline staining solution (see Recipes) to make lipids visible under long-wave UV light after drying.
Calculate the Rf value as follows:
Rf values of different fluorophore-conjugated DOPE lipids are 0.70 and 0.55 for pHrodo Red-DOPE and pHrodo Green-DOPE, respectively.
Figure 3. Thin layer chromatography (TLC). A. Materials needed for TLC preparation. B. Prepared TLC plate; L = pure lipid, P = reaction product. C. TLC plate placed upright in TLC chamber. D. TLC plate photographed under white light. E. TLC plate after primuline staining, photographed under long UV light. The position of non-reacted DOPE and pHrodo Red-DOPE is indicated by an open and filled arrowhead, respectively. O, origin; F, solvent front of the chromatograms.
Prepare the preparative TLC by using a 20 cm long TLC plate.
Draw a line 1 cm above the bottom of a 20 cm long TLC plate.
Dissolve the reaction product in 100 μL of CHCl3:MeOH (2:1, v/v).
Apply the complete volume of dissolved product evenly on the line of the TLC plate, e.g., with a glass Pasteur pipette.
Let the TLC plate dry completely for approximately 1 h in the fume hood.
Fill a TLC chamber with TLC eluent (see Recipes) up to a filling level of approximately 0.8 cm.
Close the lid and let the chamber saturate for 20 min.
Place the TLC plate upright in the chamber.
Remove the TLC plate when the solvent front is approximately 1 cm beneath the top.
Let TLC plate dry under the fume hood before taking an image under white light and under long-wave UV to make fluorophore-coupled lipids and unreacted fluorophore visible. A typical result is shown in Figure 4.
Figure 4. Preparative thin layer chromatography (TLC) of a pHrodo-labelled lipid with a reference of the pure product on the right side of the TLC plate. The chromatogram shown was dried completely before photographing under white light. The origin is indicated by the black line.
C2. Re-extraction of the labelled lipid from preparative TLC
Carefully scrape off the product bands with a spatula and place the silica in pointed-bottom glass tubes (without joint, must fit into centrifuges).
Add 1 mL of the extraction solution (see Recipes) to each tube used and vortex each tube for 1 min.
Centrifuge the tubes at 480× g for 10 min at 8 °C.
Transfer the liquid phase to a new glass tube.
Repeat steps 2–4 until the liquid phase becomes colourless.
Note: In the glass tube with the combined liquid phases, some silica might settle down at the bottom after a few minutes. The liquid phases can be transferred to a new vessel to remove silica.
To induce a phase separation, add a 1:1 mixture of CHCl3 and H2O (v/v). Transfer the organic phase (coloured bottom phase) to a new glass tube and dry down via N2 stream or in a desiccator. Proceed with quantification (Section D) or store at -20 °C.
C3. Purification via column chromatography
As an example, the purification of Alexa Fluor 488 DPPE is used. The exact gradient conditions can vary depending on the used lipid and fluorophore.
Prepare the column.
Provide the column with glass a wool plug to create a tight filter in front of the valve.
Transfer enough CM Sepharose (typically approximately 20–25 mL of suspension) into a beaker to yield a stationary phase of 10 cm height in the column (1cm diameter).
Fill the Sepharose into the column in one go and run out surplus MeOH.
Note: Be careful to never let the column run dry!
Close the valve before the top of the column runs dry. To remove air bubbles in the column, tap it gently e.g., with a cork ring while the Sepharose is settling.
After settling, cover the Sepharose under a 1–3 mm thick layer of glass beads. Gradually rinse the MeOH to CHCl3 according to the mixtures given in Table 2.
Table 2. MeOH and CHCl3 ratios for rinsing the column
VMeOH (mL) VCHCl3 (mL)
7 3
5 5
3 7
1 9
0 10 (10×)
Dissolve the reaction product in approximately 250 μL of CHCl3 and carefully add it to the top of the column by moving in a circle along the wall to ensure an even distribution. Wash your sample tube with small volumes until colourless and apply it on the column (Figure 5A).
Perform the column chromatography while gradually increasing the MeOH content of the eluent, starting from pure CHCl3 (Table 3). Collect all fractions separately in 10 mL glass tubes.
Evaporate the solvent in all sample tubes either in a desiccator or with a rotary evaporator. The reaction product can be identified via TLC (Figure 5B). Continue with quantification or store the samples at -20 °C.
Table 3. Used MeOH and CHCl3 ratios for the purification of A488-DPPE
V MeOH (mL) V CHCl3 (mL) MeOH (%) Total volume (mL)
-- 20 0 20
1 19 5 20
1.4 18.6 7 20
4.5 45.5 9 50
3.6 26.4 12 30
9 51 15 60 (A488-DPPE)
15 15 50 30 (A488-DPPE)
20 10 67 30 (A488-DPPE)
Figure 5. Purification via column chromatography. A. Used column for the purification of labelled lipids. B. TLC plate to determine the product containing fractions, exemplified for nitrobenzoxadiazole (NBD)-labelled lipid. The chromatogram shown was dried completely before photographing under ambient light.
Quantification of the reaction according to Bartlett (1959) and Ploier and Menon (2016), with small modifications to the protocol
Note: All steps described below must be performed in a fume hood using appropriate personal protection and following lab safety guidelines.
Turn on the electrical heating block (placed in fume hood) and set it to 195 °C.
Prepare the sample tubes. We recommend preparing triplicates.
Dissolve the sample in an appropriate volume of CHCl3 and transfer approximately 20 nmol (assuming 100% yield) into a glass tube.
Evaporate the solvent via N2 stream.
Prepare the standard tubes. We recommend preparing two standards per concentration.
Dilute an aliquot of the phosphate standard solution (see Recipes) to 0.4 mM in deionized water.
Transfer 0 (0 μL), 5 (12.5 μL), 10 (25 μL), 20 (50 μL), 30 (75 μL), 40 (100 μL), and 50 nmol (125 μL) phosphate to glass tubes.
Add 650 μL of perchloric acid to all tubes (including standards) and cover them loosely with glass marbles.
Heat all tubes in the heating block in the hood at 195 °C for approximately 1–2 h until the solution turns clear. Let the solution cool to room temperature.
Turn on the water bath under the fume hood and set it to 80 °C.
When samples are at room temperature, add 3.3 mL of deionized water to each test tube and vortex it.
Add 0.5 mL of molybdate solution (see Recipes) to each test tube and vortex it.
Add 0.5 mL of ascorbic acid solution (see Recipes) to each test tube and vortex it.
Boil all samples at 80 °C in the water bath for 10 min.
After cooling all the samples to room temperature, transfer each sample in duplicates to a 96-well plate.
Determine the absorption of each well using a microplate reader set to 812 nm. The reading can be repeated at 780 and/or 720 nm to adjust for the best sensitivity.
Average the duplicate and triplicate readings of each standard and sample, respectively.
Subtract the value of the blank standard (0 nmol phosphate) from all standard and sample readings. This is the corrected absorbance.
Plot standard curves (mean absorbance of wavelengths vs. nmol phosphate) and perform linear regression using e.g., Microsoft Excel, resulting in an equation of the type y = mx + b, where y is the absorbance, x is the phosphate concentration, and m is the slope; the intercept of the y-axis b is 0 after subtracting the value of the blank standard.
Use the regression line to solve for sample concentration, by comparing the sample absorbance to the standard curve obtained (Figure 6).
Figure 6. Typical plot of the results of a phosphate assay. Linear regression was applied to the averaged phosphate standard absorbance (812 nm) values of all different concentrations (blue points and dotted line). The sample concentration (red point) was determined using the equation given by linear regression and included into the graph.
Analyse your product using reliable methods for small amounts such as mass spectroscopy. A typical result is shown for pHrodo Green-labelled DOPE in Figure 7.
Figure 7. Exemplary MALDI-TOF mass spectra of pHrodo Green-labelled DOPE via MALDI-TOF mass spectrometry in negative ion mode (A) and positive ion mode (B). Since no chemical structure was published by the manufacturer and only an approximate molecular weight of ~750 g/mol was given, indicated signals correspond to the expected product peak in the range of mass per charge (m/z) 1,250. Accordingly, m/z 1,241.2 corresponds to the deprotonated product molecule and m/z 1,243.3 to the proton adduct.
Recipes
Ascorbic acid solution (50 mg m/L)
Dissolve 600 mg of L-ascorbic acid in 12 mL of deionized water. This solution cannot be stored.
Extraction solution
Mix 2 mL of chloroform, 4.4 mL of MeOH, and 2 mL of H2O and store in a sealed glass bottle at room temperature.
Molybdate solution (12 mg/L)
Dissolve 144 mg of ammonium molybdate tetrahydrate in 12 mL of deionized water. This solution cannot be stored.
Phosphate standard solution (4 mM)
Dissolve 35.598 mg of di-sodium hydrogen phosphate dihydrate in 50 mL of deionized water. This solution can be stored at -20 °C.
Primuline staining solution (0.05%, w/v)
Dissolve 50 mg of primuline in 100 mL of a mixture of water/acetone (v/v, 20:80)
Reaction solvent
Mix 6 mL of DCM with 1 mL of DMF and store in a sealed glass bottle at room temperature.
TLC eluent
Mix 30 mL of chloroform, 35 ml EtOH, 35 mL of triethylamine, and 7 mL of deionized water directly in the TLC chamber. This solution cannot be stored or used more than once.
Acknowledgments
This protocol was adapted from our previous work (Kemmer et al., 2015; Veshaguri et al., 2016; Kühnel et al., 2019; Bohr et al., 2020). We would like to thank PD Dr. Jürgen Schiller (University of Leipzig, Germany) for his continuous help with mass spectroscopy during the whole time of this project. This work was supported by an instrument grant from the Deutsche Forschungsgemeinschaft (INST 213/886-1 FUGG, INST 213/985-1FUGG; GU 1133/11-1). The graphical abstract was created using BioRender.com.
Competing interests
The authors declare that no competing interests exist.
References
Bartlett, G. R. (1959). Phosphorus assay in column chromatography. J Biol Chem 234(3): 466-468.
Berg, J., Block, S., Höök, F. and Brzezinski, P. (2017). Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks. Isr J Chem 57(5): 437-445.
Bohr, S. S., Thorlaksen, C., Kühnel, R. M., Günther-Pomorski, T. and Hatzakis, N. S. (2020). Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature. Langmuir 36(23): 6473-6481.
Clausen, M. V., Hilbers, F. and Poulsen, H. (2017). The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease. Front Physiol 8: 371.
Gerdes, B., Rixen, R. M., Kramer, K., Forbrig, E., Hildebrandt, P. and Steinem, C. (2018). Quantification of Hv1-induces Proton Translocation by a Lipid-couples Oregon Green 488-based assay. Anal Bioanal Chem 410(25): 6497-6505.
Kemmer, G. C., Bogh, S. A., Urban, M., Palmgren, M. G., Vosch, T., Schiller, J. and Günther Pomorski, T. (2015). Lipid Conjugated Fluorescent pH Sensor for Monitoring pH Changes in Reconstituted Membrane Systems. Analyst 140(18): 6313-6320.
Kühnel, R. M., Grifell-Junyent, M., Jørgensen, I. L., Kemmer, G. C., Schiller, J., Palmgren, M., Justesen, B. H. and Günther Pomorski, T. (2019). Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells. Analyst 144(9):3030-3037.
Leiding, T., Górecki, K., Kjellman, T., Vinogradov, S.A., Hägerhäll, C. and Årsköld, S. P. (2009). Precise Detection of pH Insode Large Unimolecular Vesicles Using Membrane-Impermeable Dendritic Porphyrin-Based Nanoprobes. Anal Biochem 388(2): 296-305.
Li, M., Jørgensen, S. K., McMillan, D. G. G., Krzemiński, Ł, Daskalakis, N. N., Partanen, R. H., Tutkus, M., Tuma, R., Stamou, D., Hatzakis, N. S., et al. (2015). Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in Heme-Copper Oxidase. J Am Chem Soc 137(51): 16055-16063.
Li, S., Hu, P. C. and Malmstadt, N. (2011). Imaging Molecular Transport across Lipid Bilayers. Biophys J 101(3): 700-708.
Ohlsson, G., Tabaei, S. R., Beech, J., Kvassman, J., Johanson, U., Kjellbom, P., Tegenfeldt, J. O. and Höök, F. (2012). Solute Transport on the Sub 100 ms Scale across the Lipid Bilayer Membrane of Individual Proteoliposomes. Lab Chip 12(22): 4635-4643.
Padan E. and Landau, M. (2016). Sodium-Proton (Na+/H+) Antiporters: Properties and Roles in Health and Disease. In: Sigel, A., Sigel, H. and Sigel, R. K. O. (Eds.). The Alkali Metal Ions: Their Role for Life (pp. 391-458). Springer.
Picci, G., Marchesan, S. and Caltagirone, C. (2022). Ion Channels and Transporters as Therapeutic Agents: From Biomolecules to Supramolecular Medicinal Chemistry. Biomedicines 10(4): 885.
Ploier, B. and Menon, A. K. (2016). A Fluorescence-based Assay of Phospholipid Scramblase Activity. J Vis Exp (115): 54635.
Schubert, R. (2003). Liposome Preparation by Detergent Removal. Meth Enzymol 367: 46-70.
Schwamborn, M., Schumacher, J., Sibold, J., Teiwes, N. K. and Steinem, C. (2017). Monitoring ATPase Induced pH Changes in Single Proteoliposomes with the Lipid-coupled Fluorophore Oregon Green 488. Analyst 142(14): 2670-2677.
Veshaguri, S., Christensen, S. M., Kemmer, G. C., Ghale, G., Moller, M. P., Lohr, C., Christensen, A. L., Justesen, B. H., Jørgensen, I. L., Schiller, J., et al. (2016). Direct Observation of Proton Pumping by a Eukaryotic P-type ATPase. Science 351(6280): 1469-1473.
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4,695 | https://bio-protocol.org/en/bpdetail?id=4695&type=0 | # Bio-Protocol Content
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Peer-reviewed
Synthesis of Bacteria-mimetic Gold Nanoparticles for Phagocytosis by Immune Cells
CG Cheng Gao *
MT Mian Tang *
SL Simon M. Y. Lee
RW Ruibing Wang
(*contributed equally to this work)
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4695 Views: 1105
Reviewed by: Alka MehraSameer NadafChhuttan L MeenaKarem A Court
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Original Research Article:
The authors used this protocol in Science Advances May 2022
Abstract
Cell-based carrier exhibits inherent advantages as the next generation of drug delivery system, namely high biocompatibility and physiological function. Current cell-based carriers are constructed via direct payload internalization or conjugation between cell and payload. However, the cells involved in these strategies must be firstly extracted from the body and the cell-based carrier must be prepared in vitro. Herein, we synthesize bacteria-mimetic gold nanoparticles (GNPs) for the construction of cell-based carrier in mice. Both β-cyclodextrin (β-CD)-modified GNPs and adamantane (ADA)-modified GNPs are coated by E. coli outer membrane vesicles (OMVs). The E. coli OMVs induce the phagocytosis of GNPs by circulating immune cells, leading to intracellular degradation of OMVs and subsequent supramolecular self-assembly of GNPs driven by β-CD-ADA host–guest interactions. In vivo construction of cell-based carrier based on bacteria-mimetic GNPs avoids the immunogenicity induced by allogeneic cells and restriction by the number of separated cells. Due to the inflammatory tropism, endogenous immune cells carry the intracellular GNP aggregates to the tumor tissues in vivo.
Graphical overview
Collect the outer membrane vesicles (OMVs) of E. coli by gradient centrifugation (a) and coat on gold nanoparticles (GNP) surface (b) to prepare OMV-coated cyclodextrin (CD)-GNPs and OMV-coated adamantane (ADA)-GNPs (c) via ultrasonic method
Keywords: Bacteria biomimetics Gold nanoparticle Supramolecular chemistry Cell-based carrier Drug delivery
Background
As a self component, cells exhibit inherent physiological properties in drug delivery, such as biocompatibility and homing effect (Yang et al., 2022). These help the drug payload to escape clearance by the mononuclear phagocyte system and perform the physiological function–based hitchhiking delivery. Recently, cell-based carriers were mainly constructed via direct drug internalization; this procedure was very simple, needing only in vitro cell culture with the payload (Li et al., 2021). In addition, another construction strategy was surface conjugation between cell and payload, which could be divided into covalent conjugation, receptor–ligand interaction, host–guest interaction, and physical interaction (Krueger et al., 2018; Li et al., 2018). The host cell must be artificially modified in vitro and then conjugated with payload. Thus, cell-based carriers, via either intracellular drug internalization or cell-surface attachment, were constructed in vitro before in vivo administration. This presented three challenges in clinical translation and application: 1) large-scale preparation is barely possible due to the limitation of separating sufficient number of endogenous cells; 2) in vitro drug loading processes may inevitably affect the physiological function of the carrier cells; and 3) transporting cells correspond to one single host, and would generate immune rejection when applied to other individuals.
In light of these issues, bacteria-mimetic gold nanoparticles (GNPs) were developed for in vivo construction of immune cell–based carriers and efficiently and stably hitchhiking delivery. In this design, both β-cyclodextrin (β-CD)-modified GNPs (CD-GNPs) and adamantane (ADA)-modified GNPs (ADA-GNPs) are coated by E. coli outer membrane vesicles (OMVs). Host–guest interactions involve two molecules or materials that can form complexes through unique structural relationships and noncovalent binding. Due to host–guest interactions between β-CD and ADA (Loescher and Walther, 2020; Wang et al., 2021), the host β-CD could bind with guest ADA to form a supramolecular complex. Thus, CD-GNPs bound with ADA-GNPs to produce GNP aggregates. It has been observed that the GNP aggregates show higher absorption ratio and therefore, higher temperature rise compared to single GNPs. The effect of photothermal therapy (PTT) was significantly improved in the form of GNP aggregates in comparison to GNPs. The E. coli OMVs coating induce the phagocytosis of GNPs by circulating immune cells, leading to intracellular degradation of OMVs and subsequent supramolecular self-assembly of GNPs. In addition to the increased PTT efficiency, GNP aggregation increased the nano-size of GNP into micro-size of GNP aggregate, which significantly inhibits the leakage of GNPs from immune cells (Zhang et al., 2021). This bacterial OMV-coating strategy for immune cell phagocytosis was not only applied to GNPs, but also extended to other therapeutic reagents that directly modulate immune cells or target disease tissues via immune cell hitchhiking delivery.
As different cells (platelets, stem cells, macrophages, T cells, or even tumor cells) have different physiological functions, the coating material could be specifically designed for phagocytosis according to the required type of cells as carriers for hitchhiking delivery.
Materials and reagents
Falcon tube (Falcon, catalog number: 352098)
Millipore with 10 kDa filter (Millipore, catalog number: UFC901024)
Nylon membrane filter with 0.45 pore size (Sigma-Aldrich, catalog number: Z290815-100EA)
HAuCl4·3H2O (Sigma-Aldrich, catalog number: 520918)
Sodium citrate (Aladdin, catalog number: S189183-100g)
Mono(6-mercapto-6-deoxy)-beta-cyclodextrin (Shandong Binzhou Zhiyuan Biotechnology Co., Ltd, catalog number: 81644-55-5)
1-adamantanethiol (Aladdin, catalog number: A169723-5g)
E. coli (ATCC, catalog number: 33694)
Polypropylene bacterial culture tube (Falcon, catalog number: 352057)
DMSO (Sigma-Aldrich, catalog number: D8418-100ML)
Lysogeny broth (LB) (Thermo Fisher Scientific, catalog number: 10855021)
LB medium (see Recipes)
Sodium citrate solution (5%) (see Recipes)
HAuCl4 solution (0.25 mM) (see Recipes)
Mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM) (see Recipes)
GNP solution (10 mM) (see Recipes)
1-adamantanethiol in DMSO (2 mM) (see Recipes)
Equipment
Centrifuge (Thermo Fisher Scientific, Heraeus Multifuge X3, model: 10325804)
Dynamic light scattering (Malvern, Nano-ZS)
Ultra-high-speed centrifuge (Beckman, Optima XPN-100 Ultracentrifuge)
Sonication bath (Branson, Bransonic ultrasonic cleaner, model: 5510E-DTH)
Transmission electron microscope, 120 kV (HITACHI, model: HT7800)
Microplate reader (Molecular Devices, SpectraMax, model: M5)
Vacuum oven (Thermo Fisher Scientific, Vacutherm, model: VT6205)
Software
Software associated with plate reader used (SoftMax Pro 7 Software)
Software associated with dynamic laser scanning used (Malvern Zetasizer Software v7.11)
Software associated with transmission electron microscope used (Gatan DigitalMicrograph 3.9)
Procedure
Preparation of GNPs via Turkevich method
Use HAuCl4in combination with sodium citrate as a reducing and capping agent for the nanoparticles.
Prepare HAuCl4 solution (0.25 mM; see Recipes) in a conical flask.
Put 100 mL of HAuCl4 solution (0.25 mM) into a 500 mL round-bottom flask with single neck and stir at 130 °C in an oil bath in a magnetic stirrer.
When HAuCl4 solution starts to boil, add 0.7 mL of sodium citrate solution (5%; see Recipes) into HauCl4 solution.
When the solution turns wine red, add 0.7 mL of sodium citrate solution (5%) again and repeat the addition process of sodium citrate solution twice.
Cool the reaction solution down to room temperature and transfer the entire reaction mixture to multiple 2 mL polypropylene microcentrifuge tubes. Centrifuge at 12,000× g for 30 min to collected precipitated GNPs.
Add 100 mL of ddH2O to disperse the precipitated GNPs and centrifuge at 12,000× g for 30 min again. Repeat the washing twice with deionized water and collect purified GNPs.
Characterize GNPs by transmission electron microscope (TEM) and dynamic light scattering (DLS). TEM imaging shows the morphology of Au nanoparticles and DLS analysis reflects the size distribution.
Place the precipitated GNPs in a 2 mL polypropylene microcentrifuge tube and dry GNPs in the vacuum oven at 25 °C for two days (Figure 1a).
Figure 1. Photos of different gold nanoparticles (GNP) precipitates. a) GNP solution and GNP precipitate. b) CD-GNP solution and CD-GNP precipitate. c) ADA-GNP solution and ADA-GNP precipitate. d) Isolated outer membrane vesicles (OMV) precipitate. e) OMV-coated CD-GNP solution and OMV-coated CD-GNP precipitate. f) OMV-coated ADA-GNP solution and OMV-coated ADA-GNP precipitate.
Preparation of CD-GNPs
Put 1,000 mL of ddH2O in a conical flask and add 2.3 g of mono(6-mercapto-6-deoxy)-beta-cyclodextrin to prepare mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM; see Recipes).
Dry GNPs in a vacuum oven and dissolve 1.97 g of GNP power in 1,000 mL of ddH2O to prepare GNPs solution (10 mM; see Recipes).
Add 2 mL of mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM) to 2 mL of GNPs solution (10 mM) in a beaker and stir in a magnetic stirrer for 2 h at room temperature.
Centrifuge the resultant solution at 21,000× g for 8 min in a 2 mL polypropylene microcentrifuge tube and collect the precipitated CD-GNPs.
Dry CD-GNPs in a vacuum oven at 25 °C for two days (Figure 1b).
Preparation of ADA-GNPs
Dissolve 1-adamantanethiol in DMSO (2 mM, 2 mL; see Recipes).
Add GNP solution (10 mM, 2 mL) into 1-adamantanethiol solution in a beaker and stir for 2 h.
Centrifuge the resultant solution at 21,000× g for 8 min in a polypropylene microcentrifuge tube and collect the precipitated ADA-GNPs.
Dry ADA-GNPs in a vacuum oven at 25 °C for two days (Figure 1c).
Isolation of bacterial OMVs
Inoculate E. coli in a polypropylene bacterial culture tube containing 50 mL of LB medium (see Recipes) for three days, and then centrifuge resulting bacterial culture in a 50 mL Falcon tube at 2,000× g for 5 min. Disperse the precipitated bacteria in ddH2O to achieve a OD600 value of 1.0, measured in a microplate reader at wavelength of 600 nm.
Filter 50 mL of the supernatant through a 0.45 pore size nylon membrane filter.
Transfer the resulting filtrate into Millipore 10 kDa filter and centrifuge at 5,000× g for 5 min to obtain the OMVs-rich solution in a Falcon tube.
Centrifuge the OMVs-rich solution in a high-speed polypropylene copolymer (PPCO) centrifuge tube at ultra-high speed of 150,000× g for 3 h.
Collect the precipitated OMVs in a polypropylene microcentrifuge tube and store at -80 °C (Figure 1d).
Synthesis of OMV-coated CD-GNPs
Lyophilize OMVs to get OMV power.
Mix 1 mg of OMVs and 1 mg of CD-GNPs in 50 mL of ddH2O in Falcon tubes. Divide 50 mL of the resulting solution into two 50 mL Falcon tubes.
Sonicate the Falcon tubes for 10 s at intervals of 10 s in a sonication bath at a frequency of 40 kHz (100 W) for 2 min at room temperature.
Centrifuge the subsequent solution in the Falcon tube at 5,000× g for 30 min to precipitate OMV-coated CD-GNPs (Figure 1e).
Place the solution of OMV-coated CD-GNPs in copper mesh and dry for TEM imaging, to analyze the coating layer on GNP surface.
Place the solution of OMV-coated CD-GNPs in a quartz cuvette for DLS analysis, to measure the diameter change of GNP before and after OMV coating.
Synthesis of OMV-coated ADA-GNPs
Mix 1 mg of OMVs and 1 mg of ADA-GNPs in 50 mL of ddH2O solution. Divide 50 mL of the resulting solution into two 50 mL Falcon tubes.
Sonicate Falcon tubes for 10 s at intervals of 10 s in a sonication bath at a frequency of 40 kHz (100 W) for 2 min at room temperature.
Centrifuge the subsequent solution in the Falcon tube for 30 min at 5,000× g to precipitate OMV-coated ADA-GNPs (Figure 1f).
Place the solution of OMV-coated ADA-GNPs in copper mesh and dry for TEM imaging, to analyze the coating layer on GNP surface.
Place the solution of OMV-coated ADA-GNPs in quartz cuvette for DLS analysis, to measure the diameter change of GNP before and after OMV coating.
Data analysis
Normalize peak value of diameter curve to 1 (y-axis, corresponding to the frequency of occupied particle diameter).
TEM is a microscopy technique capable of imaging at a significantly higher resolution than light microscopes. This enables the instrument to capture fine details of nano-sized nanoparticles. GNP solution was added on ultra-thin carbon film and then analyzed by TEM. The appropriate view was chosen and imaged. Data file containing figure information was generated. Finally, this profile was loaded into Digital Micrograph and the TEM figure was obtained. The data analysis step is shown in Figure 2. Figure 3a and 3b show the morphology of GNP nanoparticles and GNP aggregates. DLS is a technique in physics that can be utilized to determine the size distribution profile of nano-sized particles in suspension or in solution. GNP solution was added into a glass cell and then analyzed by DLS. An Excel file with diameter distribution and corresponding intensity were generated. Finally, these data were loaded into GraphPad Prism with x-axis of diameter value and y-axis of intensity value, and the figure of size distribution was obtained. The photo of DLS instrument and data analysis step are shown in Figure 4. DLS shows a large size distribution of GNP aggregates in comparison to that of ADA-GNPs and CD-GNPs (Figure 3c).
Figure 2. Data generation steps by TEM analysis
Figure 3. Characterization of outer membrane vesicles (OMV)-coated gold nanoparticles (GNPs). a) TEM image of GNPs. Scale bar: 500 nm. b) TEM image of GNP aggregates. Scale bar: 2 μm. c) Size distribution of the mixture of CD-GNPs and ADA-GNPs determined by DLS. d) TEM image of M-GNPs. Scale bar: 50 nm. Insert: amplified M-GNPs (the scale bar was 20 nm). e) Size distribution of GNPs, M-GNPs, and OMVs determined by DLS. f) The protein bands of M-GNPs, OMVs, and E. coli in the gel by Coomassie Blue staining.
OMV-coated CD-GNPs/OMV-coated ADA GNPs were characterized by TEM and DLS. TEM was utilized to analyze the coating layer on GNP surface; a transparent membrane layer with ~6 nm thickness was observed on GNP surface (Figure 3d). DLS was utilized to measure the diameter change of GNP before and after OMV coating; an increased size from 30 to 45 nm after coating of GNP with OMV was observed (Figure 3e).
Fit the frequency-diameter using a X-Y model with GraphPad Prism 9.0 software (Figure 4c and 4e).
Figure 4. Photo of DLS instrument and data generation steps
Recipes
HAuCl4 solution (0.25 mM)
HAuCl4·3H2O, 85 mg
ddH2O, 1,000 mL
Mono(6-mercapto-6-deoxy)-beta-cyclodextrin solution (2 mM)
Mono(6-mercapto-6-deoxy)-beta-cyclodextrin, 2.3 g
ddH2O, 1,000 mL
GNP solution (10 mM)
GNP, 1.97 g
ddH2O, 1,000 mL
1-adamantanethiol in DMSO (2 mM)
1-adamantanethiol, 336 mg
DMSO, 1,000 mL
LB medium
Tryptone, 10 g/L
Yeast extract, 5 g/L
NaCl, 10 g/L
Sodium citrate solution (5%)
Sodium citrate, 50 g
ddH2O, 1,000 mL
Acknowledgments
This research was funded by Dr. Stanley Ho Medical Development Foundation (Grant No.: SHMDF-OIRFS/2021/002), Science and Technology Development Fund (FDCT) Macau SAR (Grant No.: 0065/2021/A2), and National Natural Science Foundation of China (22071275).
The current protocol is derived from the original paper (Gao et al., 2022).
Competing interests
R.W. and C.G. have one pending patent based on the bacteria-mimetic gold nanoparticles presented in this manuscript.
References
Gao, C., Wang, Q., Li, J., Kwong, C. H. T., Wei, J., Xie, B., Lu, S., Lee, S. M. Y. and Wang, R. (2022). In vivo hitchhiking of immune cells by intracellular self-assembly of bacteria-mimetic nanomedicine for targeted therapy of melanoma. Sci Adv 8(19): eabn1805.
Krueger, T. E. G., Thorek, D. L. J., Denmeade, S. R., Isaacs, J. T. and Brennen, W. N. (2018). Concise Review: Mesenchymal Stem Cell-Based Drug Delivery: The Good, the Bad, the Ugly, and the Promise. Stem Cells Transl Med 7(9): 651-663.
Li, T., Dong, H., Zhang, C. and Mo, R. (2018). Cell-based drug delivery systems for biomedical applications. Nano Res 11(10): 5240-5257.
Li, Z., Wang, Y., Ding, Y., Repp, L., Kwon, G. S. and Hu, Q. (2021). Cell-Based Delivery Systems: Emerging Carriers for Immunotherapy. Adv Funct Mater 31(23): 2100088.
Loescher S. and Walther A. (2020). Supracolloidal Self‐Assembly of Divalent Janus 3D DNA Origami via Programmable Multivalent Host/Guest Interactions. Angew Chem Int Engl 59 (14): 5515-5520.
Wang, H., Wu, H., Yi, Y., Xue, K-F., Xu, J-F., Wang, H., Zhao, Y. and Zhang, X. (2021). Self-Motivated Supramolecular Combination Chemotherapy for Overcoming Drug Resistance Based on Acid-Activated Competition of Host–Guest Interactions. CCS Chemistry 3(8): 1413-1425.
Yang, L., Yang, Y., Chen, Y., Xu, Y. and Peng, J. (2022). Cell-based drug delivery systems and their in vivo fate. Adv Drug Deliv Rev 187: 114394.
Zhang, J., Yang, L., Huang, F., Zhao, C., Liu, J., Zhang, Y., and Liu, J. (2021). Multifunctional hybrid hydrogel enhanced antitumor therapy through multiple destroying DNA functions by a triple‐combination synergistic therapy. Adv Healthc Mater, 10(21): 2101190.
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β-lactamase (Bla) Reporter-based System to Study Flagellar Type 3 Secretion in Salmonella
FC Fabienne F. V. Chevance
KH Kelly T. Hughes
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4696 Views: 498
Reviewed by: David A. CisnerosSrujana Samhita Yadavalli Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS Genetics Jul 2022
Abstract
Export of type 3 secretion (T3S) substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of cultured cell supernatants followed by western blot analysis of the secreted substrates. In our lab, we have developed β-lactamase (Bla), lacking its Sec secretion signal, as a reporter for the export of flagellar proteins into the periplasm via the flagellar T3S system. Bla is normally exported into the periplasm through the SecYEG translocon. Bla must be secreted into the periplasm in order to fold into an active conformation, where it acts to cleave β-lactams (such as ampicillin) to confer ampicillin resistance (ApR) to the cell. The use of Bla as a reporter for flagellar T3S allows the relative comparison of translocation efficiency of a particular fusion protein in different genetic backgrounds. In addition, it can also be used as a positive selection for secretion.
Graphical overview
Utilization of β-lactamase (Bla) lacking its Sec secretion signal and fused to flagellar proteins to assay the secretion of exported flagellar substrates, into the periplasm, through the flagellar T3S system. A. Bla is normally transported into the periplasm space through the Sec secretion pathway, where it folds into an active conformation and allows resistance to ampicillin (ApR). B. Bla, lacking its Sec secretion signal, is fused to flagellar proteins to assay the secretion of exported flagellar proteins into the periplasm through the flagellar T3S system.
Keywords: β-lactamase Secretion assays Salmonella Flagellar type-III secretion Positive selection for secretion
Background
Flagella are helical, corkscrew-like appendages that, depending on their clockwise or counterclockwise rotation, push or pull bacterial cells. They act like tiny propellers allowing bacteria to move through liquids or across hydrated surfaces. For the assembly of the bacterial flagellum, a flagellar type 3 secretion (T3S) system initially exports early component substrates that build the hook-basal body (HBB) structure, which is the main component making up the flagellar motor. Upon HBB completion, the flagellar T3S system undergoes a secretion substrate specificity switch, resulting in an export selectivity for late substrate proteins, which include flagellin that assembles into the long external filament that acts as the propeller of the flagellum.
The export of T3S substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of the supernatant of cultured cells, followed by western blot analysis of the secreted substrates. This technique is very useful at assessing protein translocation but is limited to the study of substrates that are translocated outside the cells. We have developed a reporter assay using β-lactamase (Bla), lacking its Sec secretion signal, for the secretion of flagellar proteins into the periplasm through the flagellar T3S system. Bla is an enzyme that cleaves and inactivates β-lactam antibiotics, such as ampicillin (Ap), through hydrolysis of the peptide bond of the characteristic four-membered β-lactam ring. The inactivation of the antibiotic provides Ap-resistance (ApR) to the bacterium; for that, Bla needs to be transported into the periplasm where it folds into an active conformation. Such translocation into the periplasm occurs through the Sec-secretion pathway. Secreted proteins through the Sec-dependent pathway are readily recognized by an N-terminal signal sequence, which is cleaved during the process of secretion into the periplasm to yield a mature protein. Flagellar proteins are exported through the T3S pathway. Fusing Bla without its N-terminal signal sequence to the C-terminal of flagellar proteins results in the transport of the Fla-Bla fusions into the periplasm, where Bla is active and confers ApR (Lee and Hughes, 2006; Hirano et al., 2009; Erhardt and Hughes, 2010; Singer et al., 2014; Hendriksen et al., 2021; Qu et al., 2022). In cells expressing intact flagellar structures, Fla-Bla fusions are secreted into the periplasm transiently after completion of the flagellar core T3S system until outer membrane penetration. Once the flagellar structure penetrates the outer membrane, the Fla-Bla fusions are secreted into the extracellular medium. Mutants defective in rod assembly continuously secrete Fla-Bla fusion into the periplasm, which results in higher levels of ApR. By using assays to assess minimum inhibitory concentrations of ampicillin, we can estimate how much of the Fla-Bla fusion is exported and provide a quick estimate of secreted flagellar substrate levels. The use of Bla as a reporter for the flagellar T3S substrates also provides a positive selection for secretion. Using this technique, we were able, for example, to localize important sites of recognition in an early substrate’s secretion signal by the flagellar T3S apparatus located at the cytoplasmic base of flagellum (Qu et al., 2022). This system is not specific to Salmonella; Bla fusions to T3S proteins have been used in other Gram-negative pathogens, such as enteropathogenic and enterohemorrhagic Escherichia coli and Yersinia enterocolitica (Charpentier and Oswald, 2004; Diepold et al. 2015). In these systems, Bla fusions were used to measure the translocation of effector proteins in living host cells and in the extracellular medium, using a fluorescent β-lactamase substrate. Thus, fusion to Bla can be used to measure translocation of proteins either into the periplasm or the extracellular medium. We use secretion of Bla fusions in the periplasm because of the powerful selection of ApR. Here, we describe in detail the protocol used to assay the minimum inhibitory concentration to ampicillin for the assessment of the translocation efficiency of protein fusions in the periplasm and for positive selections using this system.
Materials and reagents
96-well culture plates (Olympus plastic, catalog number: 25-104)
Test tubes (borosilicate glass 13 × 100 mm) (FisherBrand, catalog number: 14-961-27)
Petri dishes (9 cm diameter)
Microcentrifuge tubes (1.5 mL)
Conical centrifuge tubes (50 mL)
Millipore water, sterilized
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-100G)
Bacto peptone (Gibco, catalog number: 211677)
Difco bile salts No. 3 (BD, catalog number: BD 213020)
Bacto proteose peptone (Gibco, catalog number: 211684)
Agar, powdered (Apex, catalog number: 20-273)
Sodium chloride (Sigma-Aldrich, catalog number: S9888-1KG)
Yeast extract (Apex, catalog number: 20-254)
Tryptone, powdered (Apex, catalog number: 20-251)
Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Fisher, catalog number: S373-500)
Potassium phosphate monobasic (KH2PO4) (Fisher, catalog number: P285-3)
Aluminum foil
Strains to be tested and containing fusions to β-lactamase (see Notes)
Lysogeny Broth-Lennox (LB) media (see Recipes)
PPBS plates (see Recipes)
10× phosphate buffer (see Recipes)
Buffered saline (see Recipes)
Ampicillin solution stocks (see Recipes)
Lysogeny Broth-Lennox (LB) plates (see Recipes)
Notes:
We routinely use Salmonella typhimurium LT2, strain TH437 (S. typhimurium LT2 wild type) as a negative control (MIC 1.5) and strain TH15737 (flgM-bla ΔflgHI fljBenxvh2 Δflk) as a positive control (MIC 100). These strains are available upon request.
We insert the Bla fusion without its signal sequence (amino acids 24–286 of Bla) just before the stop codon of the gene of interest, in the chromosome, using the lambda red recombination technique. We routinely do not add linkers, but we see no reason they could not be added. Confirm that the DNA sequence of any construct made is correct and that the protein expression is unaffected by the fusion before conducting assays.
Equipment
Conventional autoclave
-20 °C freezer
Vortex mixer
Temperature-controlled environmental shaker (set at 37 °C)
Temperature-controlled shaking water bath (set at 37 °C)
Standard micropipettes as well as matching tips
2,000 μL electronic pipette repeater (Rainin EDP3-Plus LTS 0.2–2 mL)
20 μL electronic pipette repeater (Rainin EDP3-Plus LTS 2–20 μL)
25 mL glass pipettes, sterilized
10 mL glass pipettes, sterilized
Note: Multichannel pipettes can be used instead of pipette repeaters.
Procedure
Secretion assays in liquid media using 96-well plates
Prepare the cells
Inoculate three independent single colonies for each bacterial strain to be tested into 1 mL of LB media (see Recipes) supplemented with any required supplements. Make sure to include a strain that does not express β-lactamase fusion as a negative control.
Grow the cells, under aeration, at 37 °C overnight.
Dilute the cells 200-fold by pipetting 5 μL of the overnight cell culture into 995 μL of buffered saline (see Recipes).
Prepare the ampicillin solutions
Prepare a stock of 100 mg/mL ampicillin (Ap stock; see Recipes). Make 500 μL aliquots in microcentrifuge tubes and store at -20 °C until use. Prepare fresh every three months.
Transfer 50 mL of LB media into a 50 mL conical centrifuge tube. Discard 400 μL of media using a pipette before adding 400 μL of the Ap stock in order to prepare the 800 μg/mL first solution. Mix well by vortexing. Label this tube number 1.
Label 10 additional 50 mL tubes 2–11. Add 25 mL of LB media to each of them.
Make the serial dilution by transferring 25 mL of Ap 800 μL/mL of conical centrifuge Tube 1 to conical centrifuge tube 2. Close and mix well, then perform the new transfer from tube 2 to 3, etc., until tube 10. Tube 11 contains only LB media as a control.
At the end, we get the following serial dilutions:
1
800 μg/mL
2
400 μg/mL
3
200 μg/mL
4
100 μg/mL
5
50 μg/mL
6
25 μg/mL
7
12.5 μg/mL
8
6.25 μg/mL
9
3.125 μg/mL
10
1.565 μg/mL
11
0 μg/mL
Notes:
This is just an example of a typical range used for one of our Bla fusion expression strains. A different range or more specific dilutions of ampicillin solutions can also be prepared, if necessary, in order to obtain a different range of concentrations.
Smaller volumes can be used to prepare the solutions; however, using larger volumes gives better reproducibility.
Load the ampicillin solutions and cells on the 96-well plate
Calculate the number of plates needed, depending on the number of samples. Make sure to include control samples, such as buffered saline–only, to ensure all solutions are sterile. Place the plates such that they are facing 12 by 8, as illustrated in Figure 1.
Figure 1. Suggested layout to load ampicillin solutions and samples in the plates
Number each plate and write sample numbers on the plates.
Using a 2,000 μL electronic pipette repeater, transfer 198 μL of each of the ampicillin solutions across the plates. Load solution 11 (no ampicillin) first, then solution 10 (1.565 μg/mL), etc., until the higher concentration solution.
Once all plates are loaded with the Ap solutions, use a 20 μL electronic pipette repeater to load 2 μL of the 200-fold diluted cells to each well, starting from the least concentrated Ap solution down until the most concentrated solution.
Place each plate on top of each other with their cap and tape them together and to the shaker plate in the incubator.
Incubate at 37 °C and 180 rpm for 18 h.
Notes:
Ampicillin is sensitive to light; keep the antibiotic in the dark throughout all steps. Use aluminum foil if necessary to protect the plates from the light.
While we routinely use ampicillin in our studies, carbenicillin might be a better option because of its increased stability.
Data analysis
Carefully remove the plates from the incubator and lay them in front of you with the lowest ampicillin concentration at the top.
Note in which wells bacteria have grown or not. The minimum inhibitory concentration (MIC) is defined as the first ampicillin concentration for which the cells are NOT growing. Figure 2 shows an example of typical results obtained. In this example, seven different samples were loaded and grown for 18 h at 37 °C. Sample 8 was buffered saline as a control. It is easy to see where samples are growing or not. Thus, in this example, sample 1 gives a MIC of 25; samples 2 and 3 give a MIC of 6.25; samples 4, 5, and 7 give a MIC of 50, and sample 6 gives a MIC of 12.5 μg/mL.
Record the results.
Figure 2. An example of a 96-well secretion assay. Seven different samples were loaded on this plate and grown for 18 h at 37 °C. Buffered saline was loaded on column 8. The minimum inhibitory concentration (MIC) is defined as the first ampicillin concentration for which the cells are NOT growing. Thus sample 1 gives a MIC of 25; samples 2 and 3 give a MIC of 6.25; samples 4, 5, and 7 give a MIC of 50, and sample 6 gives a MIC of 12.5 μg/mL.
Notes:
It is a good idea to conduct replicate analysis on different days and to include some internal controls (positive and negative) in each assay.
We avoid the use of plasmid expression system for this assay in order to prevent artifacts associated with varying plasmid copy numbers in different cells. Chromosomally expressed Bla fusions are effective and give consistent, reproducible data.
For the most part, three independent replicates give consistent MIC values (same value). For some genetic backgrounds, however, there might be inconsistencies in the MIC values obtained. In this case, repeat the assay with nine independent biological replicates for that particular genetic background, and take the most common value for MIC (found at least five times). If the results are still inconsistent, use solutions with a different range of ampicillin concentrations.
Secretion assays using solid medium containing bile salts
We found that the utilization of bile salts helps to provide a more stringent screen or selection (Hirano et al., 2009) and can also be used for ApR assays on solid medium (Qu et al., 2022). The protocol we developed for such assay is as follows:
Inoculate three independent single colonies for each bacterial strain to be tested into 1 mL of LB media supplemented with any required supplements.
Grow the cells, under aeration, at 37 °C until the culture reaches approximately 2 × 109 cells/mL (overnight culture).
Transfer 20 μL of the overnight culture into 2 mL of fresh LB media (100-fold dilution) and any required supplements.
Grow the freshly diluted cells for 90 min at 37 °C with aeration.
Dilute cells 1,000-fold into buffered saline, by pipetting 1 μL of the freshly grown cells into 1,000 μL of buffered saline.
Spot 4 μL of the 1,000-fold diluted onto PPBS plates (see Recipes) containing varying concentrations of Ap.
Incubate overnight at 37 °C.
Report the presence or absence of growth on all the PPBS-Ap plates with varying Ap levels. An example of a MIC assay using PPBS-Ap plates is shown in Figure 3.
Figure 3. An example of secretion assay using solid medium containing bile salts
Note: The resistance to ampicillin on PPBS-Ap plate is stronger than in Ap liquid media. The dilutions of cells that we used for the solid assay match the resistance obtained by simple streaking our mutants on PPBS plates. The advantage of using solid medium containing bile salts (PPBS) is that the selection is tight and shows only the strongest ampicillin resistant mutants. In Qu et al. (2022), we used both solid and liquid secretion assays. Using PPBS solid medium, we distinguished alleles with significant secretion of the Bla fusion. Using liquid secretion assays without bile salts, we could detect low levels of secretion of fusions with amino acid substitutions that were not observed to be secreted on the more stringent bile salts–containing solid medium.
PPBS-Ap plates used for selections
We use Bla as a reporter for flagellar T3S, not only for the quantification of secreted flagellar protein levels, but also as a positive selection for secretion. We found that the key for ApR selections is to add bile salts to the plates, which helped to provide a stringent screen (Hirano et al., 2009). A typical protocol for selection on PPBS-Ap plates is described below:
Inoculate 10 independent single colonies, from a strain containing Bla, into 1 mL of LB media supplemented with any required supplements. Also, start a strain that does not express Bla (no-Bla) as a control.
Grow overnight cultures of the 10 independent colonies and the no-Bla control strain at 37 °C with aeration.
Plate 100 μL of each independent culture and the no-Bla control strain onto the appropriate PPBS-Ap plate. (Choose a 2-fold higher Ap concentration; for example, if the strain grows on PPBS-Ap5 plates but not on PPBS-Ap10, use PPBS-Ap10 for the selection.)
Next day, pick four ApR colonies from each independent selection and purify by streaking twice onto non-selective media (LB plates; see Recipes).
Recheck each individual colony for ApR and keep one colony from each independent selection.
Map the mutations or send to genome sequencing in order to identify the mutation responsible for the ApR phenotype.
Recipes
Ampicillin solution stocks
For liquid assays:
Prepare 10 mL stock solution of 100 mg/mL of ampicillin in water.
Filter sterilize.
Aliquot in 500 μL microcentrifuge tubes and keep at -20 °C until use.
Use one single aliquot for each assay, to avoid freeze thawing.
For PPBS-Ap plates:
Prepare 100 mL stock solutions of 6 or 20 mg/mL of ampicillin in 50% ethanol/50% water, filter sterilize, and keep at -20 °C in the dark
Notes:
i. Ampicillin is sensitive to light; keep the antibiotic in the dark throughout all steps.
ii. Prepare fresh stock solutions of sodium ampicillin every three months.
Proteose peptone bile salt (PPBS) plates (1 L)
Prepare Flasks A and B as follows:
Flask A Flask B
17 g of Bacto peptone
3 g of Bacto proteose peptone
10 g of NaCl
1.5 g of Difco bile salt #3
500 mL of distilled water
12 g of agar
500 mL of distilled water
Place media flasks in an autoclave-safe bin containing a small amount of water and autoclave for 30 min. Add the desired concentration of ampicillin solution to flask A as soon as the flasks can be hand touched (approximately 55 °C). Mix Flask A and B by pouring contents of flask A into B and then back and forth two more times, to ensure the liquid is well mixed. Pour plates. Protect plates from light. Place plates in plastic bags after two days of drying at room temperature. Place in a cardboard box to protect from light and store at 4 °C. Plates can be used for at least one month.
Lysogeny LB plates (1 L)
Prepare flasks A and B as follows:
Flask A Flask B
10 g of tryptone
5 g of yeast extract
5 g of sodium chloride
12 g of agar
500 mL of distilled water
500 mL of distilled water
Place media flasks in an autoclave-safe bin containing a small amount of water and autoclave for 30 min. Once the flasks can be hand touched (approximately 55 °C), mix Flask A and B by pouring contents of flask B into flask A and then back and forth two more times to ensure the liquid is well mixed. Pour plates. Place plates in plastic bags after two days of drying at room temperature. Store at 4 °C. Plates can be used for at least three months.
Lysogeny LB media (1 L)
Weigh the following ingredients and dissolve into 1,000 mL of distilled water:
10 g of tryptone
5 g of yeast extract
5 g of sodium chloride
Aliquot into 200 mL bottles and autoclave for 30 min. Store autoclaved bottles at room temperature until use.
10× phosphate buffer (1 L)
Weigh the following ingredients and dissolve into 500 mL of distilled water:
110 g of Na2HPO4·7H2O
30 g of KH2PO4
Adjust pH to 7 and add water up to 1 L. Sterilize by autoclaving.
Buffered saline (1 L)
Weigh the following ingredients and dissolve into 1,000 mL of distilled water:
8.5 g of sodium chloride
100 mL of 10× phosphate buffer
Aliquot into 200 mL bottles and autoclave for 30 min. Store autoclaved bottles at room temperature until use.
Acknowledgments
This work was supported by PHS grant GM056141 from the National Institutes of Health (to K.T.H.). This protocol was adapted from previous work (Lee and Hughes, 2006; Hirano et al., 2009; Erhardt and Hughes, 2010; Qu et al., 2022).
Competing interests
The authors declare that no competing interests exist.
References
Charpentier, X. and Oswald, E. (2004). Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 β-lactamase as a new fluorescence-based reporter. J Bacteriol 186(16): 5486-5495.
Diepold, A., Kudryashev, M., Delalez, N. J., Berry, R. M. and Armitage, J. P. (2015). Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome. PLoS Biol 13(1): e1002039.
Erhardt, M. and Hughes, K. T. (2010). C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation. Mol Microbiol 75(2): 376-393.
Hirano, T., Mizuno, S., Aizawa, S. and Hughes, K. T. (2009). Mutations in flk, flgG, flhA, and flhE that affect the flagellar type III secretion specificity switch in Salmonella enterica. J Bacteriol 191(12): 3938-3949.
Hendriksen, J. J., Lee, H. J., Bradshaw, A. J., Namba, K., Chevance, F. F. V., Minamino, T. and Hughes, K. T. (2021). Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure. mBio 12(5): e0239221.
Lee, H. J. and Hughes, K. T. (2006). Posttranscriptional control of the Salmonella enterica flagellar hook protein FlgE. J Bacteriol 188(9): 3308-3316.
Singer, H. M., Erhardt, M. and Hughes, K. T. (2014). Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates. Mol Microbiol 93(3): 505-520.
Qu, D., Jiang, M., Duffin, C., Hughes, K. T. and Chevance, F. F. V. (2022). Targeting early proximal-rod component substrate FlgB to FlhB for flagellar-type III secretion in Salmonella. PLoS Genet 18(7): e1010313.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite
Category
Microbiology > Microbial biochemistry > Other compound
Biological Sciences > Microbiology
Molecular Biology
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AM Aswathy Mangalath
VR Vishnu Raj
RS Rasitha Santhosh
AT Anoopkumar Thekkuveettil
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4697 Views: 709
Reviewed by: Oneil Girish BhalalaAnand Ramesh PatwardhanMatthias Rieckher
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Original Research Article:
The authors used this protocol in Journal of Neuroscience Research Nov 2022
Abstract
A fascinating question in neuroscience is how sensory stimuli evoke calcium dynamics in neurons. Caenorhabditis elegans is one of the most suitable models for optically recording high-throughput calcium spikes at single-cell resolution. However, calcium imaging in C. elegans is challenging due to the difficulties associated with immobilizing the organism. Currently, methods for immobilizing worms include entrapment in a microfluidic channel, anesthesia, or adhesion to a glass slide. We have developed a new method to immobilize worms by trapping them in sodium alginate gel. The sodium alginate solution (5%), polymerized with divalent ions, effectively immobilizes worms in the gel. This technique is especially useful for imaging neuronal calcium dynamics during olfactory stimulation. The highly porous and transparent nature of alginate gel allows the optical recording of cellular calcium oscillations in neurons when briefly exposed to odor stimulation.
Keywords: Immobilization Microfluidic Sodium alginate Calcium imaging C. elegans Live recording
Background
Caenorhabditis elegans is an ideal model organism to observe dynamic changes at the cellular level using fluorescence labeling of target molecules owing to its transparent nature. Unfortunately, worms are sensitive to light and become very active when placed under a microscope. For optical calcium imaging, immobilizing the worms for a prolonged period is essential; however, this can be difficult due to movement artifacts.
Anesthetic agents, such as sodium azide or levamisole, are the most popular choice for immobilizing worms; however, they can cause physiological and behavioral variations in the organism (Fang-Yen et al., 2012; Lewis et al., 1980). As an alternative, gluing worms to a thin agar pad using cyanoacrylate adhesive has been used in calcium imaging and electrophysiological studies (Kerr et al., 2000). This method allows worms to recognize and respond to external stimuli more efficiently; however, it can be challenging to master, particularly gluing the worm only to the tail region. Often, worms become mired when glue sticks to the trunk and head and cannot be used for the experiment (Kerr et al., 2000). Additionally, it is difficult to measure calcium kinetics in the head neurons using this method because of the quick head turns of the worm.
Microfluidic chips have been employed to trap worms for extended kinetic measurements (Hulme et al., 2007). This expensive technique requires the use of precise microfluidic channels and fluid pumps. Polystyrene beads (0.1 μm size) on 5%–10% agarose pads have been used to immobilize worms for extended measurement (Kim et al., 2013). Despite its simplicity, bead-based immobilization triggers dopamine levels in worms (Dong et al., 2018; Raj and Thekkuveettil, 2022) and can compromise the interpretation of the results.
Here, we present a new protocol for immobilizing worms using sodium alginate gel. This method is quick, cost-effective, and causes minimal distress to worms. The gel creates a protective cavity around them, similar to that of microfluidic channels. The alginate gel is highly porous and enables olfactory signals to reach the worm. To prove the efficacy of this method, we applied it to a functional imaging application and showed that it can accurately measure calcium spikes in neurons during odor exposure.
Materials and reagents
Materials
Sodium alginate powder (SDFCL SD Fine Chem Ltd, Mumbai. CAS No. 9005-38-3)
Cover glass, 22 mm × 40 mm (Labtech medico P Ltd, catalog number: LT-71)
Calcium chloride (HiMedia, CAS No. 10035-04-8)
Whatman filter paper, pore size 11 μm (Whatman, catalog number: 1001-917)
Isoamyl alcohol (IAA) (Central Drug House, Bombay, CAS No. 123-51-3)
Equipment
Leica DMi8 automated fluorescent microscope (S/N 455551) with Leica DFC7000 T camera
InjectMan 4 (Eppendorf)
Software
GraphPad Prism 6 software
Las X image acquisition software
Procedure
Preparation of 5% sodium alginate
Dissolve 1 g of sodium alginate in 20 mL sterile distilled water in a 50 mL beaker to make a 5% sodium alginate solution.
Stir the solution overnight using a magnetic stirrer at room temperature until the final solution is clear and bubble-free.
Worm block preparation
On a coverslip, add a small drop of 5% sodium alginate solution at room temperature and slightly spread it to make it sufficiently thick for the worms to immerse.
Place the worms (one can add 1–4 worms per coverslip) in the sodium alginate layer.
Allow the worms to settle for 2 min with minimum thrashing movements. To avoid overflow of the solution, place a plastic ring of 0.5 cm diameter as a barrier. To the inside of the ring, add 100 μL of 100 mM CaCl2 (as the divalent ions) to polymerize the sodium alginate solution with the worm entrapped inside. Within 2–3 min, the sodium alginate will polymerize at room temperature (see Figure 1).
Figure 1. Preparation of alginate scaffold to entrap worms. Worms were placed in the 5% sodium alginate prior to the addition of 100 mM CaCl2 to polymerize sodium alginate solution. Microscopic image showing an immobilized worm.
After polymerization, remove the plastic ring and wipe off the excess CaCl2 solution using filter paper. Alginate polymerization will occur around the worm, and the worms will show restricted movements.
Worm viability within alginate gel
Observe the immobilized worms for 2 min to record their movement with and without exposure to fluorescent light.
The head bends can be measured as a sign of viability.
The worms can be kept in the alginate scaffold for more than 30 min at room temperature.
The worms can be kept in a humid chamber to avoid dehydration for repeated recording.
Olfactory stimulation
As the odor delivery system for this study, a piece of Whatman filter paper (0.3 × 0.3 mm) was placed on the tip of a glass capillary tube.
Attach the capillary tube to the Eppendorf InjectMan’s arm in order to measure and maintain a constant distance between the worm and the odor source (Figure 2).
In order to measure the calcium kinetics in ring interneuron D (RID) neurons, stimulate the strain ZM9078 (hpls587–GcaMP6) with 1/300 diluted IAA.
Saturate the Whatman filter paper with 5 μL of the 1/300 diluted IAA and keep it 1 mm away from the worm.
Move the capillary away from the worm using the joystick after 20 s of exposure.
Figure 2. Setup for olfactory stimulation. Leica DMi8 automated fluorescent microscope with the Eppendorf InjectMan. A. Whatman filter paper. B. capillary tube. C. Eppendorf InjectMan’s arm.
Image acquisition
For calcium imaging, use a Leica DMi8 automated fluorescent microscope and LasX image acquisition software to manage the imaging process (Leica).
Use 20× magnification to capture time-lapse pictures.
The exposure time for fluorescence imaging is 150 ms.
Leica DFC7000T camera time-lapse sequences are captured for up to 2 min (2 frames/s).
Image processing
Perform image processing with Fiji software. Capture images in the JPG file format.
Draw a region of interest (ROI) over the RID motor neuron in ZM9078 (hpIs587–GCaMP6) strain.
Two macro files were written for image processing.
ROI_Calulation.ijm
// This macro moves across images
// Finds the maximum pixel value
for (i=1; i<=nImages; i++) {
selectImage(i);
processImage(); //or simply put your processing commands here.
}
function processImage() {
getRawStatistics(nPixels, mean, min, max);
run("Find Maxima...", "noise="+max+" output=[Point Selection]");
getSelectionBounds(x, y, w, h);
makeOval((x-70), (y-40), 140, 90);
}
Measure_Values.ijm
// This macro moves across images
// Finds the maximum pixel value
for (i=1; i<=nImages; i++) {
selectImage(i);
// run("Set Measurements...", "area mean min integrated redirect=None decimal=3");
run("Measure");
}
Use ROI_calculation.ijm macro to draw ROI in the images.
Calculate fluorescent intensity measurement with Measure_values.ijm macro (see Figure 3).
Figure 3. Screenshots of each step of image processing using Fiji software
Calculate change in fluorescent intensity by subtracting the fluorescent intensity of subsequent images from the mean of the initial 10 s images.
Calculate the fluorescence value for each image frame from the ROI.
Data analysis
Statistical analysis
Calcium transients were plotted as ∆F/F, where ∆F is the change in the fluorescence value from its baseline fluorescence (F).
The mean fluorescence intensities of the first 10 frames (t = 200 ms) were taken as baseline fluorescence (F0).
The odor stimulus was given for 20 s (40 frames). The analysis was conducted using GraphPad Prism 6 software.
Results
Immobilization of worms in alginate scaffold
2%, 3%, or 5% alginate polymer solutions were used to immobilize the worms. The viability of the worms was checked by counting the head bends per minute. No statistically significant difference in head bends was observed in worms trapped in 5% alginate compared to 2% and 3% alginate (p ≤ 0.5; n = 5; Figure 4A). However, when we converted the data as viability measurement, the results showed that there is a 100% probability of survival in 2% alginate compared to 80% and 60% in the 3% and 5% alginate scaffold, respectively (Figure 4B). A total immobilization of the worms was observed in the 5% alginate polymer compared to the other two percentages. To see the worm behavior in 2%, 3%, and 5% alginate polymer check supplementary Videos 1, 2, and 3, respectively.
Figure 4. Viability of worms in sodium alginate gel. A. Head bends after immobilization. The head bends per minute for the 2%, 3%, and 5% alginate immobilized worms. Worms with no head bend within 1 min of observation were considered dead and not used in the study. B. Percentage of viability. The viability of the worms was 100% in 2% alginate, 80% in 3% alginate, and 60% in 5% alginate. The values are expressed as mean ± SEM; n = 5; ordinary one-way ANOVA.
Quantification of size alteration of worms in the alginate
The normal body width of C. elegans has a diameter of approximately 50 μm. We measured the worm's body width and concluded that the alginate polymer causes worm body expansion. Analysis was done in M9 buffer and different concentrations of alginate. The result showed that in the M9 buffer, the worm width is 60 μm. In comparison, in alginate immobilized worms, the width is approximately 80 μm (Figure 5).
Figure 5. Body width of immobilized worms. Body width of the immobilized worms in different concentrations of sodium alginate. The values are expressed as mean ± SEM; n = 5 or more, ordinary one-way ANOVA, p < 0.0001 (****).
Measurement of RID neuron activation during the exposure of IAA
C. elegans shows attractive behavior towards IAA. Genetically encoded Ca2+ indicator GCaMP6-expressing (strain ZM9078 flp-14p::GCaMP6::wCherry) in RID was used to visualize Ca2+ transients in the RID interneuron, which helps for the forward movement while exposed to IAA. Calcium transients in the RID neuron were observed in the worm without and with exposure to 1/300 diluted IAA by entrapping the worm in the alginate polymer. The odor exposure lasted 20 s. Data showed a significant calcium influx in the RID interneuron during IAA exposure (Figure 6).
Figure 6. Calcium imaging of alginate immobilized worm. A. Brightfield image of the immobilized ZM9078 (hpIs587 - GCaMP6) strain. B. Fluorescent image of immobilized ZM9078 strain. C. Alginate-immobilized worms were used for calcium imaging. Worms were exposed to 1/300 diluted IAA for 20 s (data is an average of n = 7 readings).
Discussion and Conclusion
Compared with other imaging techniques in C. elegans, our in vivo imaging method is straightforward and affordable to image adult C. elegans without using any anesthetic agent or gluing the worm to the agar pad. Using this new method, we analyzed the neuronal calcium firing in worms by applying IAA as an odor stimulus. Significantly enhanced fluorescent intensity was observed in the ZM9078 strain during exposure to IAA. Time-lapse image analysis was done using Fiji software (Schindelin et al., 2012). Among the various percentages of sodium alginate tested, 5% alginate provided the best immobilization concentration.
The worms can be removed from the scaffold and kept back in the food plate for repeated imaging assays. The worms could survive for more than 30 min at room temperature in a 5% alginate scaffold or in a humid chamber for a couple of hours. Dehydration and starvation would significantly impact the survival of worms within the scaffold when they are kept for a prolonged period.
The change in body width was a significant observation while standardizing the protocol. The entrapment of the worms in the alginate scaffold could impact its size. The polymerization of alginate is a fast process and could reduce the space available for the worm, which could be one of the reasons for this observation.
Furthermore, we can deliver the volatile chemical stimuli from outside because of the high porosity of the alginate scaffold. Physiological and behavioral functions of the organism are not disturbed by alginate immobilization. The major limitation of this method is that entrapping in 5% alginate causes mortality in the worm. However, head bends can be used to select the surviving worms. One can try temperature control stages attached to the microscope to reduce the movements of the worms, which will allow using a lower alginate concentration to enhance the survival rate. With our approach, immobilizing C. elegans becomes much easier without any special technical knowledge.
Acknowledgments
We thank CGC for providing the ZM9078 strain. We acknowledge SCTIMST for supporting the research. We acknowledge Saurabh S. Nair, Department of Medical Device Engineering, SCTIMST for writing the Fiji macro files. We acknowledge SCTIMST for supporting the research. We also acknowledge CSIR for the fellowship to A.M. and UGC fellowship to R.S. The protocol is adapted from Raj and Thekkuveettil (2022).
Competing interests
The authors declare no conflict of interest.
References
Dong, L., Cornaglia, M., Krishnamani, G., Zhang, J., Mouchiroud, L., Lehnert, T., Auwerx, J. and Gijs, M. A. M. (2018). Reversible and long-term immobilization in a hydrogel-microbead matrix for high-resolution imaging of Caenorhabditis elegans and other small organisms. PLoS One 13(3): e0193989.
Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I. and Avery, L. (2012). Laser microsurgery in Caenorhabditis elegans. Methods Cell Biol 107: 177-206.
Hulme, S. E., Shevkoplyas, S. S., Apfeld, J., Fontana, W. and Whitesides, G. M. (2007). A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab Chip 7(11): 1515-1523.
Kerr, R., Lev-Ram, V., Baird, G., Vincent, P., Tsien, R. Y. and Schafer, W. R. (2000). Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26(3): 583-594.
Kim, E., Sun, L., Gabel, C. V. and Fang-Yen, C. (2013). Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization. PLoS One 8(1): e53419.
Lewis, J. A., Wu, C. H., Berg, H. and Levine, J. H. (1980). The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics 95(4): 905-928.
Raj, V. and Thekkuveettil, A. (2022). Dopamine plays a critical role in the olfactory adaptive learning pathway in Caenorhabditis elegans. J Neurosci Res 100(11): 2028-2043.
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. 28; 9(7):676-682.
Supplementary information
Video recording of worms entrapped in 2%, 3%, and 5% alginate polymer: Supplementary_videos_1-3.zip.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Category
Neuroscience > Neuroanatomy and circuitry > Animal model
Neuroscience > Behavioral neuroscience
Biological Sciences > Biological techniques
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4,698 | https://bio-protocol.org/en/bpdetail?id=4698&type=0 | # Bio-Protocol Content
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Peer-reviewed
Ten-fold Robust Expansion Microscopy
HD Hugo G. J. Damstra
BM Boaz Mohar
ME Mark Eddison
AA Anna Akhmanova
LK Lukas C. Kapitein
PT Paul W. Tillberg
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4698 Views: 3043
Reviewed by: Zinan ZhouFarah HaqueSurabhi SonamXin XuLei Gao
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Cited by
Original Research Article:
The authors used this protocol in eLIFE Feb 2022
Abstract
Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.
Keywords: Expansion microscopy Super resolution Light microscopy Antibody labeling Ultrastructure Protein stain
Background
Expansion microscopy (ExM) circumvents the diffraction limit of light microscopy by physically expanding the specimen four-fold in each dimension (Chen et al., 2015; Tillberg et al., 2016). Expansion is achieved by chemically anchoring proteins and other biomolecules directly to a hyper-swelling gel, followed by aggressive proteolysis to enable uniform swelling of the gel material. Assuming sufficiently high labeling density, the resolution increase of ExM depends on the expansion factor of the gel recipe used. Recently, ExM variants have been described that seek to improve resolution by increasing the expansion factor, for example by multiple rounds of expansion such as iterative ExM (Chang et al., 2017), by decreasing the concentration of crosslinker (Chen et al., 2015), usually bisacrylamide (bis), or by using a different crosslink chemistry (Truckenbrodt et al., 2019). By systematic exploration of the expansion gel recipe space, we assessed the limits of single-round expansion and generated Ten-fold Robust Expansion Microscopy (TREx), an optimized ExM method that allows for robust ten-fold expansion in a single step. Since TREx uses the same chemistry and single-round embedding procedure as the original ExM protocol, it can be easily implemented for users already familiar with ExM. Moreover, we show ten-fold expansion sufficiently de-crowds biological specimens to visualize local protein densities using general protein and membrane stains.
Materials and reagents
4-Hydroxy-TEMPO (4HT) (Sigma-Aldrich, catalog number: 176141)
Acrylamide 40% solution (Sigma-Aldrich, catalog number: 01697)
Acrylic acid (Sigma, catalog number: 147230)
Acryloyl-X SE (AcX) (Thermo Fisher, catalog number: A20770)
Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: 215589)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647)
N,N'-Methylenebisacrylamide (bis) 2% solution (Fisher Scientific, catalog number: BP1404)
Coverslips (18 mm) (Marienfeld, catalog number: 107032)
DAPI (Thermo Fisher, catalog number: D1306)
DMSO (Thermo Fisher, catalog number: D12345)
Gluteraldehyde (GA) (Electron Microscopy Sciences, catalog number: 16100)
Guanidine HCl (Sigma, catalog number: G3272)
Methanol (MeOH) (Sigma-Aldrich, catalog number: 34860)
Multi-well culture plates (6-well and 12-well) (Sigma-Aldrich, catalog numbers: CLS3335 and CLS3336, respectively)
Paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15710)
Proteinase K (Thermo Fisher, catalog number: EO0491)
Silicone sheet material (Sigma-Aldrich, catalog number: GBL664107/GBL665501)
Sodium acrylate (Sigma-Aldrich, catalog number: 408220)
Sodium dodecyl sulfate (SDS) (Sigma, catalog number: 71736)
Tetramethyl ethylenediamine (TEMED) (Bio-Rad, catalog number: 1610800)
Triton X-100 (Sigma, catalog number: T8787)
PBS 10× (Thermo Fisher, catalog number: 70011044)
TAE 50× (Thermo Fisher, catalog number: B49)
NaCl (Thermo Fisher, catalog number: 447302500)
Tris (Sigma, catalog number: T1503)
Gelation solution (see Recipes)
Digestion buffer (see Recipes)
Gelation solution recipe for tissue slices (see Recipes)
Disruption buffer (see Recipes)
Procedure
Part I: Protocol for cultured cells
Fixation
Fix cells (18 mm coverslips, coated for optimal cell growth as desired) for 10 min at 37 °C using pre-warmed fixative. Fixative may be 4% PFA, 4% PFA + 0.1% glutaraldehyde (GA) for preservation of membranes, -20 °C MeOH, or extraction as described in Damstra et al. (2022).
Wash with PBS twice.
If no permeabilization will be performed (e.g., if no immunofluorescence IF labeling will be performed), permeabilize for 10 min with 0.1% Triton X-100 in PBS at room temperature (RT) (this is important to ensure gel spans membranes fully), followed by washing with PBS three times.
IF (optional, most standard IF methods may be used)
Block with 3% BSA in PBS for 1 h at RT.
Incubate with primary antibodies in 3% BSA in PBS for 1–3 h at RT or overnight at 4 °C.
Wash with PBS three times.
Incubate with secondary antibodies in 3% BSA in PBS for 1–3 h at RT or overnight at 4 °C.
Wash with PBS three times.
Anchoring
Incubate with 0.1 mg/mL AcX in PBS overnight at RT or 0.2 mg/mL for 1 h at RT.
For AcX stock solution, dilute 5 mg of AcX in 500 μL of anhydrous DMSO (10 mg/mL), aliquot, and store at -20 °C (dilute a 20 μL aliquot in 2 mL of PBS just prior to use).
Gelation
Assemble gelation chamber [parafilm-covered glass slide and silicone ring (13 mm diameter)] and pre-chill on ice (Figure 1).
Figure 1. Gelation chamber before (left) and during gelation (right). Gelation chamber consists of a parafilm-covered glass slide and a silicon spacer that is held in place by binder clips. The gelation chamber is formed when the coverslip that contains the cells is placed cell-side down on the silicon spacer (right) ensuring contact with the gelation solution.
Prepare monomer solution [gelation solution (see Recipes) without the APS and TEMED] and keep on ice. Monomer solution can optionally be aliquoted and stored at -20 °C. When using an aliquot, please ensure the aliquot is fully thawed at RT because acrylamide has reduced solubility at low temperatures.
Wash cells with PBS once.
Add TEMED and APS to the monomer solution to form gelation solution, vortex thoroughly, and put back on ice.
Transfer 170 μL to the chamber (pipette directly on the parafilm), tap the side of coverslip on a tissue to remove excess PBS, and put cells down on the monomer solution to close the chamber. Transfer directly to the incubator set at 37 °C for 1 h (Figure 1).
Homogenization
Gently remove gels from the gelation chamber and transfer to a 12-well plate. At this point, the gel typically remains attached to the coverslip. The gel will spontaneously separate from the coverslip during either enzymatic or non-proteolytic homogenization. For enzymatic digestion: add 2 mL of digestion buffer (see Recipes) and digest for 4 h at 37 °C. Optional: add DAPI to digestion buffer.
Depending on the experiment, a non-proteolytic disruption (3 h incubation in 5% SDS, 200 mM NaCl, and 50 mM Tris pH 7.5 at 80 °C) or a hybrid disruption and digestion approach can be used to increase the amount of protein retained after digestion (see below). For staining with general protein stains in combination with digestion, we typically wash the gel twice for 15 min with PBS, incubate with 20 μg/mL of fluorescently labeled NHS-ester or maleimide for 1.5 h at RT, and then proceed with digestion. When using disruption or a hybrid approach to increase the number of accessible residues for labeling, general protein stains can be added after the disruption step.
Expansion
Transfer gel to a 15 cm dish and completely fill with water (Milli-Q). Exchange water at least twice after 30 min and leave overnight at RT to expand (Figure 2a).
Figure 2. Illustration of gel chamber construction for expansion of tissue and example of gel expansion. a) Piece of tissue positioned on glass slide between dabs of vacuum grease. b) Coverslip pressed down gently to contact the tissue, held in place by the dabs of vacuum grease. c) Completed chamber, filled with gelation solution. d) After gelling at 37 °C, the coverslip is gently removed and flipped over, and excess gel is trimmed away from the specimen using a curved scalpel. e) After digestion and washing in water, the fully expanded gel. Scale bars: (a) 0.5 cm, (b) 5 cm.
Part II: Protocol for expanding tissue
Perfusion and post-fixation
Mice were transcardially perfused with ice-cold 4% formaldehyde in 100 mM sodium phosphate buffer, pH 7.4. Brains were dissected out and post-fixed in 4% formaldehyde between 2 h and overnight at 4 °C, followed by washing with PBS and slicing by vibratome at 100–300 μm.
Wash with PBS twice.
If no permeabilization will be performed (e.g., for IF), permeabilize for 30 min with 0.1% Triton X-100 in PBS at RT (this is important to ensure gel spans membranes fully), followed by washing with PBS three times.
IF (optional, most standard IF methods may be used)
Block with blocking buffer (2% BSA, 0.1% Triton X-100 in PBS) at RT for 30 min to 2 h.
Incubate with primary antibodies in blocking buffer at RT for several hours to several days.
Wash with blocking buffer four times for 30 min.
Incubate with secondary antibodies in blocking buffer at RT for several hours to several days.
Wash with blocking buffer three times for 30 min and with PBS once.
Anchoring
Treat tissue slices with 0.1 mg/mL AcX in PBS overnight or 0.2 mg/mL for 1 h.
For AcX stock solution, dilute 5 mg of AcX in 500 μL of anhydrous DMSO (10 mg/mL), and store at -20 °C (dilute 20 μL aliquot in 2 mL of PBS just prior to use).
Wash with PBS.
Gelation
Unlike for cultured cells, tissue slices must be incubated with gelation solution (including APS, TEMED, and the radical inhibitor 4HT to delay the onset of gelation; see Recipes) to ensure the monomer solution can completely diffuse through the tissue. Prepare monomer solution (gelation solution without APS, TEMED, or 4HT), which can optionally be aliquoted and stored at -20 °C. When using an aliquot, please ensure the aliquot is fully thawed at RT because acrylamide has reduced solubility at low temperatures.
For the gelation chamber, place four dabs of vacuum grease on a glass slide, spaced to coincide with the corners of a glass coverslip.
On ice, add gelation solution to the specimen and incubate on ice for 20 min while shaking.
Remove excess gelation solution and transfer the specimen to glass slide in the middle of the dabs of vacuum grease. To seal the chamber, place a glass coverslip on top of the dabs of vacuum grease and gently press down until the coverslip contacts the tissue.
Gently pipette gelation solution in excess from the side, making sure the specimen remains at least ~3 mm away from air or silicone grease, and transfer directly to incubator set at 37 °C for 1 h.
Homogenization
Gently remove gel from gelation chamber by teasing the coverslip off with a razor blade and tweezers.
Trim the gel close to the specimen and wash once in PBS for 5 min.
Disrupt with proteinase K as with cultured cells.
Wash with PBS four times for 30 min each.
To preserve the most total protein for NHS-ester staining, the anchoring and disruption steps may be reduced in strength, though potentially at the cost of reduced expansion isotropy between different subcellular structures. For example, both the AcX and proteinase K concentrations can be reduced ~10-fold, with the digestion step followed by high-temperature non-proteolytic disruption with SDS, by transferring the gel to an excess volume of disruption buffer and incubating for 3 h at 80 °C.
If cracks appear in the specimen upon expansion, increasing the proteinase K concentration or time in disruption buffer may help. If cracks persist, increasing the temperature of disruption buffer treatment (e.g., to 90 °C) can greatly increase the strength of disruption. The original ExM disruption method using proteinase K diluted 1:100 in buffer containing guanidium can also be tried if more aggressive digestion is needed. To add sub-cellular context, disrupted specimens may be stained with NHS-ester dyes, e.g., Atto594 or Alexa488, diluted to 20 μg/mL in 1× PBS for 1 h at RT.
Expansion
Transfer gel to a 6-well plate or 15 cm dish, wash with water for 30 min at least three times, and leave overnight at RT to expand. Keep covered to protect from acidification due to CO2 in room air.
Notes
Fine tuning expansion factor
Increasing the bisacrylamide concentration will decrease the expansion factor and make the gel more rigid; decreasing the bisacrylamide concentration will increase the expansion factor at the expense of mechanical integrity. If ten-fold expansion is not obtained, we recommend varying the bisacrylamide concentration accordingly. If ten-fold expansion is not required, bisacrylamide can be increased to improve the toughness of the gel at the expense of expansion factor. Of note, we also observed the freshness of APS can have a moderate impact on the expansion factor. We recommend aliquoting APS and not subjecting stocks to multiple rounds of freezing to prevent this source of variation.
Sodium acrylate purity
We have observed commercial sodium acrylate comes in varying purity with unknown impact on gel quality. 4M sodium acrylate can also be made by neutralizing acrylic acid with 10 N NaOH. The protocol for 20 mL of sodium acrylate (4 M) is as follows:
In a fume hood, combine 5.5 mL of acrylic acid and 4.5 mL of water (Milli-Q). Next, gradually add 7.2 mL of NaOH (10 M). Use a water bath to prevent excessive heating. After most of the highly volatile and noxious acrylic acid has been converted to non-volatile sodium acrylate, the solution can be transferred out of the fume hood.
Add 1 M NaOH gradually until the pH is 7.5–8 (usually around 1 mL). Use a pH meter to monitor pH, not pH test strips. Buffering capacity of 4 M acrylate at pH 7.75 is only ~4 mM. Add water up to a final volume of 20 mL.
Imaging methods
TREx is compatible with any microscope. Confocal and light-sheet microscopy can be used to take particular advantage of the compatibility of TREx with thick tissue specimens. Since the expanded hydrogel is mostly water, it is particularly compatible with water objectives. A good compromise of long working distance and high numerical aperture (NA) is important because expansion renders specimens both thicker and spatially diluted. We typically use 40–86× water immersion with NA 1.0–1.2, or for thicker specimen air or water immersion objectives with NA 0.8–1.0. To prevent drift during acquisition, expanded gels may be immobilized during imaging by coating the glass support, such as a well plate or imaging chamber, with poly-L-lysine, which will adhere strongly to the expanded gel. For quick acquisitions, immobilization is not strictly necessary.
General protein stains
TREx is compatible with any maleimide or NHS-ester general protein stain. The labeling pattern is modulated by the hydrophobicity of the fluorophore (Sim et al., 2021); thus, we recommend the user to test a range of dyes. We typically use Alexa 488/594 NHS (Thermo Fisher, A20100, A37572, respectively), Cyanine3/Cyanine5 NHS (Lumiprobe, 11020, 13020, respectively), or Atto 647N maleimide (Sigma-Aldrich, 05316).
Gelation chambers
This protocol is compatible with many gelation chamber designs used for different types of specimens. As a generic starting point for cultured cells, we recommend using an uncharged glass slide as the chamber bottom and adhesive silicone material as a spacer gasket. Positive charged glass can also be used—this surface will stick to the gel more strongly. For spacer gaskets, silicone isolators (e.g., Sigma GBL664107 or GBL665501) are an easy solution (Figure 3). The silicone sheet material from Digi-key comes as thin as 250 μm, allowing for faster gel expansion, and can be trimmed to make a gasket of any shape (e.g., a right trapezoid, for keeping track of gel orientation) using a laser cutter or other cutting method. The chamber top piece can be a coverslip (possibly with adherent cells) or a glass slide. For tissue slices up to at least 300 μm thick, we simply sandwich the tissue between a glass slide and a glass coverslip, with dabs of vacuum grease at the corners of the coverslip to keep it in place. Keep in mind that the portion of the gel that forms within ~3 mm of any silicone or air edge should be trimmed away and discarded, as oxygen from these materials inhibits polymerization, altering the process of gelation.
Figure 3. Gelation chamber for cultured cells assembled using adhesive silicone sheet material from Digi-key, with right trapezoid chambers cut by laser cutter
Recipes
Gelation solution
Reagent Stock Final concentration Amount
Sodium acrylate 4 M 1.1 M 271 μL
Acrylamide 5.6 M 2 M 360 μL
Bis 2% 0.005% 2.5 μL
PBS 10× 1× 100 μL
Water (Milli-Q) 236.5 μL
TEMED 10% 0.15% 15 μL
APS 10% 0.15% 15 μL
Sodium acrylate 4 M: dissolve 0.38 g/mL in Milli-Q or by neutralizing acrylic acid (see below).
Acrylamide 5.6 M equals 40% stock solution
Digestion buffer
Reagent Stock Amount per gel
TAE buffer 1,550 μL
Triton X-100 10% in PBS 100 μL
Guanidine HCl 5 M 320 μL
Proteinase K 600 U/mL 30 μL
Gelation solution recipe for tissue slices
Reagent Stock Final concentration Amount
Sodium acrylate 4 M 1.1 M 2.7 mL
Acrylamide 5.6 M 2.0 M 3.6 mL
Bis 2% 0.005% 0.025 mL
PBS 10× 1× 1.0 mL
Water (Milli-Q) 2.225 mL
TEMED 10% 0.15% 0.15 mL
APS 10% 0.15% 0.15 mL
4HT 0.1% 0.0015% 0.15 mL
Acrylamide 5.6 M equals 40% stock solution
Disruption buffer
Reagent Stock Final concentration Amount
SDS 10% 5% 10 mL
Tris pH 7.5 1 M 0.05 M 1 mL
NaCl 5 M 0.2 M 0.8 mL
Water (Milli-Q) 8.2 mL
Acknowledgments
This protocol is based on the publication of Ten-fold Robust Expansion microscopy (TREx) (Damstra et al., 2022). A.A. is supported by the Netherlands Organization for Scientific Research Spinoza Prize. L.C.K. is supported by the European Research Council (ERC Consolidator Grant 819219). B.M., M.E., and P.W.T. are supported by the Howard Hughes Medical Institute (HHMI).
Competing interests
There are no conflicts of interest or competing interests.
References
Chang, J. B., Chen, F., Yoon, Y. G., Jung, E. E., Babcock, H., Kang, J. S., Asano, S., Suk, H. J., Pak, N., Tillberg, P. W., et al. (2017). Iterative expansion microscopy. Nat Methods 14(6): 593-599.
Chen, F., Tillberg, P.W. and Boyden, E. S. (2015) Expansion microscopy. Science (347):543-548.
Damstra, H. G. J., Mohar, B., Eddison, M., Akhmanova, A., Kapitein, L. C. and Tillberg, P. W. (2022). Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). Elife 11: e73775.
Sim, J., Park, C. E., Cho, I., Min, K., Eom, M., Han, S., Jeon, H., Cho, H-J., Cho, E-S., Kumar, A., et al. (2021) Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy. Biorxiv: 443629.
Tillberg, P. W., Chen, F., Piatkevich, K. D., Zhao, Y., Yu, C. C., English, B. P., Gao, L., Martorell, A., Suk, H. J., Yoshida, F., et al. (2016). Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34(9): 987-992.
Truckenbrodt, S., Sommer, C., Rizzoli, S. O. and Danzl, J. G. (2019). A practical guide to optimization in X10 expansion microscopy. Nat Protoc 14(3): 832-863.
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Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain
SM Samsuzzoha Mondal *
HJ Honey Priya James *
FM Francesco Milano
RJ Rui Jin
TB Tobias Baumgart
(*contributed equally to this work)
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4699 Views: 919
Reviewed by: David PaulAnindita Sarkar Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Communications Aug 2022
Abstract
Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains.
Graphical overview
Keywords: BAR-proteins Amphiphysin N-BAR domain GST-fusion protein purification BAR-protein purification
Background
Bin/Amphiphysin/Rvs (BAR) family proteins have drawn wide interest due to their roles in several membrane trafficking pathways (Frost et al., 2009). During clathrin-mediated endocytosis, the N-BAR proteins endophilin and amphiphysin are recruited to the neck of endocytic buds and facilitate membrane scission (Takei et al., 1999; Milosevic et al., 2011). The N-BAR domain forms a homodimer in solution and adopts a crescent-like shape known for generating, sensing, and stabilizing highly curved endocytic membrane nanostructures (Gallop et al., 2006). Our group's recent in vitro reconstitution study has shown that endophilin can undergo phase separation via self-association and, alternatively, via multivalent interactions with its binding partners (Mondal et al., 2022). Unlike endophilin, amphiphysin (full length protein) did not show phase separation behavior, although its N-BAR domain did phase separate. These recent observations suggest that spatiotemporal organization on the membrane and curvature generation properties of BAR proteins could be regulated by a phase separation–dependent mechanism.
Purification of recombinant amphiphysin and endophilin as GST fusion proteins was first reported by DeCamilli and coworkers (David et al., 1996; Ringstad et al., 1997). The GST tag facilitates the purification of the protein of interest from E. coli lysate by affinity chromatography. Moreover, it is recognized for its ability to improve protein solubility and enhance expression efficiency (Smith and Johnson, 1988). Additionally, Gallop et al. (2005) demonstrated that using a GST tag instead of a polyhistidine tag resulted in more efficient purification of the endophilin N-BAR domain. A general strategy to purify the GST-tagged BAR proteins involves extraction of the fusion protein from bacterial cell lysate by GST affinity chromatography, removal of the GST tag by PreScission protease cleavage followed by ion exchange chromatography, and finally, size exclusion chromatography. Later on, several other groups adopted this basic purification strategy to purify various N-BAR proteins (Gallop et al., 2006; Capraro et al., 2013; Ambroso et al., 2014). Our present protocol allows the purification of various full-length N-BAR proteins, their N-BAR domains, and their mutants, with minor or no further optimization. We found that additional optimization is needed when purifying the amphiphysin N-BAR domain, since the protein tends to precipitate upon removal of the GST tag and for that reason the N-BAR domain loses its membrane curvature generation activity. The protein stability in aqueous solutions can be enhanced by the addition of cosolvents such as glycerol. Vagenende et al. (2009) reported that glycerol prevents protein aggregation by inhibiting protein unfolding and by interaction with hydrophobic surface regions of the protein to form amphiphilic interfaces. The addition of glycerol to buffers for all the chromatography steps was found to minimize precipitation, and the protein retained its curvature generation abilities. Here, we provide a step-by-step guide to purifying full-length amphiphysin and the N-BAR domain. Steps that are common and different for full-length proteins and the N-BAR domain are indicated.
Materials and reagents
Pipette tips 10 μL (Thermo Scientific, catalog number: 2139)
Pipette tips 200 μL (Fisher Scientific, catalog number: 02-707-500)
Pipette tips 1 mL (Fisher Scientific, catalog number: 02707508)
Disposable cuvettes (Fisher Scientific, catalog number: 14955127)
Polypropylene round-bottom tube (Corning, catalog number: 352059)
Petri dishes (Corning, catalog number: 353003)
0.22 μm filter (Merck Millipore, catalog number: SLGVR33RS)
10 mL syringes (Becton, Dickinson and Company, catalog number: 301029)
Collection tubes (Fisher Scientific, catalog number: 14-961-26)
Syringe (Henke Sass Wolf, catalog number: 4850001000)
Gel-loading tips (Fisher Scientific, catalog number: 05-408-150)
E. coli BL21 DE3 codon plus (Agilent Technologies, catalog number: 230280)
Plasmids: full length human amphiphysin 1 (1-695) and its N-BAR domain (1-247) sequences contained in pGEX-6P vectors were generously provided by Pietro DeCamilli’s lab. Plasmids are available from the Baumgart lab upon request.
PreScission protease (Genscript, catalog number: Z02799)
SOC medium (Corning, catalog number: 46-003-CR)
LB agar, Miller (Fisher BioReagents, catalog number: BP1425-500)
Ampicillin (Fisher BioReagents, catalog number: BP1760-25)
Chloramphenicol (Acros Organics, catalog number: 227920250)
200 proof ethanol (Decon Laboratories, catalog number: 2716)
Parafilm (Bemis, catalog number: PM-999)
LB broth, Miller (Sigma-Aldrich, catalog number: L3152)
Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Lab Scientific, catalog number: I-555)
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 11359061001)
Glycerol (Sigma-Aldrich, catalog number: G7893)
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3)
Tris(hydroxymethyl)aminomethane (Tris) (Fisher BioReagents, catalog number: BP152-1)
Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP120-500)
Dithiothreitol (DTT) (Fisher BioReagents, catalog number: BP172-5)
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-500)
Glutathione (GSH) (Acros Organics, catalog number: 120001000)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Fisher Scientific, catalog number: BP310-500)
Tris(2-carboxyethyl)phosphine (TCEP) (Thermo Scientific, catalog number: PG82089)
Sodium dihydrogen phosphate (Fisher Scientific, catalog number: BP329-500)
Disodium hydrogen phosphate (Fisher Scientific, catalog number: S374-500)
Sodium hydroxide (Fisher Scientific, catalog number: S318-500)
Acrylamide, 30% (Bio-Rad Laboratories, catalog number: 1610158)
Sodium dodecyl sulfate (SDS) (Acros Organics, catalog number: 327311000)
NovexTM sharp prestained protein ladder (Invitrogen, catalog number: 57318)
Loading buffer for SDS-PAGE (0.3 M Tris, 60% v/v glycerol, 12% w/v SDS, 0.3 M DTT, 0.6% bromophenol blue)
GelCodeTM Blue safe protein stain (ThermoFisher Scientific, catalog number: 24596)
Amicon® ultra centrifugal filters (Millipore Sigma, catalog numbers: UFC901024, and UFC903024)
LB agar plates (see Recipes)
Luria Bertani (LB) media (see Recipes)
PMSF stock solution (see Recipes)
Buffers (see Recipes)
GST lysis buffer
GST wash buffer
GST elution buffer
Cation exchange buffer A
Cation exchange buffer B
Size exclusion buffer
SDS-PAGE running buffer
Equipment
Incubator and shaker (Eppendorf, catalog number: M1335-0004)
UV Vis spectrophotometer (Agilent Technologies, catalog number: G1103A)
Sonicator (QSonica, catalog number: Q700)
Centrifuge (Sorvall, catalog number: SO-RC5B)
PTI F10S-6 × 500 rotor (Piramoon Technologies, catalog number: I590496)
SS34 8 × 50 rotor (Sorvall, catalog number: SO-34)
Centrifuge bottles, 500 mL (Nalgene, Thermo Scientific, catalog number: 3120-9500)
High-speed centrifuge tubes, 50 mL (Nalgene, Thermo Scientific, catalog number: 3114-0050)
Tabletop centrifuge (Sorvall, catalog number: SO-RT7)
Swing buckets (Sorvall, catalog number: 00436)
pH sensor (Fisher Scientific, catalog number: 13-620-631)
Akta Purifier (GE Healthcare) or Akta Pure (Cytiva)
GST column (Cytiva, catalog number: 17528202)
Cation exchange column (Cytiva, catalog number: 17115201)
Superdex 200 column (Cytiva, catalog number: 28989335)
Hot water bath (Precision Scientific, catalog number: 66643)
Autoclave (Steris, model: Amsco Lab 250)
Mini gel tank (Life Technologies, catalog number: A25977)
Electrophoresis power supply (Fisher Scientific, catalog number: 55860-4)
NanoDrop (Thermo Scientific, catalog number: ND-ONE-W)
Gel imagers: ChemiDocTM MP Imaging System (Bio-Rad, catalog number: 12003154) and Typhoon FLA 7000 (GE Healthcare)
Software
Unicorn: version 5.20 for Akta purifier (GE Healthcare) and version 7.6 for Akta Pure (Cytiva)
Procedure
Transformation of BL21 codon plus (DE3) RIPL cells
Thaw chemically competent BL21 codon plus (DE3) RIPL cells and the plasmid (pGEX-6P-1) on ice for 10 min.
Add 50 μL of the bacterial suspension into a polypropylene round-bottom sterile tube. Further, add 1 μL (~100 ng) of the desired plasmid [either full-length amphiphysin 1 (Amph1) or the N-BAR domain encoded into a pGEX-6P-1 vector] directly into the bacterial suspension. Incubate the mixture on ice for 20 min. Do not vortex.
Incubate the bacteria at 42 °C in a water bath for precisely 45 s and put the tube directly on ice for 20 min.
Add 1 mL of SOC medium and incubate at 37 °C for 1 h in a shaker at 250 rpm.
Spread 100 μL of the transformation reaction mixture onto a prewarmed LB agar plate containing ampicillin (0.1 mg/mL; see Recipes).
Incubate the plate at 37 °C for 16 h.
After transformation, the agar plates can be stored for one week at 4 °C.
Expression of full-length Amph1/Amph1 N-BAR in E. coli
Prepare the starter culture by inoculating a single colony of the transformed bacteria into 100 mL of autoclaved LB media supplemented with ampicillin (0.1 mg/mL) and chloramphenicol (0.035 mg/mL) (see Recipes). Incubate the culture at 37 °C in a shaker at 250 rpm for 12–16 h.
Note: We prepare stock solutions of ampicillin (25 mg/mL) in Milli Q water and chloramphenicol (35/mL) in absolute ethanol and store them in a -20 °C freezer. Required volume of the stock solution to the culture media can be added to achieve final working concentration of ampicillin (0.1 mg/mL) and chloramphenicol (0.035 mg/mL).
Inoculate 4 × 1 L of autoclaved LB broth containing ampicillin (0.1 mg/mL) and chloramphenicol (0.035 mg/mL) with 25 mL of starter culture. Grow the secondary culture at 37 °C in a shaker at 225 rpm.
Using a UV Vis spectrophotometer and disposable cuvettes, monitor the optical density at the wavelength of 600 nm every 1 h. Once the OD600 reaches a value of ~0.5 (approximately 3 h), reduce the incubator temperature to 18 °C and wait (another 20 min) for the OD600 to reach approximately 0.8.
Make an aqueous stock solution of IPTG by dissolving 300 mg of IPTG in 10 mL of autoclaved water. Add 2.5 mL of the stock solution to each 1 L of culture to obtain 0.3 mM final IPTG concentration. Also, add 1 mL from the 25 mg/mL stock solution of ampicillin and 0.25 mL from the 35 mg/mL stock solution of chloramphenicol to each 1 L culture. Incubate the flasks at 18 °C with shaking (225 rpm) for 12–16 h.
Divide the culture between multiple centrifuge bottles. Harvest the cells by centrifugation using a Sorvall SO-RC5B centrifuge equipped with a PTI F10S rotor, operated at 6,000× g for 20 min at 4 °C. Discard the supernatant and proceed with the next step. The pellet can be stored at -80 °C after transferring into 50 mL conical tubes and flash freezing with liquid nitrogen. Thaw the frozen bacterial cell pellet obtained from the post-induction of secondary culture on ice for 2–3 h.
Purification of full-length Amph1/Amph1 N-BAR
Cell Lysis: Resuspend the pellet in 60–80 mL of GST lysis buffer [see Recipes; use buffer without glycerol for full-length Amph1 and buffer containing 10% glycerol (v/v) for Amph1 N-BAR] supplemented with 1 mM of PMSF (see Recipes). Break any clumps by pipetting up and down. Transfer the suspension into a metallic beaker.
Sonicate the suspension using a tip sonicator at an amplitude value set between 30 and 50 for 5 min with a 1 s pulse on and 4 s pulse off cycle (25 min total sonication time). The maximum power output of the sonicator we use is 700 watts and, for a 1 s pulse at amplitude of 50, approximately 3.5 J is delivered to the probe. The sonication should be performed on ice to prevent heating of the suspension. Ensure the sonicator tip remains well immersed in the solution throughout the entire sonication process to prevent frothing. At the end of the sonication cycle, the lysed suspension should turn from light yellow to orangish and less viscous than the starting mixture. Continue the sonication for an additional 1–2 min if mixture still looks yellowish or seems to be similarly viscous as before.
Transfer the suspension into high-speed centrifuge tubes and centrifuge at 30,000× g for 1 h at 4 °C, using an SS34 8 × 50 rotor to pellet cell debris.
After centrifugation, filter the supernatant (hereafter cell lysate) through the 0.22 μm syringe filter to remove the particulate matter.
Note: Collect the cell pellet and cell lysate (~20 μL) samples for SDS-PAGE analysis.
GST affinity chromatography: Perform GST affinity chromatography using a fast protein liquid chromatographic (FPLC) system (ÄKTA purifier) with 2 × 5 mL GSTrapTM FF columns connected in a series. Pre-equilibrate the GSTrapTM FF column with five column volumes (CV) of GST lysis buffer [use buffer without glycerol for full-length Amph1 and buffer containing 10% glycerol (v/v) for Amph1 N-BAR9] prior to sample application.
Apply the filtered cell lysate from Step C4 onto the column using an injection loop at a flow rate of 1 mL/min and monitor the absorbance at 280 nm (Figure 1A and Figure 2A). Collect the flowthrough 1 (FT1) and inject it onto the column, and again save flowthrough 2 (FT2).
Wash the column with lysis buffer in order to remove the non-specifically bound protein. Washing should be performed until the UV absorption at 280 nm (UV_280) reaches a flat baseline (typically requires the passing of 100–150 mL of lysis buffer).
Wash the column with GST wash buffer (30–50 mL; see Recipes) in order to bring the salt concentration down to 150 mM (can be monitored from the conductance of the mobile phase; a rapid reduction in the conductance indicates buffer switching).
Elute the protein by passing elution buffer [see Recipes; use buffer without glycerol for full-length Amph1 and buffer containing 10% glycerol (v/v) for Amph1 N-BAR]. Start collecting elution as soon as the UV_280 signal starts increasing and collect until the signal reaches a flat baseline (~10–15 mL typically).
Note: Typically, we maintain a flow rate of 1 mL/min for loading the cell lysate, 2 mL/min for loading FT1 and for the washing steps, and 1 mL/min for the elution step. One can also maintain 1 mL/min flow rate throughout all steps.
Cleavage of GST tag by protease treatment: Thaw an aliquot (125 μg) of PreScission protease from -80 °C storage and add the protease to the eluted protein solution. Keep the mixture for overnight shaking at 4 °C in order to cleave the GST affinity tag.
Notes:
Keep a small (~20 μL) aliquot of the GST elution for running SDS-PAGE.
Typically, addition of 125 μg of PreScission protease has shown complete cleavage of GST-tag when we purified proteins at a scale of 4 × 1 L expressions. In case there is a band of protein with uncleaved GST-tag still present after overnight protease treatment, one more aliquot of protease can be added.
Collect samples (~20 μL) of FT2 and pre- and post-cleavage protein samples for SDS-PAGE analysis.
Running SDS-PAGE analysis: Mix the samples with loading buffer in a 1:5 ratio. Further, heat all the samples on a heating block set at approximately 95 °C for 5 min. Then, using gel-loading tips, load the samples into a 12% acrylamide gel along with pre-stained protein ladder. Run the gel at 200 V for 1 h using a running buffer (see Recipes). Stain the gel with GelCodeTM Blue safe protein stain and then destain with water to visualize the bands (Figure 1B and Figure 2D).
Note: Molecular weights of full-length Amph1 and its N-BAR domain are 76 kDa and 29 kDa, respectively. GST has a molecular weight of 26 kDa. The full-length protein appeared slightly closer to the 110 kDa band and the N-BAR appeared closer to the 30 kDa band of the protein marker.
Cation exchange chromatography: Equilibrate the cation exchange column (5 mL) with cation exchange buffer A [see Recipes; without glycerol for full-length Amph1 and with 10% glycerol (v/v) for the N-BAR] and cation exchange buffer B [see Recipes; without glycerol for full-length Amph1 and with 10% glycerol (v/v) for the N-BAR]. A typical column equilibration program starts with 100 % buffer A and 0% buffer B (five column volumes); then, switches to 100% buffer B and 0% buffer A (five column volumes); and finally switches back to 100% buffer A and 0% buffer B (five column volumes). Also, equilibrate the injection loop with two loop volumes of buffer A.
Inject the cleaved protein sample onto the column using an injection loop at a flow rate of 1 mL/min and collect the FT1. Repeat the same and save FT2.
Wash the column with buffer A until the absorbance at 280 nm reaches baseline.
Elute the protein with a linear gradient from 0% buffer B, 100% buffer A to 45% buffer B, and 55% buffer A over the course of a 100 mL elution volume and collect the fractions (Figure 1C and Figure 2B).
Run the FT2 and fractions on SDS-PAGE (Figure 1E and Figure 2D).
Pool the fractions containing the protein and concentrate using Amicon® ultra centrifugal filters (use molecular weight cutoff of 10 kDa for N-BAR and 30 kDa for full-length Amph1) up to a final volume of 10 mL.
Size exclusion chromatography: Equilibrate the size exclusion column and the injection loop with size exclusion buffer (see Recipes; without glycerol for full-length Amph1 and with 10% glycerol for the N-BAR).
Inject the pooled protein fractions post-cation exchange chromatography and after concentrations into the size exclusion column (Superdex 200) at a flow rate of 0.8 mL/min.
Elute the proteins and collect the fractions (Figure 1D and Figure 2C). Analyze the fractions using SDS-PAGE (Figure 1F and Figure 2E).
Note: The BAR domain proteins often elute as two separate bands in size exclusion (Figure 2C). Samples from both these size exclusion bands usually appear as the same molecular weight species in SDS-PAGE, indicating that these bands are actually different oligomeric species. The dimer usually elutes at the end due to its smaller size than the larger oligomers. We recommend that the fractions under the different elution bands should be collected and concentrated separately to avoid mixing between various oligomers.
Figure 1. Representative FPLC chromatograms and SDS-PAGE images from full-length Amph1 purification. A. Chromatogram showing loading of cell lysate (supernatant) onto GST affinity column and elution. Line colors represent the chromatograms corresponding to the parameters mentioned on top. The chromatogram between 0 and 200 mL represents the sample loading step, 200–340 mL indicates washing with GST lysis buffer, 340–380 mL indicates washing with GST wash buffer, and 380–400 mL indicates the elution step. B. SDS-PAGE after GST affinity purification. Lane 1: flowthrough 2 (FT2); lane 2: elution before protease cleavage; lane 3: elution after PreScission treatment. C. Cation exchange elution chromatogram (purple line represents UV_280 profile and green line represents the concentration of buffer B indicating the protein of interest elutes around 50 mL). D. UV_280 profile from the size exclusion chromatography step showing the elution band between 45 and 70 mL. E. SDS-PAGE after cation exchange chromatography. Lanes 1 and 2: flowthrough from cation exchange column; lanes 3–9: fractions collected under the elution peak shown in Figure 1C. F. SDS-PAGE after size exclusion chromatography. Lanes 1–8: fractions collected under the peak shown in Figure 1D.
Figure 2. Representative FPLC chromatograms and SDS-PAGE image from Amph1 N-BAR purification. A. Chromatogram from the GST affinity purification showing the UV_280 profile during loading (0–100 mL), column wash with lysis buffer (90–170 mL), column wash with GST wash buffer (170–200 mL), and elution (200–220 mL) steps. B. Cation exchange elution chromatogram (blue line represents UV_280 profile and green line represents the concentration of buffer B) indicating the protein of interest elutes around 50 mL. C. Size exclusion chromatogram showing a peak around 60 mL corresponding to the oligomeric species, and a peak around 75 mL corresponding to the homodimeric species. D. Representative SDS-PAGE image after cation exchange chromatography of Amph1 N-BAR. Lane 1: PreScission protease-treated sample before loading to cation exchange column showing the bands for cleaved GST tag (between 20 and 30 kDa markers) and N-BAR domain (~30 kDa); lane 2: flowthrough 2 (FT2) from the cation exchange column; lanes 3–9: fractions collected under the elution peaks shown in Figure 2B. E. SDS-PAGE for Amph1 N-BAR after size exclusion chromatography. Lanes 1–10: fractions collected under the elution peaks (elution volume 50–90 mL) shown in Figure 2C.
Pool and concentrate the fractions containing protein using Amicon ultra centrifugal filters (use molecular weight cutoff of 10 kDa for N-BAR and 30 kDa for full-length Amph1).
Concentration determination and storage: Determine the protein concentration using NanoDrop by measuring the absorbance at 280 nm. The extinction coefficients of full-length Amph1 and N-BAR are 55,000 M-1 cm-1 and 28,500 M-1 cm-1, respectively.
Transfer the proteins into small aliquots, flash freeze into liquid nitrogen, and store at -80 °C.
Notes
Always handle live bacterial cultures under a flame or in a biosafety cabinet, in order to maintain aseptic conditions.
Dispose of E. coli cultures as recommended by biosafety protocols.
The flash-frozen cell pellets can be stored at -80 °C for up to six months.
Recipes
LB agar plates
Add 3.7 g of LB agar in 100 mL of Milli Q water and sterilize by autoclaving. Allow the medium to reach a lukewarm temperature and add the required antibiotic ampicillin (0.1 mg/mL). Further, pour approximately 25 mL into sterile Petri dishes. Allow to solidify and then seal with parafilm. The plates can be stored at 4 °C for one month.
Luria Bertani (LB) media
Prepare 4 L of media by dissolving LB in Milli Q water (25 g/L) and autoclave to sterilize. Add the required antibiotics ampicillin (0.1 mg/mL) and chloramphenicol (0.035 mg/mL) before inoculation.
PMSF stock solution
Dissolve PMSF (mol. wt. 174 g/mol) in absolute ethanol (0.174 g solid in 10 mL) to prepare a 100 mM stock solution. The stock solution can be stored at -20 °C.
Buffers
Prepare buffers without glycerol for full-length Amph1 and with 10% glycerol for the N-BAR. Use diluted sodium hydroxide or HCl solutions to adjust the buffer pH.
GST lysis buffer (1 L)
300 mM NaCl 17.532 g
50 mM Tris 6.06 g
1 mM EDTA 4 mL of 0.25 M stock
2 mM DTT 0.308 g
pH = 8, filter and store at 4 °C
GST wash buffer (0.25 L)
150 mM NaCl 2.19 g
50 mM Tris 1.515 g
1 mM EDTA 1 mL of 0.25 M stock
2 mM DTT 0.077 g
pH = 8, filter and store at 4 °C
GST elution buffer (0.25 L)
150 mM NaCl 2.19 g
50 mM Tris 1.515 g
20 mM GSH 1.536 g
1 mM EDTA 1 mL of 0.25 M stock
2 mM DTT 0.077 g
pH = 8, filter and store at 4 °C
Cation exchange buffer A (1 L)
150 mM NaCl 8.77 g
20 mM sodium phosphate (including both dibasic and monobasic salts)
Sodium dihydrogen phosphate 1 g
Disodium hydrogen phosphate 3.11g
1 mM EDTA 4 mL of 0.25 M stock
1 mM DTT 0.154 g
pH = 7, filter and store at 4 °C
Cation exchange buffer B (0.5 L)
1 M NaCl 29.22 g
20 mM sodium phosphate (including both dibasic and monobasic salts)
Sodium dihydrogen phosphate 0.5 g
Disodium hydrogen phosphate 1.56 g
1 mM EDTA 2 mL of 0.25 M
1 mM DTT 0.08 g
pH = 7, filter and store at 4 °C
Size exclusion buffer (1 L)
150 mM NaCl 8.766 g
20 mM HEPES 4.766 g
1 mM TCEP 0.287 g
pH = 7.4, filter and store at 4 °C
SDS-PAGE running buffer (1 L)
Tris 3.0 g
Glycine 14.4 g
SDS 1 g
pH 8.3
Acknowledgments
The authors acknowledge Pietro DeCamilli’s lab for providing us with plasmids for full-length amphiphysin, N-BAR domain, and PreScission protease. This research was financially supported by the National Institutes of Health, grant No GM 097552.
Competing interests
The authors declare no competing interests.
Ethics
No animal/human subject is used in this protocol.
References
Ambroso, M. R., Hegde, B. G. and Langen, R. (2014). Endophilin A1 induces different membrane shapes using a conformational switch that is regulated by phosphorylation. Proc Natl Acad Sci U S A 111(19): 6982-6987.
Capraro, B. R., Shi, Z., Wu, T., Chen, Z., Dunn, J. M., Rhoades, E. and Baumgart, T. (2013). Kinetics of endophilin N-BAR domain dimerization and membrane interactions. J Biol Chem 288(18): 12533-12543.
David, C., McPherson, P. S., Mundigl, O. and de Camilli, P. (1996). A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals. Proc Natl Acad Sci U S A 93(1): 331-335.
Frost, A., Unger, V. M. and De Camilli, P. (2009). The BAR domain superfamily: membrane-molding macromolecules. Cell 137(2): 191-196.
Gallop, J. L., Butler, P. J. and McMahon, H. T. (2005). Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission. Nature 438(7068): 675-678.
Gallop, J. L., Jao, C. C., Kent, H. M., Butler, P. J., Evans, P. R., Langen, R. and McMahon, H. T. (2006). Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J 25(12): 2898-2910.
Milosevic, I., Giovedi, S., Lou, X., Raimondi, A., Collesi, C., Shen, H., Paradise, S., O'Toole, E., Ferguson, S., Cremona, O., et al. (2011). Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission. Neuron 72(4): 587-601.
Mondal, S., Narayan, K., Botterbusch, S., Powers, I., Zheng, J., James, H. P., Jin, R. and Baumgart, T. (2022). Multivalent interactions between molecular components involved in fast endophilin mediated endocytosis drive protein phase separation. Nat Commun 13(1): 5017.
Ringstad, N., Nemoto, Y. and De Camilli, P. (1997). The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc Natl Acad Sci U S A 94(16): 8569-8574.
Smith, D. B. and Johnson, K. S. (1988). Single-Step Purification of Polypeptides Expressed in Escherichia coli as Fusions with Glutathione S-Transferase. Gene 67(1): 31-40.
Takei, K., Slepnev, V. I., Haucke, V. and De Camilli, P. (1999). Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol 1(1): 33-39.
Vagenende, V., Yap, M. G. S. and Trout, B. L. (2009). Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol. Biochemistry 48(46): 11084-11096.
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Molecular Biology > Protein > Protein-protein interaction
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47 | https://bio-protocol.org/en/bpdetail?id=47&type=1 | # Bio-Protocol Content
Improve Research Reproducibility
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This is an In Press version of the protocol that has not yet been assigned to an issue.
Peer-reviewed
Total RNA Extraction from C. elegans
Fanglian He
In Press
Published: Mar 20, 2011
DOI: 10.21769/BioProtoc.47 Views: 30231
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Abstract
This protocol describes total RNA extraction from worms with or without using commercial RNA extraction kits.
Materials and Reagents
C. elegans
Trizol (Life Technologies, Gibco®, catalog number: 15596-026 )
DEPC treated H2O (Life Technologies, Ambion®)
Turbo DNase (Life Technologies, Ambion®, catalog number: AM2238 )
RNeasy Mini kit (Life Technologies, Gibco®, catalog number: 15596-026 )
Ethanol
Chloroform
Isopropanol
Liquid nitrogen
RNase-free EDTA
KH2PO4
Na2HPO4
NaCl
MgSO4
M9 buffer (see Recipes)
Equipment
15-ml Corning tubes (Corning)
RNase-free eppendorf tubes (Eppendorf)
Filter tips (Eppendorf)
Dissecting microscope
Water bath
Procedure
Wash worms off plates with M9 buffer and collect them in 15-ml Corning tubes.
Wash 2 – 3 x with M9 buffer to get rid of bacteria.
Add 10 ml Trizol for every ml of packed worms (typically - add just 1 ml Trizol for ≤ 100 µl worms).
Note: At this step, you can freeze tubes in liquid nitrogen immediately and store at -80 °C until you are ready to proceed.
Vortex tubes for 30 sec, then place in liquid nitrogen, let thaw at 37 °C, and repeat several times (3 – 6 x). Then freeze at -80 °C until ready to prepare.
Thaw frozen worms/Trizol mixture and vortex 30 sec then put on ice for 30 sec. Repeat this 6 - 7 x.
Most worms (not 100%) should appear disrupted under a dissecting microscope.
Move to RNase-free eppendorf tubes (alternatively, move before freezing at step 2).
Let stand at room temperature (RT) for 5 min.
Chloroform extraction (working in hood).
Add 2 ml chloroform per 1 ml of packed worms (typically 200 µl).
Invert 15 sec, let sit 3 min RT for phase separation.
Spin 15 min at 12,000 x g at 4 °C. RNA is in the aqueous supernatant.
Isopropanol precipitation (working in hood).
Transfer top aqueous phase to new RNase-free eppendorf tube.
Add 0.7 volumes (of what is already in tube) isopropanol (typically 400 - 500 µl).
Gently invert several times to mix.
Leave at RT for 10 min.
Spin at 12,000 x g for 10 min at 4 °C.
A small white RNA pellet at the bottom of tube should be visible. Carefully pipet out supernatant.
Wash pellet with ice cold 75% EtOH (use DEPC-treated H2O to make EtOH solution).
Spin 4,000 rpm at 4 °C for 5 min.
Pipet out EtOH. When almost all the ethanol has evaporated (faint halo around pellet), resuspend in 25-100 µl DEPC-H2O by pipetting and incubate at 60 °C (water bath) for 10 min (if you have been using larger tubes, now transfer to RNAse-free eppendorf tube).
Set up Turbo DNase reaction.
Dilute the 10x Turbo buffer in the RNA sample to 1x.
Add 10 units of Turbo DNase per ml of sample (1 µl per 100 µl is sufficient).
Incubate at 37 °C for 30 min.
Add RNase-free EDTA (pH 8.0, use DEPC water) to a final concentration of 5 mM and incubate at RT 10 min.
Take 260/280 nm and 260 nm absorption reading. If pure, 2.0 ratio. Expect 1-4 mg/gram of worms.
Store frozen at -20 °C (or -80 °C for long-term storage).
Notes
Steps 10-12 can be replaced by using Qiagen RNeasy mini kit as described below:
Transfer top aqueous phase to new 1.5 ml RNase-free eppendorf tube.
Slowly add an equal volume of 70% EtOH and mix by inverting tubes.
Transfer the mixture to a Qiagen RNeasy spin column and follow manufacture's instructions (see Qiagen RNeasy Mini Handbook).
Recipes
1 liter M9 buffer
3 g KH2PO4
6 g Na2HPO4
5 g NaCl
Add H2O to 1 liter. Sterilize by autoclaving.
After solution cools down, and 1 ml autoclaved/sterile 1 M MgSO4
Article Information
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© 2011 The Authors; exclusive licensee Bio-protocol LLC.
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4,700 | https://bio-protocol.org/en/bpdetail?id=4700&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
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Peer-reviewed
Isolation and Quantification of Mandelonitrile from Arabidopsis thaliana Using Gas Chromatography/Mass Spectrometry
AA Ana Arnaiz
JV J. Lucas Vallejo-García
SV Saul Vallejos
ID Isabel Diaz
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4700 Views: 413
Reviewed by: Xiaofei LiangPrajita PandeySneha Ray
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Original Research Article:
The authors used this protocol in Plant Physiology Aug 2022
Abstract
Mandelonitrile is a nitrogen-containing compound, considered an essential secondary metabolite. Chemically, it is a cyanohydrin derivative of benzaldehyde, with relevant functions in different physiological processes including defense against phytophagous arthropods. So far, procedures for detecting mandelonitrile have been effectively applied in cyanogenic plant species such as Prunus spp. Nevertheless, its presence in Arabidopsis thaliana, considered a non-cyanogenic species, has never been determined. Here, we report the development of an accurate protocol for mandelonitrile quantification in A. thaliana within the context of A. thaliana–spider mite interaction. First, mandelonitrile was isolated from Arabidopsis rosettes using methanol; then, it was derivatized by silylation to enhance detection and, finally, it was quantified using gas chromatography–mass spectrometry. The selectivity and sensitivity of this method make it possible to detect low levels of mandelonitrile (LOD 3 ppm) in a plant species considered non-cyanogenic that, therefore, will have little to no cyanogenic compounds, using a small quantity of starting material (≥100 mg).
Keywords: Benzaldehyde cyanohydrin Mandelonitrile Hydroxynitrile lyase Arabidopsis thaliana Spider mite Cyanogenic glycosides Cyanohydrin
Background
Cyanogenic glycosides (CNglcs) are secondary plant metabolites arisen from amino acids and found in over 3,000 plant species, including economically important crops as Prunus spp., Manihot spp., Malus spp., or Sorghum spp. (Gleadow and Møller, 2014). CNglcs play crucial roles in different physiological processes such as germination, transport of essential nutrients, turnover processes, oxidative stress regulation, or defense responses (Møller, 2010; Kadow et al., 2012; Flematti et al., 2013; Neilson et al., 2013). The metabolism of CNglcs is complex and derives in cyanohydrin production, which in turn can be converted into hydrogen cyanide and ketones or aldehydes in the presence of α-hydroxynitrile lyase enzymes (HNLs). Plant cyanohydrins have been demonstrated to act as direct defense molecules, with a highly toxic action against herbivores and pathogens (Park et al., 2002; Beran et al., 2019). In addition, they can also work as signaling molecules, triggering the defense response (Bernal-Vicente et al., 2020). However, some herbivores, mainly lepidopteran insects, have developed the ability to counteract or take advantage of these cyanogenic molecules, and alternatively may degrade or use these compounds for their benefit (Zagrobelny and Møller, 2011; Zagrobelny et al., 2014).
The focus of this protocol is one of these CNglcs derivatives, mandelonitrile. Mandelonitrile is a cyanohydrin derived from CNglc metabolism, as prunasin or amygdalin among others (Gleadow and Møller, 2014), that occurs naturally in cyanogenic plants. It is characterized by having hydroxyl and cyano groups attached to the same carbon atom (Figure 1). Some works have reported the quantification of mandelonitrile in different cyanogenic plant species such as Prunus cerasus L. and Aronia melanocarpa Ell using high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC/MS) techniques, respectively (Hirvi and Honkanen, 1985; Chandra and Nair, 1993).
Figure 1. Example of a cyanogenesis pathway in plants with mandelonitrile as the central player. HCN: hydrogen cyanide.
Chassagne et al. (1996) detected amygdalin and its corresponding parental ion, benzaldehyde cyanohydrin (or mandelonitrile), in Passiflora fruits using GC/MS. More recently, mandelonitrile has been quantified in peach micro-propagated shoots by ultra-performance liquid chromatography (UPLC) coupled to a mass spectrophotometer (Bernal-Vicente et al., 2020). However, as far as we know, mandelonitrile levels haven’t been determined in the model plant A. thaliana until now, maybe because it is considered a non-cyanogenic plant (CNglcs have not been detected in A. thaliana wild-type plants). Despite this, an alternative pathway to cyanohydrin production has recently been described—the 4-hydroxy-indole-3-carbonylnitrile (4-OH-ICN) route—as a rare metabolic re-invention leading to alternative cyanogenic compounds, to expand A. thaliana defenses and also contribute to the production of cyanohydrins (Rajniak et al., 2015; Pastorczyk et al., 2020). In addition, an HNL has also been found in A. thaliana, and its encoding gene has been cloned and the recombinant protein has been purified and crystalized (Andexer et al., 2007). Biochemical studies revealed that Arabidopsis HNL recognized a broad range of substrates, was enantioselective, and transformed aliphatic and aromatic aldehydes and/or ketones into the corresponding R-cyanohydrins, including mandelonitrile (Andexer et al., 2007).
Therefore, the study of the relationship between mandelonitrile levels and the role of HNL in Arabidopsis has allowed the establishment of their defense role against the two-spotted spider mite Tetranychus urticae (Arnaiz et al., 2022). Consequently, mandelonitrile can be considered a target molecule for the study of Arabidopsis–spider mite interaction in the plant defense process. This is why the development of a protocol to determine the levels of mandelonitrile in A. thaliana plants under controlled conditions and after spider mite infestation is of crucial interest. In addition, this protocol was established since it was not possible to adapt previous protocols already described for other plant species. In fact, no results were obtained using HPLC techniques and with the available quantity of infested A. thaliana sample as starting plant material, suggesting the need to develop a specific quantification procedure for such a non-cyanogenic species. GC/MS was selected for mandelonitrile detection as it allowed to detect the target molecule in standard samples. Moreover, the spectrum information related to mandelonitrile-derivatized found in the databases helped us to confirm our obtained results. Additionally, the derivatization process of mandelonitrile makes it a very volatile molecule and for that reason, it was better to use GC/MS than HPLC for analysis. Finally, it is likely that this protocol will work in other plant species by applying some modifications, e.g., quantity of starting material, solvent volume, or incubation and sonication times.
Materials and reagents
Biological material
Arabidopsis thaliana Columbia-0 (Col-0) ecotype seeds were purchased at the Arabidopsis Biological Resource Center
Tetranychus urticae, London strain (Acari: Tetranychidae). Dr. Miodrag Grbic (UWO, Canada) provided the spider mite colony (see Note 1)
Materials
Microcentrifuge tube with safe lock 1.5 mL (Astik’s, catalog number: PCRP-015-500)
Pipette tips (Astik’s, catalog numbers: TIPP-1K0-10K, TIPP-200-10K, and TIPP-010-10K)
Peat moss (Klasmann-Deilmann, catalog number: L1070K0600)
Vermiculite (Valimex S.L., catalog number: 100M1006)
Plastic alveolus tray (Projar, catalog number: 4400000S28)
Cling film (KINGSWAY, catalog number: KING756046)
Aluminum foil (Alujet-Universal, catalog number: 293-4176)
Mortar and pestle (LGB, catalog number: MORN-075-001)
Tweezers (Clink, catalog number: FORS-003-002)
Micro spatula (Clink, catalog number: SPNS-150-005)
Centrifuge tubes with screw cap, 15 mL (Astik’s, catalog number: PTGP-E15-025)
Crimp neck vials ND11 (VWR, catalog number: 548-8008A)
Crimp caps with septa for crimp neck vials ND11 (VWR, catalog number: 548-8004A)
Minisart® syringe filters PTFE 0.2 μm (Sartorius, catalog number: 17573-ACK)
Gas chromatography column DB-5MS UI (Agilent, catalog number: 122-5562UI)
Chemicals and solvents
Sodium dodecyl sulfate (SDS) (PanReac AppliChem ITW Reagents, catalog number: A2263.0100)
Sodium hypochlorite (PanReac AppliChem ITW Reagents, catalog number: 212297.1211)
Methanol (VWR, catalog number: 20864.32)
Mandelonitrile (Sigma-Aldrich, catalog number: 116025)
Ethanol absolute (VWR, catalog number: 20821.365)
Acetonitrile (VWR, catalog number: 83639.320)
2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)acetamide (MSTFA) (Supelco, catalog number: 1.11805)
Silicone oil for oil baths (VWR, catalog number: 24610.363)
Autoclaved distilled water
Arabidopsis seeds sterilization solution (see Recipes)
Equipment
Single-channel pipettes PIPETMAN® L P1000 (Gilson, catalog number: FA10006M)
Single-channel pipettes PIPETMAN® L P200 (Gilson, catalog number: FA10005M)
Single-channel pipettes PIPETMAN® L P20 (Gilson, catalog number: FA10003M)
Single-channel pipettes PIPETMAN® L P2 (Gilson, catalog number: FA10001M)
Elmasonic S30H (Elma Schmidbauer GmbH, catalog number: 1001955)
Heating oven Binder E028 (BINDER GmbH, catalog number: 9010-0001)
Sorvall X4F Pro centrifuge (Thermo Scientific, catalog number: 75009026)
Digital rotary evaporator (VWR, catalog number: 531-1365P)
Precision balance LPW 2103i (VWR, catalog number: 611-3283)
Agilent 7890B gas chromatography system coupled to an Agilent 7010 triple quadrupole mass spectrometer (Agilent)
Freezer -80 °C
Sanyo MLR-350-H Panasonic growth chamber
Laminar flow hood
4 °C chamber
Software
MassHunter Workstation software version B.07.01 (Agilent)
Microsoft office Excel
Statistical analysis software such as GraphPad Prism 9, OriginPro 9.0, or Statgraphics
Procedure
A. thaliana seeds sterilization
Place approximately 80 seeds of A. thaliana Col-0 into a 1.5 mL tube.
In a laminar flow hood, add 1 mL of 70% ethanol to the tubes and mix by inversion for 2 min.
Remove ethanol using a micropipette, add 1 mL of Arabidopsis seeds sterilization solution (see Recipes) to the tube, and mix by inversion for 12 min.
Remove the sterilization solution using a micropipette and perform five washing cycles with autoclaved distilled water.
Leave the seeds in water until use (see Note 2).
A. thaliana seed sowing
Prepare a plastic seed tray with autoclaved peat moss: vermiculite (2:1) for 20 alveolus of 6 × 6 × 8 cm.
Place five sterilized seeds per alveolus avoiding corners.
Cover the planted seed tray with a plastic film and leave it at 4 °C for five days to synchronize seed germination.
Place the seed tray (that has been stratified at 4 °C for five days) into the growth chamber at 23 ± 1 °C, > 70% relative humidity, and a 16:8 h day/night photoperiod.
Grow plants for 2–3 weeks (see Note 3).
A. thaliana plants infestation
Infestation assay protocol with spider mite T. urticae was previously reported by Cazaux et al. (2014).
Divide plants into two groups: one for control plants and the other one for infestation with T. urticae.
Perform the infestation. Place 20 adult female mites per plant and cover the rosette with a ventilated plastic cup to avoid mite scape. Leave in the chamber for 24 h at 25 ± 1 °C, > 70% relative humidity, and a 16:8 h day/night photoperiod.
Remove the plastic cup carefully, cut the rosette, and quickly freeze it in liquid nitrogen.
Store plant material at -80 °C until use.
Mandelonitrile extraction
The mandelonitrile isolation protocol was adapted from Madala et al. (2014) as follows.
Crush plant material until it reaches a fine powder using a mortar and pestle (use liquid nitrogen to prevent thawing of the material).
Weigh the plant material using a precision balance (plant material ≥ 100 mg; see Note 4).
In a small mortar, add 3 mL of methanol and the frozen plant material. Homogenate with a pestle and transfer the homogenate to a 15 mL Falcon tube (see Note 5).
Sonicate for 20 min at room temperature (see Note 6).
Incubate at 60 °C for 10 min using a heating oven.
Centrifuge at 11,500× g for 5 min at room temperature.
Collect the supernatants in a 10 mL flask (see Note 7) and remove the methanol on the rotary evaporator at 50 °C. Make sure that samples are completely dry (see Note 8).
GC/MS analyses
Derivatization by silylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA):
Weigh 50 μL of mandelonitrile in a 10 mL flask to prepare the standard curve (see Note 7).
Add 100 μL of MSTFA to the mandelonitrile sample and to the Col-0 samples.
Mix well to achieve a homogeneous solution.
Incubate at 60 °C in an oil bath for 30 min protected from light (see Note 9).
Cool down the samples and the standard at room temperature.
Samples and standard curve preparation for GC analyses:
Prepare standard solutions of mandelonitrile (derivatized with MSTFA) in acetonitrile solvent at 2.5, 5, 10, 25, 50, 100, 250, and 500 ng/μL concentrations using 5 mL vials.
Prepare samples by adding 900 μL of acetonitrile to the derivatized Col-0 samples.
Filter the samples and the standards through a 0.2 μm syringe filter and place the sample and the standards in crimp neck vials for GC analyses.
GC analyses:
Inject 1 μL of the standard solution or the sample in a DB-5MS UI column of 60 m × 0.25 mm with 0.25 μm film thickness with a pressure of 17.7 psi, septum purge flow of 3 mL/min, and spitless time of 1.5 min (see Note 10).
Set the injector temperature to 250 °C.
Set the oven temperature to 60 °C, hold for 3 min, and then raise 10 °C/min up to 300 °C and maintain at 300 °C for 6 min.
Set the transfer line temperature to 280 °C.
Set the collision gas (nitrogen) and the quench gas (helium) with a flow rate of 1.5 mL/min and 2.25 mL/min, respectively.
Set the operation mode as electron ionization mode at 280 °C and 70 eV with multiple reaction monitoring (MRM) to monitor ion transitions (see Notes 11 and 12).
The total ion and the retention time zoom chromatograms between minutes 15 and 18 are shown in Figure 2A and 2B, respectively.
Figure 2. Full scan and total ion chromatograms (TICs) of mandelonitrile standard. A. Full scan chromatogram of mandelonitrile indicating the selected ion products. B. TIC of mandelonitrile at 25 ng/mL. C. Amplification of the retention times between 15 and 18 min.
Data analysis
Three independent experiments of A. thaliana spider mite infestation were performed. Therefore, three independent mandelonitrile A. thaliana extractions, derivatizations, and quantifications were done, always including the standard molecule.
The product ion 190 (m/z) was designated as a quantifier ion; therefore, this product ion was selected for analyzing the standard and the samples. MassHunter Workstation software version B.07.01 was used to analyze the results and to export the data in csv format, to analyze using Excel software.
First, the standard curve was analyzed (Figure 3) by graphical representation of the intensity obtained from the mass spectrometer (from the 190-product ion peaks) vs. the mandelonitrile concentration.
Figure 3. Standard curve of mandelonitrile derivatized
Next, the total ion chromatograms (TICs) of the samples were compared to the standard in order to find the derivatized mandelonitrile peak in the Arabidopsis samples. As shown in Figure 4, the TICs from Arabidopsis Col-0 samples present different peaks, both in the control and in the mites-infested samples, including the mandelonitrile ones marked with a red arrow in each chromatogram.
Figure 4. Total ion chromatograms of mandelonitrile and Arabidopsis Col-0 samples with their amplification zone of retention times between 15 and 18 min. Red arrows indicate the corresponding peak of mandelonitrile derivatized.
Subsequently, the corresponding chromatograms of the product ion 105 (m/z) that was selected as the qualifier ion (Figure 5), and the chromatograms of the product ion 190 (m/z) that was selected as quantifier ion (Figure 6) were analyzed.
Figure 5. Multiple reaction monitoring (MRM) chromatograms of 205–105 m/z ion transition of mandelonitrile and Arabidopsis Col-0 samples. Retention time zoom between 15 and 18 min is shown, and red arrows indicate the corresponding peak of ion product 105 (m/z) of mandelonitrile derivatized.
Figure 6. Multiple reaction monitoring (MRM) chromatograms of 205–190 m/z ion transition of mandelonitrile and Arabidopsis Col-0 samples. Retention time zoom between 15 and 18 min are shown, and red arrows indicate the corresponding peak of ion product 190 (m/z) of mandelonitrile derivatized.
Finally, the mandelonitrile concentration data in the A. thaliana samples were obtained and represented (Figure 7). Normality and homoscedasticity of data were analyzed using OriginPro 9.0 software. When the data fulfilled both assumptions, one-way ANOVA followed by Tukey’s test were run.
Figure 7. Mandelonitrile quantification in A. thaliana in control and infested with mites. Data are represented as nanograms of mandelonitrile/grams of fresh weight. Data are means ± SE of three biological replicates (p < 0.05, one-way ANOVA followed by Tukey’s test).
Notes
T. urticae colony is reared on beans (Phaseolus vulgaris) in a growth chamber at 25 °C ± 1 °C, > 70% relative humidity, and with a 16:8 h day/night photoperiod.
Do not leave seeds in water for more than 60 min before sowing. It is better to program the sowing to do it as soon as seed sterilization is completed.
Keep an eye on seed germination every two days. When seeds start germinating, perform holes in the plastic film to allow plants to acclimate to the growth chamber environment. Two or three days after making the holes, remove the plastic film. Water the trays every 2–3 days with 15 mL per alveolus (it is important to ensure that there is no humidity excess that causes condensation on the underside of the leaves). When plants have grown, remove them from each alveolus with the help of tweezers, to leave only one plant per alveolus (preferably the most centrally located plant).
Usually, with 10 plants, approximately 200 mg of material is obtained. It is recommended to use 100–300 mg of plant material for the mandelonitrile extraction step.
It is not necessary to use 15 mL Falcon tubes; other containers, such as glass flasks, can be used.
The ultrasonic frequency of the sonicator is 37 kHz. This frequency is a preset ultrasonic frequency of the sonicator model used.
10 mL flasks were used, but smaller vials can also be used taking into account the availability of adapters for the rotary evaporator.
Samples have an oily appearance.
The use of an oil bath is not mandatory; other options can be used (water bath, incubation chamber, oven) while the sample is incubated at 60 °C. MSTFA is light-sensitive. Therefore, derivatization conditions were established using mandelonitrile; assessing if derivatization was complete was done by nuclear magnetic resonance.
It is recommended to include samples with only solvent (acetonitrile) between the standard and the samples to be analyzed to ensure that the system is thoroughly cleaned, in case there are any remains of the retained compound.
It is not necessary to use a 60 m column; shorter columns (25–30 m) can also be used. Just keep in mind that if the length of the column is reduced, it may be necessary to increase the total run time.
The precursor ion presents an m/z of 205. The ion transitions selected as quantifier and qualifier were the product ions with an m/z of 190 and 105, respectively. The collision energy of the product ions was 5 eV and 20 eV for the 190- and 105-product ion, respectively. The product ion with an m/z of 116 was also identified in the analyses. Still, since it has a very low peak intensity, it was not used as a quantifier or a qualifier.
It is important to analyze the three ions at the same time (triple quad) to rule out false positives or negatives. If one of the three ions does not come out right in a sample, that sample will have to be repeated.
Method validation was performed by Arnaiz et al. (2022), with a limit of detection and quantification of 5 ± 1 ng/mL and 12 ± 2 ng/mL, respectively.
Recipes
Arabidopsis seeds sterilization solution
1% SDS, 5% sodium hypochlorite
Acknowledgments
This work was supported by funds of “La Caixa” Foundation (LCF/PR18/51130007) and Ministerio de Universidades-European Union in the frame of NextGenerationEU RD 289/2021 (Universidad Politécnica de Madrid).
This protocol was derived from the original research published in Arnaiz et al. (2022).
Competing interests
The authors declare no financial and non-financial competing interests.
Ethics
No human and/or animal subjects were used in these protocols.
References
Andexer, J., von Langermann, J., Mell, A., Bocola, M., Kragl, U., Eggert, T. and Pohl, M. (2007). An R-selective hydroxynitrile lyase from Arabidopsis thaliana with an alpha/beta-hydrolase fold. Angew Chem Int Ed Engl 46(45): 8679-8681.
Arnaiz, A., Santamaria, M. E., Rosa-Diaz, I., Garcia, I., Dixit, S., Vallejos, S., Gotor, C., Martinez, M., Grbic, V. and Diaz, I. (2022). Hydroxynitrile lyase defends Arabidopsis against Tetranychus urticae. Plant Physiol 189(4): 2244-2258.
Beran, F., Köllner, T. G., Gershenzon, J. and Tholl, D. (2019). Chemical convergence between plants and insects: biosynthetic origins and functions of common secondary metabolites. New Phytol 223(1): 52-67.
Bernal-Vicente, A., Petri, C., Hernández, J. A. and Diaz-Vivancos, P. (2020). Biochemical study of the effect of stress conditions on the mandelonitrile-associated salicylic acid biosynthesis in peach. Plant Biol (Stuttg) 22(2): 277-286.
Cazaux, M., Navarro, M., Bruinsma, K. A., Zhurov, V., Negrave, T., Van Leeuwen, T., Grbic, V. and Grbic, M. (2014). Application of two-spotted spider mite Tetranychus urticae for plant-pest interaction studies. J Vis Exp 4(89): 51738.
Chandra, A., and Nair, M. G. (1993). Quantification of benzaldehyde and its precursors in Montmorency cherry (Prunus cerasus L.) kernels. Phytochem Anal 4(3): 120-123.
Chassagne, D., Crouzet, J. C., Bayonove, C. L. and Baumes, R. L. (1996). Identification and quantification of passion fruit cyanogenic glycosides. J Agric Food Chem 44 (12): 3817-3820.
Flematti, G. R., Waters, M. T., Scaffidi, A., Merritt, D. J., Ghisalberti, E. L., Dixon, K. W. and Smith, S. M. (2013). Karrikin and cyanohydrin smoke signals provide clues to new endogenous plant signaling compounds. Mol Plant 6(1): 29-37.
Gleadow, R. M., and Møller, B. L. (2014). Cyanogenic glycosides: Synthesis, physiology, and phenotypic plasticity. Annu Rev Plant Biol 65: 155-185.
Hirvi, T., and Honkanen, E. (1985). Analysis of the volatile constituents of black chokeberry (Aronia melanocarpa Ell.). J Sci Food Agric 36(9): 808-810.
Kadow, D., Voss, K., Selmar, D. and Lieberei, R. (2012). The cyanogenic syndrome in rubber tree Hevea brasiliensis: tissue-damage-dependent activation of linamarase and hydroxynitrile lyase accelerates hydrogen cyanide release. Ann Bot 109(7): 1253-1262.
Madala, N. E., Steenkamp, P. A., Piater, L. A. and Dubery, I. A. (2014). Metabolomic insights into the bioconversion of isonitrosoacetophenone in Arabidopsis thaliana and its effects on defense-related pathways. Plant Physiol Biochem 84: 87-95.
Møller, B. L. (2010). Functional diversifications of cyanogenic glucosides. Curr Opin Plant Biol 13(3): 338-347.
Neilson, E. H., Goodger, J. Q. D., Woodrow, I. E. and Møller, B. L. (2013). Plant chemical defence: at what cost? Trends Plant Sci 18(5):250-258.
Park, D. S., Grodnitzky, J. A. and Coats, J. R. (2002). QSAR evaluation of cyanohydrins’ fumigation toxicity to house fly (Musca domestica) and lesser grain borer (Rhyzopertha dominica). J Agric Food Chem 50(20): 5617-5620.
Pastorczyk, M., Kosaka, A., Piślewska-Bednarek, M., López, G., Frerigmann, H., Kułak, K., Glawischnig, E., Molina, A., Takano, Y. and Bednarek, P. (2020). The role of CYP71A12 monooxygenase in pathogen-triggered tryptophan metabolism and Arabidopsis immunity. New Phytol 225(1): 400-412.
Rajniak, J., Barco, B., Clay, N. K. and Sattely, E. S. (2015). A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defense. Nature 525(7569): 376-379.
Zagrobelny, M. and Møller, B. L. (2011). Cyanogenic glucosides in the biological warfare between plants and insects: the Burnet moth-birdsfoot trefoil model system. Phytochemistry 72(13): 1585-1592.
Zagrobelny, M., Olsen, C. E., Pentzold, S., Fürstenberg-Hägg, J., Jørgensen, K., Bak, S., Møller, B. L. and Motawia, M. S. (2014). Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore. Insect Biochem Mol Biol 44: 44-53.
Article Information
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Plant Science > Plant molecular biology
Molecular Biology
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A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma
BM Bharati Matta
JB Jenna Battaglia
BB Betsy J. Barnes
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4701 Views: 1304
Reviewed by: Alessandro DidonnaKomuraiah MyakalaMarco Di Gioia
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Original Research Article:
The authors used this protocol in Frontiers in Immunology Jul 2022
Abstract
Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 μL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 μL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.
Graphical overview
Keywords: NETosis ELISA Smear assay Immunofluorescence Quantification
Background
Neutrophils are the most abundant leukocyte in the human blood and are the first cells to respond to an external stimulus such as a pathogen and/or inflammatory trigger (Kaplan and Radic, 2012; Matta et al., 2022). They migrate to the site of infection where they protect the host by phagocytosing, killing, and digesting pathogens (Kaplan and Radic, 2012; Matta et al., 2022). Another powerful tool neutrophils use to defend the body is the release of neutrophil extracellular traps (NETs). NETs can trap microorganisms and kill them by releasing granular antimicrobial peptides and proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G(3); however, when this process gets dysregulated or NET clearance is diminished, it can lead to tissue damage, autoimmunity, and other inflammatory conditions (Lande et al., 2011; Kaplan and Radic, 2012; Corsiero et al., 2016; Bruschi et al., 2020; Matta et al., 2022).
Formation of NETs involves the citrullination of histones, chromatin decondensation, and disintegration of the nuclear membrane followed by the release of nuclear content along with proteins such as MPO and NE (Kaplan and Radic, 2012; Matta et al., 2022). Traditionally, NETs are detected by using either anti-MPO or anti-citrullinated histone H3 (CitH3), or neutrophil elastase as coating antibody separately followed by anti-DNA detecting antibody (Zuo et al., 2020 and 2021). We have developed and validated a more sensitive ELISA, where we use a cocktail of anti-MPO and anti-CitH3 antibodies as coating antibodies, followed by anti-DNA as the detecting antibody. We further optimized the best blocking and substrate combination suited for increased sensitivity for ELISA. To the best of our knowledge, we are the first one to develop the smear assay to visualize NETs in small amounts of patient plasma. This method requires minimal time, reagents, specialized equipment, and/or cost. These techniques can be applied to detection of NETs in serum/plasma of patients with autoimmune diseases and other inflammatory conditions where NETs have been shown to play a crucial role. But apart from that, we propose that the application of these techniques can be extended to other bodily fluids as well, where accumulation of NETs has been shown to play a role in disease pathogenesis. For example, NETs can be quantified in the synovial fluid of joints to study rheumatoid arthritis (Corsiero et al., 2016) or excretion of NETs in urine in certain inflammatory conditions such as urinary tract infections (Yu et al., 2019). Since the smear assay requires only 1 μL of sample, this assay can be applied to samples that can be obtained only in smaller quantities; for example, NETs have shown to be involved in many ocular diseases such as glaucoma, age-related macular degeneration, dry eye, and diabetic retinopathy for which only a very small volume of ocular fluid can be obtained (Martínez-Alberquilla et al., 2022). Another example is bronchiolar lavage, where NETs have been shown to be elevated in patients with pneumonia-related acute respiratory distress syndrome (Bendib et al., 2019), primary graft dysfunction after lung transplantation (Sayah et al., 2015), and COVID-19 (Yaqinuddin et al., 2020). Moreover, these techniques can also be applied to pediatric autoimmune and inflammatory diseases such as pediatric lupus, where there are limitations on sample volume (Garcia-Romo et al., 2011).
Materials and reagents
Poly-L-Lysine adhesive microscope slides (Newcomer Supply, catalog number: 5010)
ImmulonTM MicrotiterTM 96-well plates (Thermo Scientific, catalog number: 3855)
Mouse monoclonal antibody [2C7] to myeloperoxidase (MPO) (Abcam, catalog number: 25989)
Rabbit polyclonal antibody to histone H3 (anti-CitH3 citrulline R2 + R8 + R17) (Abcam, catalog number: 5103)
Sytox Green nucleic acid stain (Invitrogen, catalog number: S7020)
Cell Death Detection ELISA PLUS Anti-DNA POD kit (Roche, catalog number: 11774425001)
Goat anti-mouse Alexa Fluor 488 (2 mg/mL) (Invitrogen, catalog number: A11029)
Goat anti-rabbit Alexa Fluor 594 (2 mg/mL) (Molecular Probes, catalog number: A11012)
DAPI (4’,6-Diamidino-2-Phenylindole, dihydrochloride) (Sigma-Aldrich, catalog number: D9542-1MG)
TMB (3,3’,5,5’-tetramethylbenzidine) enhanced one component HRP membrane substrate (Sigma-Aldrich, catalog number: T9455)
2N sulfuric acid stop solution (Reagents, catalog number: CS106300-500A)
Normal rat serum control (Fisher Scientific, catalog number: 10-710-C)
Tween 20 (Thermo Fisher, catalog number: J20605.AP)
Triton X-100 (Thermo Fisher, catalog number: HFH10)
Bovine serum albumin (BSA) (Fisher catalog number: BP9703-100)
Formaldehyde (Sigma, catalog number: 252549)
Na2CO3 (Sigma, catalog number: 497-19-8)
NaHCO3 (Sigma, catalog number: 144-55-8)
VectaMount AQ mounting medium (Vector Labs, catalog number: H-5501)
Dulbecco’s Phosphate Buffer saline (PBS without calcium and magnesium chloride (Sigma, catalog number: D8537-500ML)
Wash buffer (see Recipes)
Blocking buffer (see Recipes)
Dilution buffer (see Recipes)
Coating buffer (see Recipes)
Smear dilution buffer (see Recipes)
Smear blocking buffer (see Recipes)
Fixing buffer (see Recipes)
Equipment
Pipettes, multi-channel pipettes for washings and tips
Synergy Neo2 Multi-Mode microplate reader (BioTek, model: BTNEO2)
EVOS M7000 imaging system (Invitrogen, catalog number: AMF7000)
ZEISS Confocal LSM 880 Airyscan
NanoDrop 2000 (Thermo Fisher Scientific, catalog number: ND-2000)
Software
ImageJ—Java-based program (V1.8) (https://imagej.nih.gov/ij/download.html)
GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/)
MyAssays online program (https://www.myassays.com/download-and-install-myassays-desktop.html)
Procedure
NETs ELISA
Coat 96-well ultra-high-binding plates with 50 μL of anti-MPO and anti-CitH3 unlabeled primary antibodies diluted to a concentration of 5 μg/mL each in coating buffer. Cover the plate with the plate sealer. Leave plate overnight at 4 °C.
The next morning, wash wells three times with 200 μL of PBS at room temperature.
Block plate with 200 μL of blocking buffer for 2 h at room temperature.
Wash the plate five times with 200 μL of PBS.
Add 50 μL of undiluted plasma sample to each coated well and incubate at room temperature for 2 h. Set up wells for blanks where no plasma samples are added.
Carefully wash the plate five times with 200 μL of wash buffer; do not invert plate during washes.
Add 50 μL of anti-DNA-POD antibody (from Cell Death Detection ELISA PLUS Anti-DNA POD kit) prepared 1:100 in dilution buffer to each well and incubate at room temperature for 2 h for the detection of NETs.
Wash the plate five times with 200 μL of wash buffer.
Add 100 μL of TMB (allow to come to room temperature before using) to each well and incubate plate in the dark for 3 min.
Add 100 μL of 2 N sulfuric acid stop solution to each well and measure absorbance using a Synergy Neo2 Multi-Mode microplate reader, or any ELISA microplate reader set to a wavelength of 450 nm.
Smear assay
Draw a circle using a histology liquid repellent pen on a poly-L-Lysine glass slide (2–3 circles per slide).
Drop 1 μL of plasma sample to each circle and smear in a circular motion until the volume is equally distributed. Let plasma completely dry.
Fix samples with 100 μL of smear fixing buffer (4% formaldehyde) for 10 min.
Carefully tilt the slide to remove the liquid to be collected in the formaldehyde waste container, dab the sides of the slides with a paper towel, and carefully wash each circle three times with 100 μL of PBS.
Block the slides with 100 μL of smear blocking buffer (5% BSA/PBS) for 1 h at room temperature.
Add 50–100 μL of anti-MPO and anti-CitH3 primary antibodies diluted to smear dilution buffer (1 μg/mL in 0.3% Triton X-100 + 0.1% BSA solution) to each circle and leave overnight in a moist slide chamber.
The following morning, wash slides three times with PBS and add 50–100 μL of goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 secondary antibodies diluted 1:500 in dilution buffer to each circle and incubate in the dark for 1 h.
Wash three times with 200 μL of PBS and stain slides with 5 mg/mL of DAPI diluted 1:200 in PBS for 15 min in the dark.
In another set of slides for quick nonspecific visualization, after fixing and blocking, NETs can be stained directly with nuclear dyes Sytox Green diluted 1:200 and 5 mg/mL of DAPI diluted 1:200 in PBS for 15 min in the dark.
Wash an additional three times with 200 μL of wash buffer after staining. Add a drop of VectaMount AQ mounting medium and secure a glass coverslip onto the slide making sure that there are no bubbles.
Allow the slides to dry before imaging on EVOS M7000 imaging system and/or ZEISS Confocal LSM 880.
Stain secondary antibody–only controls the similar way, except that no primary antibodies are added.
Data analysis
For ELISA data analysis, a standard curve is prepared using serial dilutions of three pooled phorbol 12-myristate 13-acetate (PMA)-stimulated healthy donor plasma samples. DNA is quantified using the NanoDrop 2000. NETs in serially diluted PMA-induced plasma samples were quantified using CitH3 and MPO + CitH3 ELISA methodology. Four-parameter logistic standard curves were created for each antibody coating, and data were extrapolated using MyAssays online software. A detailed protocol can be found in the published manuscript (Matta et al., 2022) in methodology under section “Preparing in-house standard for the quantification of NET remnants by ELISA.”
For quantification of NETs in smear assay we used ImageJ Java-based program (V1.8) to convert the images to grayscale 8-bit images, whose intensities were then quantified and graphed. A detailed protocol can be found in the published manuscript (Matta et al., 2022) in methodology under section “NET Quantification using ImageJ.”
All statistical analyses should be performed and graphed using GraphPad Prism 8. A two-tailed parametric t-test should be used for comparisons between samples with normal distribution. P < 0.05 should be considered statistically significant. A two-tailed correlation analysis should be performed using Pearson correlation coefficients assuming Gaussian distribution.
Notes
There is a disparity between ELISA and smear assay when using fresh vs. frozen plasma samples. ELISA can detect NETs in circulation from samples stored at -80 °C for long periods of time (> 6 months), as it can detect both intact and fragmented NETs. But as the smear assay is visualizing intact NETs, we found that NET structures are generally maintained in plasma samples up to six months when stored at -80 °C. Beyond six months, we could detect fragmented NETs with smear assay that compromised their quantification. According to our preliminary studies, we suggest adding EDTA/EGTA immediately after plasma isolation to prolong the stability of intact NET structures for quantification. Although we used the anti-DNA-POD antibody from Cell Death Detection ELISA PLUS Anti-DNA POD kit for the detection of NETs in ELISA, we believe that any other anti-DNA-peroxidase-conjugated antibody can be used for detection.
Recipes
Wash buffer
1% BSA + 0.05% Tween 20 in PBS
Blocking buffer
5% BSA + 5% normal rat serum in PBS
Dilution buffer
5% BSA + 0.05% Tween 20 in PBS
Coating buffer
15 mM Na2CO3, 35 mM NaHCO3, pH 9.6
To 50 mL of deionized water, add 80 mg of Na2CO3 and 150 mg of NaHCO3
Smear dilution buffer
0.3% Triton X-100 + 0.1% BSA in PBS
Smear blocking buffer
5% BSA in PBS
Fixing buffer
4% formaldehyde in PBS
Acknowledgments
This work was supported by grants from the National Institutes of Health NIAMS 1 R01 AR 076242-03, Department of Defense CDMRP LRP W81XWH-18-1-0674, and Lupus Research Alliance to BJB.
We would also like to thank members of “The NETwork to Target Neutrophils in COVID-19” for inspiration to develop new methodologies to detect NETosis.
This protocol was derived from the original work of Matta et al. (2022).
Graphical overview made with the help of https://biorender.com/.
Competing interests
The authors declare no conflicts of interest.
Ethics
The studies involving human participants were reviewed and approved by The Feinstein Institutes for Medical Research IRB. The patients/participants provided their written informed consent to participate in this study.
References
Bendib, I., de Chaisemartin, L., Granger, V., Schlemmer, F., Maitre, B., Hue, S., Surenaud, M., Beldi-Ferchiou, A., Carteaux, G., Razazi, K., et al. (2019). Neutrophil Extracellular Traps Are Elevated in Patients with Pneumonia-related Acute Respiratory Distress Syndrome. Anesthesiology 130(4): 581-591.
Bruschi, M., Bonanni, A., Petretto, A., Vaglio, A., Pratesi, F., Santucci, L., Migliorini, P., Bertelli, R., Galetti, M., Belletti, S., et al. (2020). Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis. J Rheumatol 47(3): 377-386.
Corsiero, E., Pratesi, F., Prediletto, E., Bombardieri, M., and Migliorini, P. (2016). NETosis as source of autoantigens in rheumatoid arthritis. Front immunol 7: 485.
Garcia-Romo, G. S., Caielli, S., Vega, B., Connolly, J., Allantaz, F., Xu, Z., Punaro, M., Baisch, J., Guiducci, C., Coffman, R. L., et al. (2011). Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med 3(73): 73ra20.
Kaplan, M. J. and Radic, M. (2012). Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol 189(6): 2689-2695.
Lande, R., Ganguly, D., Facchinetti, V., Frasca, L., Conrad, C., Gregorio, J., Meller, S., Chamilos, G., Sebasigari, R., Riccieri, V., et al. (2011). Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med 3(73): 73ra19.
Martínez-Alberquilla, I., Gasull, X., Pérez-Luna, P., Seco-Mera, R., Ruiz-Alcocer, J. and Crooke, A. (2022). Neutrophils and neutrophil extracellular trap components: Emerging biomarkers and therapeutic targets for age-related eye diseases. Ageing Res Rev 74: 101553.
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. Front immunol 13:951254.
Sayah, D. M., Mallavia, B., Liu, F., Ortiz-Munoz, G., Caudrillier, A., DerHovanessian, A., Ross, D. J., Lynch, J. P., 3rd, Saggar, R., Ardehali, A., et al. (2015). Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med 191(4): 455-463.
Yaqinuddin, A., Kvietys, P. and Kashir, J. (2020). COVID-19: Role of neutrophil extracellular traps in acute lung injury. Respir Investig 58(5): 419-420.
Yu, Y., Kwon, K. and Pieper, R. (2019). Detection of Neutrophil Extracellular Traps in Urine. Methods Mol Biol 2021: 241-257.
Zuo, Y., Yalavarthi, S., Shi, H., Gockman, K., Zuo, M., Madison, J. A., Blair, C., Weber, A., Barnes, B. J., Egeblad, M., et al. (2020). Neutrophil extracellular traps in COVID-19. JCI Insight 5(11): e138999.
Zuo, Y., Zuo, M., Yalavarthi, S., Gockman, K., Madison, J. A., Shi, H., Woodard, W., Lezak, S. P., Lugogo, N. L., Knight, J. S., et al. (2021). Neutrophil extracellular traps and thrombosis in COVID-19. J Thromb Thrombolysis 51(2): 446-453.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Immunology > Immune cell function > Neutrophil
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Published: Vol 13, Iss 12, Jun 20, 2023
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Original Research Article:
The authors used this protocol in Veterinary Parasitology Oct 2021
Abstract
The nematode Haemonchus placei is a pathogenic parasite, the most seriously affecting ruminant’s health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) from H. placei transitory infective larvae (xL3). ESP from xL3 were obtained from the in vitro infective larvae (L3) maintained in Hank’s medium at 37 °C with 5% CO2 for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays.
Graphical overview
Keywords: Haemonchus placei Nematode Secreted-excreted products Cell proliferation assays Relative gene expression
Background
Domestic ruminants are severely affected by the complex of gastrointestinal nematodes (GINs). Some parasites belonging to this group (i.e., Ostertagia and Haemonchus) are particularly highly pathogenic due to their blood-feeding habits. The genus Haemonchus spp. is the most pathogenic parasitic nematode, particularly in tropical regions where its prevalence reaches > 80% (Liébano-Hernández et al., 2011). Traditionally, the control of GINs is based on the use of anthelmintic drugs; however, anthelmintic resistance has dramatically increased during the last decades, risking animal health and production. In order to reduce the use of chemical anthelmintic drugs, different strategies of control and prevention have been explored. One of them is assessing the use of specific proteins as potential immunizing agents, mainly those that are involved in the host–parasite interaction. The genus Haemonchus has been used as a biological model for understanding the invasive process to the host tissues. During invasion of the gastric mucosa tissues, different excretory and secretory products (ESP), mainly proteins, are produced; some of them could have important implications as potential immune protective antigens. The in vitro development from Haemonchus spp. infective larvae (L3) to the transitory infective larvae (xL3) is an easy and economic technique that allows the release of potential antigenic molecules with important immunoprotective value. The invasion mechanism of larvae to the host tissue and their further development require the activity of enzymes to use nutrients and thus achieve the adult stage for surviving in the abomasum (stomach) of ruminants. Recently, numerous studies have intensified the identification of ESP to induce immune protection, thus hampering the larvae’s invasive process. In addition, the importance of ESP during tissue invasion by parasitic nematodes is associated to inflammatory and regulatory immune mechanisms such as cytokines (e.g., IL4, IL5, IL6, IL8, IL10), immunoglobulins (IgA, IgG, and IgE), and other important immune cells like eosinophils and neutrophils. The study of antigens and immune cells requires the use of different experimental in vitro and in vivo processes, to improve the identification of ESP with possible biological functions. The present methodology describes reproducible experimental procedures for obtaining and studying the nematode’s ESP and the host’s immune response. The following protocol describes methods related to parasitology, immunology, and molecular procedures to obtain ESP from xL3of the nematode speciesHaemonchus placeiand to evaluate them in vitro activity.
Materials and reagents
T-75 cell culture flask (Thermo Fisher, catalog number: 156800)
Gauze (any brand can be used)
McMaster egg counting chamber (JorVetTM, catalog number: J0335M)
Plastic container, 18 cm × 38 cm (any brand can be used)
Foam rubber (any brand can be used)
Aluminum foil (any brand can be used)
Distilled water (any brand can be used)
Polypropylene funnel 150 mm diameter (Corning®, catalog number: 6120P-150)
10 mL culture tube (Pyrex®, catalog number: 99445-13)
Plastic hose assembly (Fisher Scientific, catalog number: 02-594-1F)
Pasteur plastic pipette, 1 mL (MediLab, catalog number: 121227/01)
Optical lens wipes (Carolina Biol. Supply Co., catalog number: 634000)
15 mL plastic centrifuge tube (Corning®, catalog number: 430052)
50 mL plastic centrifuge tube (Corning®, catalog number: 430828)
6-well plate, flat-bottom, clear and sterile, TC-treated (Corning®, catalog number: 3506)
Petri dishes 100 mm × 15 mm, sterile (Falcon®, catalog number: 351029)
Diameter syringe filters 0.2 μm pore (Corning®, Catalog number: 431219)
Vacutainer tubes with EDTA (Becton Dickinson, catalog number: 367863)
Hypodermic green needles, 21 G × 32 mm (Nipro®, catalog number: A21)
Pasteur pipettes (Sigma, catalog number: S5893)
0.2 mL PCR Tubes with flat cap (Axygen®, catalog number: PCR-02-C)
96 well-microplates, flat-bottom, clear and sterile, TC-treated (Corning®, catalog number: 3628)
24-well plate, flat-bottom, clear and sterile, TC-treated (Corning®, catalog number: 3527)
1.5 mL microcentrifuge plastic tuber (Axygen®, catalog number: MCT-150-A)
Plastic pestle (Bel-ArtTM, catalog number: F19923-0001)
Recently collected fecal samples from infected cattle withH. placei(300–500 g)
Sodium chloride (NaCl) of commercial food grade (any brand can be used)
Sucrose (Sigma-Aldrich, catalog number: MFCD00006626)
Sodium hypochlorite (NaClO) (Cloralex-Grupo AlEn-México)
Antibiotic-antimycotic (100×) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062)
Hanks’ Balanced Salts without sodium bicarbonate (Sigma, catalog number: H6136)
Ethanol molecular grade (Hycel, catalog number: 1822-500)
Peripheral blood sample from three non-infected cattle (36 mL)
LymphoprepTM (Alere Technologies, Axis-Shield, catalog number: 1114547)
RPMI 1640 medium with L-glutamine and HEPES (Gibco, catalog number: 23400062)
Fetal bovine serum (FBS) (By Products, catalog number: 90020)
Trypan blue stain (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061)
Neubauer Improved bright line (MARIENFELD, catalog number: 0640011)
CellTiter 96® Aqueous One Solution Cell Proliferation assay (Promega, catalog number: G3582)
QIAzol lysis reagent (Qiagen®, catalog number: 79306)
Chloroform (J.T. Baker, catalog number: 9180)
Isopropyl alcohol (Sigma-Aldrich, catalog number: W292907)
Nuclease-free water (Promega, catalog number: P1193)
Agarose (Bio-Rad, catalog number: 1613100)
Ethidium bromide 10 mg/mL (Bio-Rad, catalog number: 1610433)
RQ1 RNase-Free DNase kit (Promega, catalog number: M6101)
ImProm-II Reverse Transcription System kit (Promega, catalog number: A3800)
GoTaq® qPCR Master Mix 2× (Promega, catalog number: A6001)
Custom RT2 Profiler PCR array (Qiagen, catalog number: CAPB13410R)
Trizma base (Sigma, catalog number: 93362)
Glacial acetic acid (Meyer, catalog number: 0040)
EDTA 0.5 M pH 8.0 (Invitrogen, catalog number: AM9260G)
Potassium chloride (KCl) (Sigma, catalog number: P3911)
Sodium phosphate dibasic (Na2HPO4) (Sigma, catalog number: RDD038)
Potassium phosphate monobasic (KH2PO4) (Sigma, catalog number: 221309)
1× sterile phosphate buffered saline (PBS), pH 7.4 (see Recipes)
Buffer 50× TAE (see Recipes)
3% agarose gel (see Recipes)
0.187% NaClO solution (see Recipes)
Hank’s balanced salts (medium) (see Recipes)
40% sucrose solution (see Recipes)
Saturated sodium chloride solution (see Recipes)
Equipment
Centrifuge (Thermo Fisher Scientific, Sorvall ST 8R, catalog number: 75007204)
Microscopy (Zeizz, Primo Star Hal/Led Full-Köhler ERc5s, catalog number: 415500-0057-000)
0.1–10 μL, 2–20 μL, 20–200 μL, and 100–1,000 μL micropipettes (any brand can be used)
Shaker (Labnet, OrbitTM 1000, catalog number: S2030-1000-B)
Incubator (ECOSHEL, CI-80, CO2incubator)
Mixer (Benchmark Scientific, VortexTM, catalog number: BV101-P)
Microplate absorbance reader (Bio-Rad, iMarkTM, catalog number: 1681135)
Nanophotometer (Implen, NP80)
PowerPAc chamber (Bio-Rad, 300, catalog number: 165-5050)
Thermal cycler 6000 (Qiagen, Corbett Rotor-Gene 6000)
Transilluminator Imagine System (UVP, EC3, catalog number: 81-020901)
Nucleic acid electrophoresis chamber (any brand can be used)
Granatary scale 610 × 0.1 g (any brand can be used)
Freezer of -80 °C (any brand can be used)
pH meter (any brand can be used)
Software
Rotor-Gene Q—Pure detection Software (version 1.7)
GeneGlobe Data Analysis Center of Qiagen® (https://geneglobe.qiagen.com/analyze/)
Procedure
Obtaining H. placei L3 antigens
Recovery of H. placei’s L3 stages using parasitological techniques:
Note: Basic knowledge of copro-parasitological techniques and at least one isolated H. placei strain is required to perform this methodology. Any further information can be consulted in Thienpont et al. (2003).
McMaster technique: Perform the parasitological diagnosis of infected bovine with H. placei by McMaster technique to estimate the number of eggs per gram (EPG) from fecal samples (Liébano-Hernández et al., 2011; Cedillo-Borda et al., 2020).
i. Collect 10 g of fecal samples from infected bovine by rectal route using a clean plastic bag. Using nitrile gloves, introduce the index finger into the anal sphincter of the bovine, performing circular movements in a clockwise direction. The rectal stimulus will allow the transit of the feces from the rectum, until they cross through the anal sphincter. Collect the expelled feces with a plastic bag.
ii. Weigh 2 g of feces on a granatary scale and place them in a 50 mL plastic tube.
iii. Add 28 mL of saturated sodium chloride solution at 1:24 density (see Recipes) and homogenize the feces with a plastic paddle. Put a piece of gauze (5 cm × 5 cm) on the surface of the homogenized feces.
iv. Take out 5 mL of the homogenized sample through the folds formed in the introduced gauze with a Pasteur pipette. Then, fill both compartments of the McMaster chamber.
v. Perform egg count through the McMaster chamber. Consider the number of EPG into the McMaster chamber lines from top to bottom and from left to right between chambers.
vi. Quantify the total EPG using the formula shown in Figure 1.
Figure 1. Representative figure of McMaster technique
Coproculture and Baermann technique (Liébano-Hernández et al., 2011; Cedillo-Borda et al., 2020):
i. Collect bovine fecal samples directly from the rectum (from 300 to 500 g) using nitrile gloves as indicated in step A.1.a.i.
ii. Place the fecal sample in a plastic container (38 cm diameter by 18 cm depth), add distilled water (dH2O) at 25 °C to cover the sample (~350 mL), and macerate the feces with a pistil (20 cm long × 5 cm wide) until obtaining a paste.
iii. Add foam rubber and homogenize with the fecal sample. Cover with aluminum foil and incubate at room temperature (25 °C) for seven days. Mix the feces culture every 48 h to ease the development from egg to L3.
iv. After seven days, place 100 g of fecal/foam tuber mixture in a piece of non-sterile gauze (15 cm × 15 cm) and wrap in a ball.
v. Place each gauze ball using the hands into a Baermann funnel (plastic funnel of 14 cm diameter connected with PVC hose to a 10 mL tube) and add dH2O (at 25 °C) to cover the gauze ball.
vi. After 12 h, remove all tubes with the larvae on the bottom and store them at 4 °C for 2 h to precipitate the L3.
vii. Remove the supernatant using plastic Pasteur pipettes and recover the L3 pellets.
viii. Mix all the L3in new plastic tubes. Taking advantage from the hydrotropism and phototropism of the L3, filter the L3using the optical lens paper over the Baermann funnel for their precipitation (Thienpont et al., 2003).
ix. After 24 h at room temperature, remove the tubes from the Baermann funnels, recover L3 at the bottom of the tube using a Pasteur pipette (3 mL), and place the pellet in a T-75 cell culture flask with 15 mL of dH2O. Store in a refrigerator at 4 °C (Figure 2).
Figure 2. Representative figure of the fecal culture and Baermann technique
Cleaning of H. placei L3 by density gradient and centrifugation (Liébano-Hernández et al., 2011; Cedillo-Borda et al., 2020):
i. Centrifuge the L3 at 1,000× g for 3 min at room temperature. Discard the supernatant and add 2 mL of dH2O.
ii. Add 6 mL of 40% sucrose solution (see Recipes) in a 15 mL plastic tube.
iii. Place carefully the L3 pellet in the tube containing the sucrose solution. Centrifuge the plastic tube at 1,000× g for 5 min.
iv. A larval ring will be formed in the plastic tube. Carefully transfer the larval ring (maximum 2 mL) to a new plastic tube using a glass Pasteur pipette.
v. Add 10 mL of dH2O and centrifuge at 1,000× g for 3 min at room temperature. Discard the supernatant and repeat this step twice.
vi. Suspend the larval pellet in dH2O. Store the L3 at room temperature or remove the second molt of L3 (Figure 3).
Figure 3. Schematic representation of L3 cleaning by density gradient
Removal of H. placei L3 sheath (Liébano-Hernández et al., 2011; Cedillo-Borda et al., 2020):
i. Centrifuge the L3 pellet at 1,000× g for 3 min. Discard the supernatant.
ii. Prepare 6 mL of 0.187% NaClO solution (see Recipes) in a 15 mL plastic tube. Homogenize L3 from 5 to 10 min.
iii. Confirm the elimination of H. placei L3 sheath by observing a 10 μL aliquot under an optical microscope (10×).
iv. Add 7 mL of dH2O and centrifuge at 1,000× g for 3 min. Discard the supernatant and repeat this step twice or more.
v. Suspend the unsheathed larval pellet in dH2O (Figure 4).
Figure 4. Representation of the technique for removing H. placei L3 sheath
Obtaining excreted-secreted products (ESP) from H. placei:
Note: Count the third-infective larvae considering 10 aliquots of 5 μL. Then, make a conversion taking the total volume from the aliquots and the final volume of the larvae stock.
Example:
50 μL (total aliquots volume) = 300 L3 (counted larvae)
25 mL (final volume) = X (Total larvae stock) = 150,000 L3
Obtaining ESP from in vitro culture (Figure 5)
i. Add 18,000 unsheathed L3 to a 50 mL tube with 20 mL of 1× PBS (see Recipes) supplemented with 1% antibiotic-antimycotic 100×.
ii. Then, place the 50 mL tube with L3 vertically and incubate for 30 min at room temperature.
iii. Centrifuge the L3 at 1,000× g for 5 min. Remove the supernatant and recover the L3 at the bottom of the tube.
iv. Place 3,000 L3 in a 6-well plate with 3 mL of Hank’s balanced salt medium (see Recipes) per well.
v. Incubate the 6-well plate at 37 °C with 5% CO2 for five days, in order to stimulate and collect the ESP into the culture medium.
vi. Collect 1 mL per well of the supernatant from the plate; the first collection of ESP should be at 16 h and then every 24 h. Replace the volume removed with new Hank’s medium.
ESP concentration and confirmation
i. Centrifuge the ESP from H. placei xL3 at 12,000× g for 40 min at 4 °C to recover the supernatant containing the ESP; then, filter the ESP using 0.2 μm syringe filters and store at -80 °C until use.
Figure 5. Schematic representation of obtaining excreted and secreted products from H. placei
ii. Confirm ESP through SDS-PAGE (Sambrook and Russell, 2001) at 5%–12% and protein estimation (Bradford, 1976) (Figure 6).
Figure 6. Summary of SDS-PAGE assays
Peripheral blood mononuclear cells (PBMCs) proliferation and relative gene expression assays
Obtaining PBMCs:
Blood samples
i. Select three young cattle (male or female) of similar age (between six to eight months) and housed under worm-free conditions.
ii. Identify the jugular vein and clean the puncture site with dH2O and 70% alcohol.
iii. Collect ~12 mL of blood from each bovine in EDTA tubes and mix by inverting the tubes repeatedly (Figure 7).
Figure 7. Representative figure of PBMCs isolation
PBMCs isolation (protocol modified from human mononuclear cells)
i. Dilute each blood sample in PBS 1× pH 7.4 in a 1:1 proportion (3 mL of blood sample to 3 mL of PBS) and mix slowly by inversion to avoid the lysis of PBMCs.
ii. Place 3 mL of Lymphoprep reagent in a sterile 15 mL plastic tube and carefully add 6 mL of mixed blood/PBS. Avoid mixing the Lymphoprep reagent with the mixed blood/PBS by placing the tip of the micropipette on the side of the tube close to the Lymphoprep reagent and carefully adding the mix. Two phases should be seen.
iii. Centrifuge at 800× g for 20 min at room temperature in a Sorvall ST 8R centrifuge with oscillating rotor and slow deceleration. A white PBMCs ring will be formed in the plasma/Lymphoprep reagent interface.
iv. Remove the white PBMCs ring using a 3 mL glass Pasteur pipette and transfer all cells in 15 mL tubes with PBS. Immediately dilute the PBMCs with PBS 1× pH 7.4 in a 1:2 proportion and homogenize the mix slowly.
PBMCs quantification
i. Determine the volume of PBMCs suspension in RPMI 1640+HEPES supplemented media, according to the total number of flat-bottom plates per trial, including replicates of each treatment. Mix the PBMCs suspension carefully by inversion. Consider that each well should contain 50 μL as final volume if using 96-well plates.
ii. Dilute 45 μL of 0.4% trypan blue and 5 μL of PBMCs (1:10 proportion) in a 0.2 mL tube and mix slowly using a glass Pasteur pipette.
iii. Place 20 μL of PBMCs/trypan blue mix into a Neubauer chamber and quantify under an optical microscope (40×).
iv. Count the cells using four quadrants (Figure 8). Cells localized on the outer margins of each corner should not be included. Quantify dead (blue) and living cells (white) separately in each quadrant.
Figure 8. Schematic figure of the PBMCs quantification
v. Obtain the PBMCs concentration per milliliter using the following formula:
Proliferation assays and H. placei ESP from larval stages:
Proliferation assays
Notes:
1) Use a concentration from 2.5 to 5 μg/mL of positive controls (i.e., phytohemagglutinin, lipopolysaccharide, or Concanavalin A) and from 0.02 to 2 μg/mL for ESP suspended with culture medium. Untreated cells as negative controls are required, and a final volume of 100 μL should be adjusted per treatment.
2) An evaluation with different periods of incubation post-treatment (i.e., 6, 12, 24, 48, and 72 h) is recommended.
3) Perform at least three repetitions and replicates from each donor animal.
i. Place 300,000–500,000 PBMCs containing 50 μL of medium per well in flat-bottom 96-well plates. Add 50 μL of serial dilutions (previously prepared) of each treatment (e.g., controls and ESP). Incubate the 96-well plates at 37 °C with 5% CO2 (Figure 9).
ii. To count the PBMCs proliferation, add 20 μL of CellTiter 96® Aqueous One Solution to each well. Incubate at 37 °C with 5% CO2 for 2 h.
iii. Cell quantification is carried out at 490 and 690 nm using a microplate reader for data analysis.
Figure 9. Proliferation assays representation
Data analysis of proliferation assays
i. Subtract the absorbance of 490 nm from the 690 nm value. This data will allow the elimination of background from the excess of cell debris, fingerprints, and other non-specific absorbance.
ii. Obtain the proliferation values through the normalization of untreated cells, expressed as percentage, as follows:
Relative gene expression method and analysis of immune genes:
Notes:
1) Perform the study with at least three repetitions per replicate of each donor animal.
2) Determine ESP concentrations according to the proliferation assays.
3) Evaluate different periods of incubation post-treatments (i.e., 6, 12, 24, 48, and 72 h).
4) The evaluation of immune genes activated by ESP using relative gene expression requires untreated cells as negative controls.
RNA purification (protocol modified from TRizolTM):
i. Place from 3 × 106 to 5 × 106 PBMCs in a 24-well plate containing 1.5 mL of RPMI 1640 + HEPES culture medium per well. Add each treatment—ESP or control groups (negative and positive)—using a micropipette. Incubate at 37 °C with 5% CO2.
ii. With a micropipette, collect the stimulated PBMCs from each well treated after incubation. Place the cells in 1.5 mL plastic tubes and centrifuge at 250× g for 5 min at room temperature. Remove the supernatant.
iii. Add 1 mL of TRizol reagent to 1.5 mL tubes with PBMCs and homogenize slowly at 4 °C with plastic pestle.
iv. Mix the samples using a vortex and incubate for 5 min at room temperature.
v. Add 0.2 mL of cold chloroform per 1 mL of TRizol reagent and mix by inverting the tubes repeatedly for 15 s. Incubate for 5 min at room temperature.
vi. Centrifuge samples at 12,000× g for 20 min at 4 °C. Transfer the aqueous phase corresponding to the RNA to a new tube. This phase will have a transparent appearance.
vii. Add 0.5 mL of isopropyl alcohol to the aqueous phase and mix slowly. Incubate for 15 min at room temperature.
viii. Centrifuge at 12,000× g for 15 min at 4 °C. Total RNA will remain as a pellet at the bottom of the tube.
ix. Carefully discard the supernatant with a micropipette or by decantation.
x. Suspend the RNA with 75% of cold ethanol.
xi. Homogenize the samples using a vortex and centrifuge at 7,500× g for 5 min at 4 °C. Discard the supernatant with a micropipette.
xii. Leave the RNA to air dry for 20 min inside the fume hood.
xiii. Add 30 μL of RNase-free water by carefully pipetting.
xiv. Estimate the RNA purity and concentration using a Nanophotometer at a ratio A260/A280 value. Set 1–2 μL of nuclease-free water as blank and then a similar quantity of RNA. Use a 0.5–10 μL micropipette. The result corresponds to the RNA sample purity between 1.8–2.0 values, where lower ratios indicate contamination with proteins.
xv. Confirm the integrity of RNA by setting a volume of 8 μL on electrophoresis with a 3% agarose gel (see Recipes) and carry out at 60 V for 30 min in buffer 1× TAE (see Recipes). Then, visualize the RNA integrity (28S and 18S rRNA) using a transilluminator Imagine System (Sambrook and Russell, 2001; Cedillo-Borda et al., 2020).
RNA decontamination:
i. Perform RNA decontamination in 0.2 mL plastic tubes using the reaction DNase as follows (Table 1):
Table 1. Components of the RNA decontamination reaction
Component Volume (μL)
RNA in water (300 ng is recommended) 3
RQ1 RNase-Free DNase 10× Reaction Buffer 1
RQ1 RNase-Free DNase 1
Nuclease-free water 5
ii. Incubate at 37 °C for 30 min.
iii. Add 1 μL of RQ1 DNase Stop Solution and incubate at 65 °C for 10 min to stop the reaction.
Reverse Transcription reaction (protocol modified from ImProm-II Reverse Transcription System Kit®):
i. Add 1 μL of random primers and incubate at 70 °C for 10 min. Immediately place the tube with the sample on ice.
ii. Working on ice, add the following components (Table 2) to the same PCR tube.
Table 2. Components for the reverse transcription reaction
Component Volume (μL)
imProm-IITM 5× Reaction Buffer 4
MgCl2(final concentration 1.5–8.0 mM) 2
dNTP mix (final concentration 0.5 mM each dNTP) 1
Recombinant RNasin® Ribonuclease Inhibitor 0.3
ImProm-IITM Reverse Transcriptase 1
Nuclease-free water (to 15 μL) 6.7
iii. Incubate at 25 °C for 5 min, at 42 °C for 60 min, and then at 70 °C for 15 min.
qPCR assay:
i. Perform qPCR assays using the commercial custom RT2 Profiler PCR and NCBI primer BLAST. The genes are selected from the National Centre of Biotechnology (https://www.ncbi.nlm.nih.gov/) show in Table 3.
Table 3. Gene information used in the PCR design
Custom PCR array design (CAPB13410R)
Genes GenBank Access
IL2 NM_180997
IL4 NM_173921
IL5 NM_173922
IL6 NM_173923
IL8 NM_173925
IL10 NM_174088.1
IL13 NM_174089.1
IFNγ NM_174086.1
FCεR1A NM_001100310.1
TGFβ1 NM_001166068.1
Housekeeping design using the NCBI Primer BLAST
Genes GenBank Access
β-2 microglobulin
Fw
Rv
XM_002691119.4
CCATCCAGCGTCCTCCAAAGATTC
CTGCTCCGATTTAATCTTCTCCCCA
β-actin
Fw
Rv
XM_027528015.1
CATCGCGGACAGGATGCAGAAA
CCTGCTTGCTGATCCACATCTGCT
gapdh
Fw
Rv
NM_001190390.1
TTGTCTCCTGCGACTTCAACAGCG
CACCACCCTGTTGCTGTAGCCAAAT
ii. Perform the PCR assay using the reagents indicated in Table 4 (Estrada-Reyes et al., 2017).
Table 4. Components of qPCR assays
Component Volume (μL)
cDNA (from 300 ng of RNA) 2
GoTaq® qPCR Master Mix 10.3
Nuclease-free water 12.68
iii. Place the commercial custom RT2 Profiler PCR in the Rotor-Gene 6000 and perform the PCR with the following conditions (Table 5):
Table 5. Conditions for real-time PCR
Cycles Stages Temperature (°C) Time
1 Initial denaturation 95 10 min
40 Denaturation 95 15 s
Annealing-elongation 60 45 s
1 Dissociation temperature 65–95 Rising ramp
Data analysis:
i. Obtain the threshold cycle (CT) from each gene using a threshold value of 0.05.
ii. Record the CTvalue in an Excel spreadsheet and analyze on the Qiagen® Gene Globe Data Analysis Center web platform, to normalize through the ΔΔCT method (Double delta CT).
iii. Enter https://geneglobe.qiagen.com/mx/analyze/ and select the PCR analysis tool option. A registered user account is required to access the web platform (Figure 10).
iv. Select and upload the data recorded in a datasheet with the CT values.
v. Select the control group, groups under study, and housekeeping genes. Select at least two housekeeping genes to the expression analysis.
Figure 10. Process to use the Qiagen® GeneGlobe Data Analysis Center web platform
vi. The Qiagen® Gene Globe Data Analysis Center platform shows the fold change values. Optionally, download graphs to represent the fold change values. Figure 11 shows an example of the relative expression interpretation.
Figure 11. Fold change values on the Qiagen® GeneGlobe Data Analysis Center web platform
Recipes
Note: All the solutions should be adjusted with HCl and NaOH 1 N.
Buffer 50× TAE
242 g of Trizma base (FW = 121.14)
57.1 mL of glacial acetic acid
100 mL of 0.5 M EDTA (pH 8.0)
Adjust the final volume to 1 L with deionized water.
3% agarose gel (size 10 cm × 7 cm)
1.06 g of agarose
45 mL of buffer 1× TAE
1.5 μL of ethidium bromide
0.187% NaClO solution
0.183 mL of NaClO
5.813 of distilled water
Hank’s balanced salts (medium)
49.2 mL (9.8 g/L) of Hanks’ medium
246 mL of antibiotic-antimycotic
8 μL of bovine erythrocyte (previously treated with VyM) (Rojas Martinez et al., 2016)
40% sucrose solution
20 g of sucrose
50 mL of distilled water
Phosphate buffered saline, pH 7.4
8 g of NaCl 137 mM
0.2 g of KCl 2.7 mM
1.44 g of Na2HPO4 10 mM
0.24 g of KH2PO4 2 mM
1,000 mL of distilled water
Saturated sodium chloride solution
400 g of NaCl
Adjust the final volume to 1 L with distilled water.
Acknowledgments
This protocol was derived from an original research paper (Maza-Lopez et al., 2021, https://doi.org/10.1016/j.vetpar.2021.109512), which received financial support from Consejo Nacional de Ciencia y Tecnología - Secretaría de Educación Pública (CONACYT-SEP, Grant number 287598).
Competing interests
The authors declare no conflict of interest.
Ethics
The criteria for care and handling of experimental animals were set forth in the Official Mexican Standard NOM-033-Z00-1995, NOM-051-ZOO-1995, and NOM-062-ZOO-1999.
References
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Estrada-Reyes, Z., López-Arellano, M. E., Torres-Acosta, F., López-Reyes, A., Lagunas-Martínez, A., Mendoza-de-Gives, P., González-Garduño, R., Olazarán-Jenkins, S., Reyes-Guerrero, D. and Ramírez-Vargas, G. (2017). Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after Haemonchus contortus infection. Parasite Immunol 39(6). doi: 10.1111/pim.12427.
Liébano-Hernández, E., López-Arellano, M. E., Mendoza-De-Gives G. P. and y Aguilar-Marcelino, L. (2011). Manual de diagnóstico para la identificación de larvas de nematodos gastrointestinales de rumiantes. Publicación Especial Vol. 2. México: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP): 1-44.
Maza-López, J., Contreras-Ochoa, C. O., Reyes-Guerrero, D. E., Encarnación-Guevara, S., Hernández-Ortíz, M., Olmedo-Juárez, A. and López-Arellano, M. E. (2021). Analysis of the immunomodulatory activity of excreted and secreted products from Haemonchus placei transition infective larvae (xL3). Vet Parasitol 298: 109512.
Rojas Martínez, C., Rodríguez-Vivas, R. I., Figueroa Millán, J. V., Acosta Viana, K. Y., Gutiérrez Ruiz, E. J. And Álvarez Martínez, J. A. (2016). In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite. Exp Parasitol 170: 214-219.
Sambrook, J. and Russel, D. W. (2001) Molecular cloning, a laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press. Vol. 1. P. pp. 5.4-5.13; Vol. 3. Appendix A8.40-A.849.
Thienpont, D., Rochette, F. and Vanparijs, O.F.J. (2003) Diagnosing helminthiasis by coprological examination. 3rd edition. Janssen Animal Health. P. 110. pp. 17-43.
Article Information
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© 2023 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
Immunology > Immune cell isolation > Leukocyte
Medicine
Molecular Biology > RNA > qRT-PCR
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4,703 | https://bio-protocol.org/en/bpdetail?id=4703&type=0 | # Bio-Protocol Content
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A Standardized Protocol for Early-life Stress-induced Social Defeat in Mice
ZY Zhi Yang
DW Denian Wang
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4703 Views: 649
Reviewed by: HSIU CHUN CHUANGMohammed Mostafizur RahmanJordi Boix-i-Coll
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Original Research Article:
The authors used this protocol in Cerebral Cortex Mar 2023
Abstract
Neuropsychiatric diseases, like depression, have a considerable and persistent impact on human health; however, little is known about their underlying pathogenesis. Social defeat is a model for stress-induced psychopathologies that could present with behaviors resembling those observed in humans with depression. However, previous animal models of social defeat mainly focus on adults. Here, we re-design the protocol of early-life stress-induced social defeat paradigm, which is based on a classic resident–intruder model. Briefly, each two-week-old experimental mouse of C57BL/6 strain is introduced into the home cage of an unfamiliar CD1 aggressor mouse for 30 min per day for 10 consecutive days. Later, all experimental mice are raised individually for another month. Finally, the mice are identified as defeated through social interaction and open field tests. This model has been shown to be etiological and predictive and provide high validity and could be a powerful tool to investigate the underlying pathogenesis of early onset depression.
Graphical overview
Keywords: Neuropsychiatric diseases Social defeat Early-life stress model Social interaction Stress-induced anxiety
Background
Mood and anxiety disorders have a growing prevalence, potentially affecting one out of four people during their lifetime (Anacker et al., 2018; Du et al., 2021). This could result in a great burden for the affected individuals, their families, and the society. Establishing high-validity animal models is of great importance for exploring these diseases (Becker and Wojtowicz, 2007; Anacker and Hen, 2017; Anacker et al., 2018). It has been reported that social stress plays a central role in the pathogenesis of different psychiatric disorders (Padilla-Coreano et al., 2016). Thus, scientific researchers have strived to establish animal models of social defeat that would potentially provide significant and basic information for understanding several of the physiological and behavioral abnormalities induced by social stress between individuals (Snyder et al., 2001; Golden et al., 2011; Dioli et al., 2017).
Several forms of animal models have been established, including learned helplessness, chronic unpredictable mild stress (CUMS), chronic restraint stress, chronic social defeat stress (CSDS), and others (Toyoda, 2017). Each protocol has its advantages and disadvantages. For example, the CUMS model is widely regarded as the most classic animal model of depression in use. One of its main advantages is its ability to avoid adaptive responses in animals caused by single and repetitive stimuli. However, this model also has some drawbacks, such as a lengthy modeling time and a large workload required. Our protocol is based on the CSDS model; briefly, it is established based on intermittent physical interaction between a dominant resident and a submissive intruder (Schloesser et al., 2010; Golden et al., 2011; Anacker et al., 2018).
Modifications have been made to the CSDS model according to different study aims, mainly regarding species and strains being chosen, acute or chronic exposure to stress, and the terminologies used for identifying social defeat (Anacker et al., 2016; Castilla-Ortega et al., 2016; Du et al., 2021). Here, we introduce a general procedure of a social defeat paradigm, with a focus on early-life stress. In order to enhance the aggression level of the resident mice, we chose older CD1 mice and younger C57BL/6 mice as aggressors and intruders, respectively. The C57BL/6 strain has been reported to have a greater metabolic susceptibility to social defeat stress; we chose male mice as they show territorial behavior more universally and stably than female mice. The behavioral features of social defeat are evaluated by subsequent social interaction test (SIT) and open field test (OFT). SIT is a simple test in which behaviors and staying time in the interaction and corner zones are video recorded. Compared to a normal C57 mouse, which is motivated to explore, a mouse subjected to social defeat would like to avoid the CD1 mouse in the wire mesh (Golden et al., 2011). OFT is a common measure of exploratory behavior and general activity of mice. The recording of outcomes like center time is likely to gauge some aspects of emotionality, including anxiety and depression. This model could be used for exploring specific molecular mechanisms of early-life stress-induced social defeat and depression and may open opportunities for the development of new antidepressant drugs.
Materials and reagents
C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd). Mice have free access to water and food on a 12:12 h light/dark cycle. Male and female mice were bred to generate pup litters for the experiments. In general, experiments are run in cohorts of 20–40 mice at one time.
Male CD1 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd). Mice were single housed with free access to water and food on a 12:12 h light/dark cycle. CD1 mice should be single housed for at least a week before aggression screening.
Surgical scissors and suture (RWD, catalog number: SP0009-M)
Isoflurane (RWD, catalog number: R510-22-10)
Cleaning solution (Oxytech, catalog number: D-50/500)
Equipment
Arena (Wuhan Yihong Technology Co., Ltd, catalog number: YH-OF-M/R): 48 cm × 48 cm × 40 cm with an empty wire mesh enclosure (10 cm × 10 cm × 15 cm) alongside the middle of one of the arena walls. The 10 cm wide area surrounding the wire mesh cage is called the social interaction zone; the opposing corners in the arena are called corner zones (Figure 1A).
Arena (Wuhan Yihong Technology Co., Ltd, catalog number: YH-OF-M/R): 48 cm × 48 cm × 40 cm with a center zone (20 cm × 20 cm) and four corner zones (10 cm × 10 cm) (Figure 1B)
Video recording equipment (EthoVision XT) (Figure 1C)
Stopwatch for timing
Figure 1. Schematic and physical objects of the social interaction arena (A), open field arena (B), and video-tracking equipment (C)
Software
EthoVision XT software (Noldus, https://www.noldus.com/ethovision-xt) or other video-tracking packages.
GraphPad Prism Software (version 7.0) or other statistical software.
Procedure
Aggression screening
Introduce a novel C57BL/6 mouse (8–16 weeks old) into the home cage of single-housed CD1 mouse (4–6 months old).
Record attack latencies of CD1 mice during a 3 min testing session.
Repeat the procedure for three consecutive days, using different C57BL/6 mouse for each CD1 mouse each day.
Select CD1 mice that attacked within 60 s for at least two consecutive days as aggressors.
Resect the gonads of selected CD1 aggressors (refer to Sophocleous and Idris, 2019) using surgical scissors and sutures and isoflurane, to avoid harmful behaviors against experimental mice in subsequent experiments.
Reuse CD1 aggressors for different pup litters for no longer than three months.
Early-life social defeat paradigm
Randomly assign mouse pup litters to control and experimental groups at postnatal day 15 (PD 15).
Introduce each pup of the experimental group into the home cage of an unfamiliar CD1 aggressor for a session of 30 min.
Place the pup litters back in their home cages with dams.
Repeat the aggression with different resident home cages every day from PD 15 to PD 24.
Keep pups in the control group undisturbed from PD 15 to PD 24.
Raise all experimental mice individually in their cage after the 10 day social defeat for a month.
Social interaction test (SIT)
Conduct the SIT on PD 55 between 10:00 and 14:00.
No target trial: introduce experimental and control mice into the arena while the wire mesh enclosure is empty for 300 s.
Analyze the time that experimental mice spend in the social interaction and corner zones with EthoVision XT.
Remove the experimental mice from the arena and place them into their home cage for 1 min.
Target trial: place an unfamiliar CD1 mouse into the wire mesh enclosure of the arena.
Introduce experimental mice into the arena for another 300 s.
Analyze the time that experimental mice spend in social interaction zone and corner zones with EthoVision XT.
Clean the arena and wire mech enclosure with odorless cleaning solution and wipe dry.
Open field test (OFT)
Conduct the OFT on PD 56 between 10:00 and 14:00.
Introduce each experimental mice into the arena for 10 min.
Analyze the time that experimental mice spend in the central and corner zones of the open field arena with EthoVision XT.
Analyze the total distance traveled by experimental mice with EthoVision XT.
Clean the arena with odorless cleaning solution and wipe dry.
Data analysis
Analyze all data with GraphPad Prism Software (version 7.0) or any other type of statistical software.
Assess the normality of the distribution with Kolmogorov–Smirnov test.
Present data of different groups as mean ± standard error of mean.
Use an unpaired 2-tailed Student’s t-test/two-way analysis of variance (ANOVA) for comparisons among groups.
Define statistical significance as p < 0.05.
Acknowledgments
W.D.N. designed the experiments. W.D.N. and Y.Z. performed the experiments. Y.Z. wrote the article and W.D.N. revised the manuscripts. We thank L.H.C. for his helpful review of this manuscript. We thank S.J.R. for their previous work on animal models of social defeat.
This research was supported by the National Natural Science Foundation of China (82102301 to W.D.N.), National Post-doctoral Program for Innovative Talents of China (BX20190226 to W.D.N.).
Competing interests
The authors declare that they have no conflicts of interest.
Ethics
All procedures involving animals were approved by the Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee of Sichuan University. All efforts were made to minimize the suffering of mice.
References
Anacker, C. and Hen, R. (2017). Adult hippocampal neurogenesis and cognitive flexibility - linking memory and mood. Nat Rev Neurosci 18(6): 335-346.
Anacker, C., Luna, V. M., Stevens, G. S., Millette, A., Shores, R., Jimenez, J. C., Chen, B. and Hen, R. (2018). Hippocampal neurogenesis confers stress resilience by inhibiting the ventral dentate gyrus. Nature 559(7712): 98-102.
Anacker, C., Scholz, J., O'Donnell, K. J., Allemang-Grand, R., Diorio, J., Bagot, R. C., Nestler, E. J., Hen, R., Lerch, J. P. and Meaney, M. J. (2016). Neuroanatomic Differences Associated With Stress Susceptibility and Resilience. Biol Psychiatry 79(10): 840-849.
Becker, S. and Wojtowicz, J. M. (2007) A model of hippocampal neurogenesis in memory and mood disorders. Trends Cogn Sci 11(2): 70-76.
Castilla-Ortega, E., Serrano, A., Blanco, E., Araos, P., Suarez, J., Pavon, F. J., Rodriguez de Fonseca, F. and Santin, L. J. (2016). A place for the hippocampus in the cocaine addiction circuit: Potential roles for adult hippocampal neurogenesis. Neurosci Biobehav Rev 66: 15-32.
Dioli, C., Patricio, P., Trindade, R., Pinto, L. G., Silva, J. M., Morais, M., Ferreiro, E., Borges, S., Mateus-Pinheiro, A., Rodrigues, A. J., et al. (2017). Tau-dependent suppression of adult neurogenesis in the stressed hippocampus. Mol Psychiatry 22(8): 1110-1118.
Du Preez, A., Onorato, D., Eiben, I., Musaelyan, K., Egeland, M., Zunszain, P. A., Fernandes, C., Thuret, S. and Pariante, C. M. (2021). Chronic stress followed by social isolation promotes depressive-like behaviour, alters microglial and astrocyte biology and reduces hippocampal neurogenesis in male mice. Brain Behav Immun 91: 24-47.
Golden, S. A., Covington, H. E., 3rd, Berton, O. and Russo, S. J. (2011). A standardized protocol for repeated social defeat stress in mice. Nat Protoc 6(8): 1183-1191.
Padilla-Coreano, N., Bolkan, S. S., Pierce, G. M., Blackman, D. R., Hardin, W. D., Garcia-Garcia, A. L., Spellman, T. J. and Gordon, J. A. (2016). Direct Ventral Hippocampal-Prefrontal Input Is Required for Anxiety-Related Neural Activity and Behavior. Neuron 89(4): 857-866.
Schloesser, R. J., Lehmann, M., Martinowich, K., Manji, H. K. and Herkenham, M. (2010). Environmental enrichment requires adult neurogenesis to facilitate the recovery from psychosocial stress. Mol Psychiatry 15(12): 1152-1163.
Snyder, J. S., Kee, N. and Wojtowicz, J. M. (2001). Effects of adult neurogenesis on synaptic plasticity in the rat dentate gyrus. J Neurophysiol 85(6): 2423-2431.
Sophocleous, A. and Idris, A. I. (2019). Ovariectomy/Orchiectomy in Rodents. Methods Mol Biol 1914: 261-267.
Toyoda, A. (2017) Social defeat models in animal science: What we have learned from rodent models. Anim Sci J 88(7): 944-952.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Neuroscience > Behavioral neuroscience > Animal model
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Triplet-primed PCR and Melting Curve Analysis for Rapid Molecular Screening of Spinocerebellar Ataxia Types 1, 2, and 3
ML Mulias Lian
MZ Mingjue Zhao
GP Gui-Ping Phang
IR Indhu-Shree Rajan-Babu
SC Samuel S. Chong
Published: Vol 13, Iss 12, Jun 20, 2023
DOI: 10.21769/BioProtoc.4704 Views: 443
Reviewed by: Alessandro DidonnaEsteban Paredes-OssesNeha Nandwani
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Original Research Article:
The authors used this protocol in The Journal of Molecular Diagnostics May 2021
Abstract
There are more than 40 types of spinocerebellar ataxia (SCA), most of which are caused by abnormal expansion of short tandem repeats at various gene loci. These phenotypically similar disorders require molecular testing at multiple loci by fluorescent PCR and capillary electrophoresis to identify the causative repeat expansion. We describe a simple strategy to screen for the more common SCA1, SCA2, and SCA3 by rapidly detecting the abnormal CAG repeat expansion at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Each of the three separate assays employs a plasmid DNA carrying a known repeat size to generate a threshold melt peak temperature, which effectively distinguishes expansion-positive samples from those without a repeat expansion. Samples that are screened positive based on their melt peak profiles are subjected to capillary electrophoresis for repeat sizing and genotype confirmation. These screening assays are robust and provide accurate detection of the repeat expansion while eliminating the need for fluorescent PCR and capillary electrophoresis for every sample.
Keywords: Spinocerebellar ataxia Repeat expansion disorder CAG repeat Triplet-primed PCR Heptaplex Rapid screening
Background
The autosomal dominantly inherited spinocerebellar ataxias (SCAs) are neurodegenerative diseases that mainly cause progressive degeneration of the cerebellum and, sometimes, the spinal cord (Klockgether et al., 2019). There are more than 40 types of SCA, which can be caused by deletions, missense variants, and repeat expansion in different genes (Klockgether et al., 2019). The SCAs have an average prevalence of 2.7/100,000 globally, with SCA3 being the most common, followed by SCA2, SCA6, and SCA1 (Hersheson et al., 2012; Ruano et al., 2014). SCA1, SCA2, and SCA3 are caused by large abnormal CAG trinucleotide repeat expansions in ATXN1, ATXN2, and ATXN3 genes, respectively (Chung et al., 1993; Orr et al., 1993; Kawaguchi et al., 1994; Pulst et al., 1996).
Molecular genetic testing is necessary to identify the causative gene underlying the SCA. Detection of repeat expansions in SCA1, SCA2, and SCA3 has been accomplished using standard PCR or triplet-primed PCR (TP-PCR), followed by capillary electrophoresis (Dorschner et al., 2002; Cagnoli et al., 2006; Melo et al., 2016; Cagnoli et al., 2018). However, considering the genetic heterogeneity of the SCAs and low prevalence of each SCA type, fluorescent PCR followed by capillary electrophoresis sizing at various SCA loci for every sample is a relatively costly screening method, due to high costs of the labeled primers and equipment used.
Melting curve analysis (MCA) has been shown to be robust in identifying samples carrying repeat expansion in fragile X syndrome, myotonic dystrophy type 1, and Huntington disease (Lian et al., 2015; Rajan-Babu et al., 2015; Zhao et al., 2017). Used as an intermediate step between TP-PCR and capillary electrophoresis, MCA filters out the majority of samples that are expansion-negative while identifying only potential expansion-positive samples for further capillary electrophoresis sizing at a certain SCA locus. Using a similar approach, TP-PCR MCA assays can be easily adapted to identify repeat expansion at other loci by employing primers that are specific to the loci of interest.
Materials and reagents
Axygen® 10 μL microvolume, Gilson-style P2/P10 filter tips (Axygen®, catalog number: TF-300)
Axygen® 20 μL universal fit filter tips (Axygen®, catalog number: TF-20)
Axygen® 200 μL universal fit filter tips (Axygen®, catalog number: TF-200)
Axygen® 1,000 μL universal fit filter tips (Axygen®, catalog number: TF-1000)
MicroAmpTM Fast 8-tube strip (Applied Biosystems, catalog number: 4358293)
MicroAmpTM Optical 8-cap strip (Applied Biosystems, catalog number: 4323032)
0.2 mL 8-strip PCR UltraFlux® i Tubes, flat cap (SSIbio, catalog number: 3247-00)
MicroAmpTM Optical 96-well reaction plate (Applied Biosystems, catalog number: N8010560)
Plate septa, 96 well (Applied Biosystems, catalog number: 4315933)
0.2 mL PCR tubes (Fisher Scientific, catalog number: MBP#3412-PK)
1.5 mL MaxyClear Snaplock microcentrifuge tube (Axygen®, catalog number: MCT-150-C)
0.6 mL MaxyClear Snaplock microcentrifuge tube (Axygen®, catalog number: MCT-060-C)
15 mL high clarity PP centrifuge tube (Falcon®, catalog number: 352097)
Water (HPLC) (Sigma-Aldrich, catalog number: 270733-1L)
SYBRTM Green I nucleic acid gel stain, 10,000× concentrate in DMSO (InvitrogenTM, catalog number: S7563)
Primers:
ATXN1-specific forward primer, SCA1-F (5′-AGCCAGACGCCGGGACAC-3′)
Ned-labeled SCA1-F
ATXN1-specific TP primer, SCA1-TP (5′-GTTTCGGCGTTACGAGTGGAGTG(CTG)5-3′)
ATXN1-specific reverse primer, SCA1-R (5′-CTGCGGAGAACTGGAAATGTGG-3′)
ATXN2-specific forward primer, SCA2-F (5′-GTGCGAGCCGGTGTATGG-3′)
Hex-labeled SCA2-F
ATXN2-specific TP primer, SCA2-TP (5′-GTTTCGGCGTTACGAGTGGACGG(CTG)5-3′)
ATXN2-specific reverse primer, SCA2-R (5′-CTTGCGGACATTGGCAGCC-3′)
ATXN3-specific forward primer, SCA3-F (5′-CTGCTCTTGCATTCTTTTAATACCAGTGAC-3′)
Fam-labeled SCA3-F
ATXN3-specific TP primer, SCA3-TP (5′-GTTTCGGCGTTACGAGTGGAGTC(CTG)5-3′)
ATXN3-specific reverse primer, SCA3-R (5′-CACATGGATGTGAACTCTGTCCTG-3′)
Tail primer (5′-GTTTCGGCGTTACGAGTGGA-3′)
Deoxynucleoside triphosphate set (dNTPs), PCR grade (Roche Applied Science, catalog number: 11969064001)
HotStarTaq DNA Polymerase (Qiagen, catalog number: 203205)
Q-solution (Qiagen, catalog number: 203205)
PCR Buffer (Qiagen, catalog number: 203205)
10 ng/μL genomic DNA
1 pg/μL pATXN1(CAG)35(14+1+18) and pATXN2(CAG)25(15+9), 10 pg/μL pATXN3(CAG)41, and 50 ng/μL pATXN1(CAG)35(14+1+18), pATXN2(CAG)25(15+9), and pATXN3(CAG)41 plasmid DNA (see sections A and E)
Hi-DiTM formamide (Applied BiosystemsTM, catalog number: 4311320)
GeneScanTM 500 ROXTM dye size standard (Applied Biosystems, catalog number: 401734)
Agarose (1st BASE, catalog number: BIO-1000-500g)
Ethidium bromide solution (Sigma-Aldrich, catalog number: E1510-10ML)
1 kb DNA ladder (New England Biolabs Ltd., catalog number: N3232S)
ZymocleanTM Gel DNA Recovery kit (Zymo Research, catalog number: D4007)
illustraTM GFXTM PCR DNA and Gel Band Purification kit (GE Healthcare Life Sciences, catalog number: G13/28-9034-70)
TOPOTM TA CloningTM kit, Dual Promoter, without competent cells (InvitrogenTM, catalog number: 450640)
Max efficiency DH5a competent cells (InvitrogenTM, catalog number: 18258012)
S.O.C medium (InvitrogenTM, catalog number: 15544034)
Ampicillin ready-made solution (Sigma-Aldrich, catalog number: A5354-10ML)
LB broth (InvitrogenTM, catalog number: 10855001)
10× Tris-EDTA (TE) buffer, pH 8.0, ultra-pure grade, 1 L (1st BASE, catalog number: BUF-3020-10X1L)
ZyppyTM Plasmid Miniprep kit (Zymo Research, catalog number: D4019)
BigDye® Terminator v3.1 Cycle Sequencing kit (Applied BiosystemsTM, catalog number: 4337455)
Absolute ethanol (Fisher Chemical, catalog number: E/0650DF/17)
Sodium acetate solution (3 M), pH 5.2 (Thermo ScientificTM, catalog number: R1181)
Glycerol (Sigma-Aldrich, catalog number: G5516)
DNA AWAYTM surface decontaminant (Thermo ScientificTM, catalog number: 7010)
Equipment
Pipetman P2, 0.2–2 μL (Gilson, model number: F144054M)
Pipetman P10, 1–10 μL (Gilson, model number: F144055M)
Pipetman P20, 2–20 μL (Gilson, model number: F144056M)
Pipetman P200, 20–200 μL (Gilson, model number: F144058M)
Pipetman P1000, 100–1,000 μL (Gilson, model number: F144059M)
microONE Mini Personal Centrifuge (TOMY, model number: Capsulefuge PMC-880)
Genie 2 Vortex Mixers (Scientific Industries, Inc., model number: G-560E)
StepOnePlusTM Real-Time PCR System (Applied Biosystems, catalog number: 4376600)
GeneAmp® PCR System 9700 Fast Thermal Cycler (Applied Biosystems, catalog number: 4339386)
Eppendorf tabletop centrifuge (Eppendorf, model number: 5810)
Genetic Analyzer (Applied BiosystemsTM, model number: 3130xl)
SUBCELL GT Horizontal Electrophoresis System (Bio-Rad, catalog number: A2-OK)
NanoDropTM 1000 spectrophotometer (Thermo ScientificTM, model: ND-1000)
Water bath (Memmert, model number: MEM-EW-WB14L4)
Innova 4330 refrigerated incubator shaker (New Brunswick Scientific, model: Innova 4330)
Biofuge Fresco microcentrifuge (Heraeus, catalog number: 75005521)
Shake ‘n’ Stack (Hybaid Limited, catalog number: 50125590)
Scalpel
Software
StepOneTM Real-Time PCR Software (Applied Biosystems)
GeneMapper® Software (Applied Biosystems)
Sequencing Analysis Software (Applied Biosystems)
Microsoft Excel (Microsoft)
Procedure
TP-PCR master mix preparation for amplification and melting curve analysis of the ATXN1, ATXN2, and ATXN3 CAG repeat loci
This protocol outlines the steps to perform TP-PCR amplification of the ATXN1, ATXN2, and ATXN3 CAG repeats and melting curve analysis (MCA) of the generated TP-PCR products. Each TP-PCR assay utilizes a plasmid control DNA [pATXN1(CAG)35(14+1+18), pATXN2(CAG)25(15+9), and pATXN3(CAG)41 in SCA1, SCA2, and SCA3 TP-PCR MCAs, respectively], to generate threshold melt peak temperature (Tm), which is used to classify test samples as expansion-negative (if Tm is lower than the plasmid control’s Tm) or potentially expansion-positive (if Tm is higher than the plasmid control’s Tm).
Prepare the TP-PCR master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction):
9.60 μL of HPLC water
7.50 μL of 5× Q-solution
2.50 μL of 10× HotStarTaq PCR buffer
0.25 μL of 10× SYBRTM Green I Nucleic Acid Gel Stain
1.25 μL of 10 μM ATXN1-specific forward primer (SCA1-F), ATXN2-specific forward primer (SCA2-F), or ATXN3-specific forward primer (SCA3-F)
1.25 μL of 10 μM Tail primer
1.25 μL of 1 μM ATXN1-specific TP primer (SCA1-TP), ATXN2-specific TP primer (SCA2-TP), or ATXN3-specific TP primer (SCA3-TP)
0.20 μL of 25 mM dNTPs
0.20 μL of HotStarTaq DNA polymerase
Vortex to mix and spin the tube briefly.
Aliquot 24 μL of the master mix into the MicroAmpTM Fast 8-tube strip tubes.
Add 1 μL of test sample genomic DNA (10 ng/μL) or relevant plasmid DNA [1 pg/μL pATXN1(CAG)35(14+1+18) and pATXN2(CAG)25(15+9) for SCA1 and SCA2 TP-PCR MCAs, respectively, and 10 pg/μL pATXN3(CAG)41 for SCA3 TP-PCR MCA] into the appropriate tubes. To the non-template control tube, add 1 μL of HPLC water.
Apply the MicroAmpTM Optical 8-cap strip. Sample labeling can be done on the edge instead of in the middle of the cap to avoid blocking of light to the reaction tubes.
Spin the tubes briefly.
Set the following program on the StepOnePlusTM system, transfer the tubes into the instrument, and start the amplification and melt curve analysis (melt peaks are automatically generated upon completion of the run by the on-board StepOneTM software).
Stage Step Temperature and duration
Amplification Hold 95 °C, 15 min
Cycling (35 cycles) 98 °C, 45 s
60 °C, 1 min
72 °C, 2 min
Hold 72 °C, 5 min
Melt curve Hold 95 °C, 1 min
Hold 60 °C, 1 min
Temperature ramp (at a rate of 0.8% in continuous mode) 60 °C to 95 °C
Hold 40 °C, 1 min
Generation of TP-PCR melting curve analysis profiles
This protocol outlines the steps to create melt peak plots to identify samples with potential expansion.
Open the results in Microsoft Excel.
For each sample and plasmid control, plot “Melt Region Derivative Data” (i.e., the -dF/dT values) against “Melt Region Temperature Data” by choosing “X Y (Scatter)” from the Charts option under the Insert Tab, followed by selecting “Scatter with Smooth Lines” to create the melt peak plots.
Look for the temperature that gives the highest derivative data to determine the Tm.
Observe the melt peak profiles to identify samples that display a higher Tm relative to the plasmid control used.
Identify samples with potential expansion for further repeat size verification by capillary electrophoresis.
Extension labeling of TP-PCR products for capillary electrophoresis
This protocol outlines the steps to perform extension fluorescent labeling using an aliquot of the TP-PCR products, followed by capillary electrophoresis for determination of the ATXN1, ATXN2, and ATXN3 CAG repeat sizes in samples with potential expansion.
Prepare the extension labeling master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction):
8.84 μL of HPLC water
6.00 μL of 5× Q-solution
2.00 μL of 10× HotStarTaq PCR buffer
0.80 μL of 10 μM Ned-labeled SCA1-F, Hex-labeled SCA2-F, or Fam-labeled SCA3-F
0.16 μL of 25 mM dNTPs
0.20 μL of HotStarTaq DNA polymerase
Vortex to mix and spin the tube briefly.
Aliquot 18 μL of the master mix into the PCR strip tubes.
Add 2 μL of TP-PCR products into the appropriate tubes.
Spin the strip tubes briefly.
Set the following program on the thermal cycler, transfer the tubes into the instrument, and start the amplification.
Stage Step Temperature and duration
Amplification Hold 95 °C, 15 min
Cycling (5 cycles) 98 °C, 45 s
60 °C, 1 min
72 °C, 2 min
Hold 72 °C, 5 min
Hold 4 °C
Remove the tubes and spin them briefly.
Prepare the capillary electrophoresis master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction):
9.00 μL of Hi-DiTM formamide
0.50 μL of GeneScan 500 ROX dye size standard
Vortex to mix and spin the tube briefly.
Aliquot 9.5 μL of the master mix into the MicroAmpTM Optical 96-well reaction plate.
Aliquot 4 μL of the labeled TP-PCR products into the appropriate wells.
Seal the plate with a rubber septum and spin the plate briefly.
Denature the content at 95 °C for 5 min.
Place the plate on ice.
Set the injection time and voltage in the Genetic Analyzer to be 15 s and 1 kV, respectively, and the run time to 40 min (the rest of the parameters set at default values) and start the capillary electrophoresis.
Analyze the results using the GeneMapper® software.
Examine the electropherograms to assess the ATXN1, ATXN2, and ATXN3 genotypes, repeat counts, and the number and placement of any CGG interruptions.
Flanking PCR master mix preparation and generation of control ATXN1, ATXN2, or ATXN3 plasmids for TP-PCR melting curve analysis
This protocol outlines the steps to prepare ATXN1, ATXN2, or ATXN3 plasmid controls for use in TP-PCR MCA. Briefly, it covers how to set up flanking PCR to amplify across the ATXN1, ATXN2, and ATXN3 CAG repeat sequence, followed by isolation, purification, and cloning of fragments of interest, transformation, colony PCR, and isolation of plasmid DNA.
Prepare the flanking PCR master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction; for each sample, set up 2–3 reactions):
11.60 μL of HPLC water
7.50 μL of 5× Q-solution
2.50 μL of 10× HotStarTaq PCR buffer
1.00 μL of 10 μM SCA1-F, SCA2-F, or SCA3-F
1.00 μL of 10 μM SCA1-R, SCA2-R, or SCA3-R
0.20 μL of 25 mM dNTPs
0.20 μL of HotStarTaq DNA polymerase
Vortex to mix and spin the tube briefly.
Aliquot 24 μL of the master mix into the PCR tubes.
Add 1 μL of genomic DNA (10 ng/μL) into the appropriate tubes. To the non-template control tube, add 1 μL of HPLC water.
Spin the tubes briefly.
Set the following program on the thermal cycler, transfer the tubes into the instrument, and start the amplification.
Stage Step Temperature and duration
Amplification Hold 95 °C, 15 min
Cycling (5 cycles) 98 °C, 45 s
60 °C, 1 min
72 °C, 2 min
Hold 72 °C, 5 min
Hold 4 °C
Remove the tubes and spin them briefly.
Pool the PCR products of each sample and load them into a 2% agarose gel prepared with ethidium bromide (0.5 μg/mL) alongside with 4 μL of the 1 kb DNA ladder.
Electrophorese the gel and visualize the results in an imaging system.
Locate and excise the band of interest from the gel using a razor blade, scalpel, or other device.
Isolate and purify the fragments of interest using a gel band purification kit such as the ZymocleanTM Gel DNA Recovery kit or illustraTM GFXTM PCR DNA and Gel Band Purification kit, following the manufacturer’s instructions.
Quantitate the purified PCR products using NanoDrop.
Store the purified PCR products at -20 °C until further use.
Thaw the DH5αTM competent cells on ice, set the water bath to 42 °C, and thaw the S.O.C medium at room temperature.
Prepare the ligation reaction by adding 1 μL of salt solution and 1 μL of pCR®II-TOPO® TA vector to 4 μL of purified PCR product.
Add the ligation reaction to the thawed DH5αTM competent cells (50 μL per tube).
Gently tap the tubes to mix.
Incubate the cells on ice for 30 min.
Immerse two-thirds of the tubes into the water bath pre-set to 42 °C for 20 s to heat-shock the cells.
Place the tubes on ice for 2 min.
Add 0.95 mL of the S.O.C medium to each tube.
Shake the tubes at 250 rpm for 1 h at 37 °C.
Centrifuge the tubes at 16,060× g for 5 min.
Remove 800 μL of the supernatant and discard.
Resuspend the pellet with the remaining supernatant.
Spread the content from each tube on an LB agar plate containing 100 μg/mL ampicillin using a sterilized spreader.
To prepare LB agar plates containing ampicillin, dissolve agar powder in LB medium following manufacturer’s instructions and autoclave to sterilize. Cool the agar to approximately 50 °C and add 1 mL of 100 mg/mL ampicillin before pouring them into Petri dishes and allowing them to cool and set. Store the prepared LB agar plates at 4 °C.
Incubate the plates overnight at 37 °C.
Randomly pick 10–20 colonies from each plate using micropipette tips.
Suspend each colony into a PCR tube containing 10 μL of 1× TE buffer and mark the colony numbers on the LB plates and the PCR tubes.
Perform colony PCR by setting up TP-PCR (see section A; use fluorescently labeled SCA1-F, SCA2-F, or SCA3-F and omit SYBRTM Green I Nucleic Acid Gel Stain) using 1 μL of the picked colony as the template, followed by capillary electrophoresis (see section C).
Examine the electropherograms to identify colonies that have the DNA inserts with expected ATXN1, ATXN2, or ATXN3 CAG repeat size and structure.
Create starter culture by inoculating the remaining 9 μL of the colonies of interest into 3–5 mL of LB broth containing 100 μg/mL ampicillin.
Shake the tubes at 250 rpm overnight at 37 °C.
Expand the culture by transferring the starter culture to 50–100 mL of LB broth containing 100 μg/mL ampicillin.
Shake the tubes at 250 rpm overnight at 37 °C.
Prepare bacterial glycerol stocks for future use by adding 400 μL of the culture into 400 μL of glycerol and gently pipetting them up and down to mix. Store the bacterial glycerol stocks at -80 °C.
Isolate plasmid DNA using kits such as the ZyppyTM Plasmid Miniprep kit, following the manufacturer’s instructions.
Quantitate the extracted plasmid DNAs using NanoDrop and store them at -20 °C.
Use 1–10 pg of plasmid DNA as the template for TP-PCR MCA assay.
The CAG repeat size and structure of the plasmid DNA can also be verified by Sanger sequencing (see section E).
Sanger sequencing for verification of ATXN1, ATXN2, or ATXN3 CAG repeat size and structure of plasmid DNA controls
This protocol outlines the steps to perform Sanger sequencing of the plasmid DNA for verification of the ATXN1, ATXN2, and ATXN3 CAG repeat sizes.
Prepare the sequencing reaction master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction):
HPLC water (top up to 20 μL per reaction)
8 μL of 2.5× BigDye® Terminator Mix v3.1
3.2 pmol M13 or T7 primers
Vortex to mix and spin the tube briefly.
Aliquot 16 μL of the master mix into the PCR tubes.
Add 4 μL of plasmid DNA (50 ng/μL) into the appropriate tubes.
Spin the tubes briefly.
Set up the following program on the thermal cycle, transfer the tubes into the instrument, and start the amplification.
Stage Step Temperature and duration
Amplification Hold 96 °C, 1 min
Cycling (25 cycles) 96 °C, 10 s
60 °C, 5 s
60 °C, 4 min
Hold 4 °C
Remove the tubes and spin them briefly.
Prepare the precipitation reaction master mix by dispensing the following reagents into a microcentrifuge tube (volumes stated are for a single reaction):
14.50 μL of HPLC water
3.00 μL of 3M sodium acetate
62.50 μL of 100% ethanol
Vortex to mix and spin the tube briefly.
Aliquot 80 μL of the master mix into the 1.5 mL microcentrifuge tube.
Transfer all 20 μL of the sequencing products into the appropriate tubes.
Vortex to mix and spin the tubes briefly.
Leave the tubes at room temperature away from light for 15 min.
Spin the tubes at 13,000 rpm for 20 min.
Aspirate and discard the supernatant.
Add 250 μL of 70% ethanol into each tube.
Spin the tubes at 13,000 rpm for 5 min.
Aspirate and discard the supernatant.
Repeat steps E16–E18.
Air dry the tubes.
Add 12 μL of Hi-DiTM formamide into each tube.
Vortex the tubes to dislodge and suspend the pellets.
Load all 12 μL of the content into the MicroAmpTM Optical 96-well reaction plate.
Seal the plate with a rubber septum and spin the plate briefly.
Denature the content at 95 °C for 5 min.
Place the plate on ice.
Start the capillary electrophoresis run in the Genetic Analyzer using sequencing module and all parameters set at default values.
Analyze the results using the Sequencing Analysis software.
Data analysis
The SCA1, SCA2, and SCA3 TP-PCR employs locus-specific flanking and TP primers and a common Tail primer, which together generate multiple products, each differing by one CAG repeat (Lian et al., 2021). The shorter TP-PCR products generated from an allele with smaller CAG repeat size produce melt peaks with lower Tm, whereas the longer TP-PCR products generated from an allele with larger CAG repeat size yield melt peaks with higher Tm. Plasmid control DNA carries repeat size that is sufficiently lower than the upper boundary of the normal repeat size range and is included in each TP-PCR MCA run for generation of threshold Tm to classify samples based on their Tm. The melt peak plots of non-SCA samples, SCA-positive samples, and plasmid DNA controls after SCA1, SCA2, and SCA3 TP-PCR MCAs are shown in Figure 1 (left panel). Non-SCA samples carrying non-expanded (normal) alleles are left-shifted with lower Tm compared with that of the plasmid DNA control and will not be further subjected to capillary electrophoresis sizing. On the other hand, SCA-positive samples are right-shifted relative to the control, due to their higher Tm. Any sample whose Tm is higher than the relevant plasmid’s Tm is potentially expansion-positive and will be further subjected to capillary electrophoresis for repeat size confirmation (Figure 1, right panel). Due to the unique design of the primers, the first peak in the SCA1 and SCA2 TP-PCR electropherogram represents TP-PCR product containing five repeats, while the first peak in the SCA3 TP-PCR electropherogram represents product containing 11 repeats. Each successive peak represents product containing an additional repeat, and the last peak represents the largest TP-PCR product generated. The repeat number in a sample can be determined by counting the number of peaks. In situations where CGG interruptions are present in ATXN1 and ATXN2 alleles, inefficient annealing of the TP primer to the interrupted sequences results in little to no amplification, resulting in a gap between two discrete clusters of peaks.
Figure 1. Example of melt peak profiles (left) and electropherograms (right) of non-spinocerebellar ataxia (SCA) and SCA-positive samples, together with the plasmid control DNA, after SCA1, SCA2, and SCA3 TP-PCR MCAs. In the left panel, non-SCA samples are left-shifted with lower melt peak temperatures compared with that of the relevant plasmid DNA control, while SCA-positive samples are right-shifted relative to the control. -dF/dT is shown on the x-axis and temperature is shown on the y-axis. In the right panel, numbered black arrows indicate the number of CAG repeats in non-expanded (normal) alleles, and numbered red arrows indicate the number of CAG repeats in expanded alleles. Relative fluorescence unit (RFU) is shown on the x-axis and size is shown on the y-axis.
Notes
Clean lab bench before start of experiments with 70% ethanol and DNA AWAYTM.
Mix reagents before use to ensure that they are uniform and spin them briefly to collect the content at the bottom of the tube.
Use the HotStarTaq PCR buffer and Q-solution provided with the HotStarTaq DNA polymerase.
Use aerosol barrier pipette tips. Change tips after each use.
Open and close all sample tubes carefully. Avoid splashing PCR products.
Note that SYBR Green is light sensitive. Wrap tubes containing SYBR Green with aluminum foil or use amber tubes to avoid direct light exposure.
We recommend that plasmid DNA controls are included in each TP-PCR MCA run to differentiate the melt peaks of samples with repeat expansion from those of expansion-negative samples. The result of the plasmid DNA generated from one run should not be used to differentiate samples used in other runs.
Include at least one positive control (e.g., sample with a known expansion) as well as a negative or non-template control in each assay, to ensure reliable amplification/repeat sizing and absence of amplicon or DNA contaminants in the reagents used, respectively.
We recommend using POP-7TM polymer.
The genomic DNA and plasmid DNA should be diluted with 1× TE buffer to prevent DNA degradation.
Acknowledgments
This work was supported by internal departmental funds. This protocol was derived from the original research paper (Lian et al., 2021).
Competing interests
A patent application was submitted on June 2, 2021, directed to a method of detecting a repeat expansion sequence. The authors declare that they have no other competing interests.
References
Cagnoli, C., Brussino, A., Mancini, C., Ferrone, M., Orsi, L., Salmin, P., Pappi, P., Giorgio, E., Pozzi, E., Cavalieri, S., et al. (2018). Spinocerebellar Ataxia Tethering PCR: A Rapid Genetic Test for the Diagnosis of Spinocerebellar Ataxia Types 1, 2, 3, 6, and 7 by PCR and Capillary Electrophoresis. J Mol Diagn 20(3): 289-297.
Cagnoli, C., Stevanin, G., Michielotto, C., Gerbino Promis, G., Brussino, A., Pappi, P., Durr, A., Dragone, E., Viemont, M., Gellera, C., et al. (2006). Large pathogenic expansions in the SCA2 and SCA7 genes can be detected by fluorescent repeat-primed polymerase chain reaction assay. J Mol Diagn 8(1): 128-132.
Chung, M. Y., Ranum, L. P., Duvick, L. A., Servadio, A., Zoghbi, H. Y. and Orr, H. T. (1993). Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nat Genet 5(3): 254-258.
Dorschner, M. O., Barden, D. and Stephens, K. (2002). Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis. J Mol Diagn 4(2): 108-113.
Hersheson, J., Haworth, A. and Houlden, H. (2012). The inherited ataxias: genetic heterogeneity, mutation databases, and future directions in research and clinical diagnostics. Hum Mutat 33(9): 1324-1332.
Kawaguchi, Y., Okamoto, T., Taniwaki, M., Aizawa, M., Inoue, M., Katayama, S., Kawakami, H., Nakamura, S., Nishimura, M., Akiguchi, I., et al. (1994). CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet 8(3): 221-228.
Klockgether, T., Mariotti, C. and Paulson, H. L. (2019). Spinocerebellar ataxia. Nat Rev Dis Primers 5(1): 24.
Lian, M., Rajan-Babu, I. S., Singh, K., Lee, C. G., Law, H. Y. and Chong, S. S. (2015). Efficient and highly sensitive screen for myotonic dystrophy type 1 using a one-step triplet-primed PCR and melting curve assay. J Mol Diagn 17(2): 128-135.
Lian, M., Zhao, M., Phang, G. P., Soong, Y. T., Yoon, C. S., Lee, C. G., Law, H. Y. and Chong, S. S. (2021). Rapid Molecular Screen of Spinocerebellar Ataxia Types 1, 2, and 3 by Triplet-Primed PCR and Melting Curve Analysis. J Mol Diagn 23(5): 565-576.
Melo, A. R., Ramos, A., Kazachkova, N., Raposo, M., Bettencourt, B. F., Rendeiro, A. R., Kay, T., Vasconcelos, J., Bruges-Armas, J. and Lima, M. (2016). Triplet Repeat Primed PCR (TP-PCR) in Molecular Diagnostic Testing for Spinocerebellar Ataxia Type 3 (SCA3). Mol Diagn Ther 20(6): 617-622.
Orr, H. T., Chung, M. Y., Banfi, S., Kwiatkowski, T. J., Jr., Servadio, A., Beaudet, A. L., McCall, A. E., Duvick, L. A., Ranum, L. P. and Zoghbi, H. Y. (1993). Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet 4(3): 221-226.
Pulst, S. M., Nechiporuk, A., Nechiporuk, T., Gispert, S., Chen, X. N., Lopes-Cendes, I., Pearlman, S., Starkman, S., Orozco-Diaz, G., Lunkes, A., et al. (1996). Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet 14(3): 269-276.
Rajan-Babu, I. S., Law, H. Y., Yoon, C. S., Lee, C. G. and Chong, S. S. (2015). Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis. Expert Rev Mol Med 17: e7.
Ruano, L., Melo, C., Silva, M. C. and Coutinho, P. (2014). The global epidemiology of hereditary ataxia and spastic paraplegia: a systematic review of prevalence studies. Neuroepidemiology 42(3): 174-183.
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Autolysin Production from Chlamydomonas reinhardtii
JF Justin Findinier
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4705 Views: 530
Reviewed by: Ansul LokdarshiSamed DelicNoelia Foresi
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Original Research Article:
The authors used this protocol in The Plant Cell Mar 2023
Abstract
Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.
Graphical overview
Workflow for autolysin production from Chlamydomonas reinhardtii
Keywords: Enzyme Autolysin Cell wall Chlamydomonas CRISPR Transformation
Background
Chlamydomonas’ genetic transformation has been possible for random insertional mutagenesis for over three decades (Kindle, 1990). In the last six years, methods have been developed for the precise editing of Chlamydomonas genome using the CRISPR technology by transfection of the ribonucleoprotein complex (RNP) directly through electroporation (Shin et al., 2016). Results have made clear that the cell wall is a strong barrier to entry of the RNP, as editing efficiency is usually much higher in cell wall–less strains or after digestion of the cell wall. Cell wall degradation can be performed using a commercially available enzyme called subtilisin/alcalase, but it has been shown to be only half as efficient as autolysin (Hwang et al., 2018), an enzyme produced by Chlamydomonas during mating in order to digest the cell wall and allow cell fusion of the two partners (Matsuda and Kubo, 2004). Autolysin can be extracted from a mixture of mating cells by centrifugation, preserved by freezing at -80 °C, and used later on any given walled strain to prepare for transformation. Several protocols are available in the literature as well as on the Chlamydomonas Resource Center website, but their efficiency has proven inconsistent in our hands. Here, we present a very simple protocol that requires minimal monitoring of cell growth and density, with overall > 70% efficiency. This will allow researchers interested in using the CRISPR technology to produce good quality autolysin and obtain mutated strains in their gene of interest, regardless of their favorite wildtype strain.
Materials and reagents
90 mm Petri dishes
Inoculating loop
Sterile 50 mL centrifuge tubes
Cell scraper
Pipette tips
1.7 mL microcentrifuge tubes
50 mL conical tubes
50 mL Luer-lock syringe
0.45 μm PES filter (VWR, catalog number: 76479-020)
Permanent marker pen
Microscope slides with eight wells (75 mm × 25 mm) (MP Biomedicals, catalog number: 096040805)
Plastic spectrophotometer cuvette (VWR, catalog number: 97000-586)
1 L filtration unit (VWR, catalog number: 10040-440)
Mating type (MT)+ wildtype strain CC-125 (available from the Chlamydomonas Resource Center, www.chlamy.org)
MT- wildtype strain CC-124 (available from the Chlamydomonas Resource Center, www.chlamy.org)
Note: Other pairs of MT+/MT- strains can be used; CC-620/621 are usually recommended due to their high mating efficiency.
Cell wall–less wildtype strain CC-503 (cw92, available from the Chlamydomonas Resource Center, www.chlamy.org)
Ammonium chloride (NH4Cl) (Sigma, catalog number: A9434)
Sodium chloride (NaCl) (Sigma, catalog number: 746398)
Calcium chloride dihydrate (CaCl2·2H2O) (Sigma, catalog number: C3881)
Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma, catalog number: 63140)
Tris base (J.T. Baker, catalog number: 4099-07)
Potassium phosphate monobasic (KH2PO4) (J.T. Baker, catalog number: 3246-07)
Potassium phosphate dibasic (K2HPO4) (J.T. Baker, catalog number: 3252-07)
Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA·2H2O) (Sigma, catalog number: E5134)
Iron (II) sulfate heptahydrate (FeSO4·7H2O) (Sigma, catalog number: F-8048)
Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma, catalog number: Z0251)
Boric acid (H3BO3) (Sigma, catalog number: B6768)
Manganese (II) chloride tetrahydrate (MnCl2·4H2O) (Sigma, catalog number: 221279)
Copper (II) chloride dihydrate (CuCl2·2H2O) (Sigma, catalog number: 467847)
Sodium molybdate dihydrate (Na2MoO4·2H2O) (J.T. Baker, catalog number: 3764)
Cobalt (II) chloride hexahydrate (CoCl2·6H2O) (Sigma, catalog number: C2644)
Glacial acetic acid (Sigma, catalog number: 320099)
Triton X-100 (Sigma, catalog number: T-9284)
Granulated agar (Fisher Scientific, catalog number: BP9744-5)
0.1% Triton X-100 (see Recipes)
Sucrose (Sigma, catalog number: S0389)
Stock solutions
Salts +N solution (20×) (see Recipes)
Salts -N solution (20×) (see Recipes)
2 M Tris Base (100×)
Phosphate buffer (200×) (see Recipes)
Hutner’s trace elements (200×) (see Recipes, also available from the Chlamydomonas Resource Center, www.chlamy.org)
TAP (Tris-Acetate-Phosphate) liquid medium (see Recipes)
TAP 1.5% agar medium (see Recipes)
TAP -N liquid medium (see Recipes)
TAP sucrose 40 mM (see Recipes)
Equipment
Pipettes
Centrifuge and microcentrifuge
Light microscope
Laminar flow hood
Glass beaker
Spectrophotometer
Vacuum pump
Procedure
Cell growth
Spread MT+ (CC-125) and MT- (CC-124) cells on TAP 1.5% agar medium using an inoculating loop (approximately two loop-full of cells spread over the whole surface).
Incubate for 7–10 days in bright light (lid facing down, 22 °C, ~100 μmoles m-2·s-1) (Figure 1).
Figure 1. Plates of Chlamydomonas CC-124 (MT-) and CC-125 (MT+) cells grown on TAP agar plate at harvesting time. Two loop-full of cells were spread over the plate and grown under bright light (100 μmoles m-2·s-1) for seven days.
Gametogenesis
Start this step in the late afternoon (5:00–6:00 PM).
For each plate, add 10 mL of TAP -N liquid medium to a sterile 50 mL centrifuge tube (use one tube per plate).
Using a cell scraper, transfer the cells from the agar to the liquid.
Use a pipette to resuspend cell clumps as much as possible (Figure 2).
Figure 2. Cell suspension after transfer from TAP agar plates to TAP -N liquid medium. Cells were scraped from plate and resuspended in liquid medium (here, MT- CC-124 on the left and MT+ CC-125 on the right). Pipetting is used to break cell clumps as much as possible.
Leave horizontally in bright light (~100 μmoles m-2·s-1) overnight without shaking.
Mating and autolysin production
In the morning, transfer 10 μL of cells to a microscope slide and check for motility.
Note: Over 50% of cells should be moving actively.
Mix the MT+ and MT- strains in the 50 mL centrifuge tubes and incubate for 2 h in the dark without shaking.
Centrifuge at 3,000× g for 5 min.
Transfer supernatant to a clean glass beaker and discard the tube containing the pellet.
Filter the supernatant into a sterile 50 mL centrifuge tube using a 50 mL syringe and a 0.45 μm PES filter.
Set aside 2 mL of filtered supernatant for Section D and place the rest to freeze immediately in a -80 °C freezer.
Testing autolysin preparation efficiency
Four days prior to autolysin production, inoculate CC-125 (cell walled) and CC-503 (cell wall–less) strains in 5 mL of TAP liquid medium in a 50 mL conical tube.
Note: Any cell wall–less strain (cw15 or cw92 mutant) can be used as a control for lysis. After detergent treatment (step D10), it should show a white pellet and a green supernatant. CC-5325/CC-4533, which is annotated cw15, synthesizes a cell wall and should not be used for that purpose (Zhang et al., 2022).
Harvest the 5 mL of late log CC-125 and CC-503 cells and adjust each OD750nm to 0.3 with fresh TAP -N medium.
To evaluate lysis efficiency by chlorophyll absorbance, transfer 1 mL to a cuvette and measure OD662nm of the initial cell suspensions (OD1, zero with TAP -N medium).
Centrifuge 4 × 1 mL of CC-125 and 2 × 1 mL of CC-503 at 1,500× g for 5 min in 1.7 mL centrifuge tubes.
Discard the supernatants.
Resuspend the two pellets of CC-503 in 1 mL each of TAP -N (positive control), two pellets of CC-125 in 1 mL each of TAP -N liquid medium (negative control), and the two other CC-125 pellets in 1 mL each of the autolysin preparation supernatant set aside in step C6. We now have six tubes for three conditions: cell wall–less CC-503, cell-walled CC-125 in TAP -N, and cell-walled CC-125 treated with the prepared autolysin.
Incubate horizontally for 45 min in bright light (~100 μmoles m-2·s-1) without shaking.
Centrifuge all the tubes at 1,500× g for 5 min.
Discard the supernatants.
For each condition, resuspend tube 1 in 1 mL of water and tube 2 in 1 mL of 0.1% Triton X-100.
Vortex for 2 min.
Centrifuge at 1,500× g for 5 min.
Autolysin efficiency can be quickly assessed from the color of the pellets and supernatants (Figure 3). After Triton X-100 treatment, the supernatant in the CC-125 cells treated with efficient autolysin is green, and we can proceed with the quantification of lysis efficiency. If it is still translucent, the autolysin preparation failed, and all tubes should be discarded.
Figure 3. Expected results from autolysin and detergent treatment. Cells incubated with the prepared autolysin or in control conditions were treated with the detergent Triton X-100 to induce cell lysis. Cell wall–less cells (CC-503) display a green supernatant showing cell lysis and release of chlorophyll, while the pellet becomes white (B). Cell-walled cells (CC-125) incubated in control conditions are resistant to the detergent and the cell pellet stays green (D). Cells treated with good autolysin undergo at least partial lysis and display a green supernatant (F).
Transfer the supernatant to a cuvette and measure OD662nm (OD2, zero with 0.1% Triton X-100).
Calculate gross efficiency by the ratio between the OD662nm after (OD2) and before (OD1) detergent treatment:
Efficiency (%) = OD2662nm/OD1662nm
Using the autolysin preparation
Thaw the frozen autolysin in a beaker filled with water at room temperature for ~1 h.
Note: We recommend using the freshest preparation possible. Avoid using autolysin stored for more than two months, as it will significantly lose activity over time.
Centrifuge cells to transform at 1,500× g for 5 min.
Discard the supernatant.
Resuspend the pellet in autolysin solution at ~5 × 106 cells/mL.
Incubate in bright light (~100 μmoles m-2·s-1) without shaking for 45 min.
Centrifuge at 1,500× g for 5 min.
Discard the supernatant.
Resuspend the pellet in 25 mL of TAP sucrose 40 mM.
Centrifuge at 1,500× g for 5 min.
Discard the supernatant.
Proceed with the transformation protocol.
Data analysis
All samples not treated with Triton X-100 are expected to show a clear supernatant and a dark green pellet (Figure 3A, 3C, and 3E). After Triton X-100 treatment, the cell wall–less strain CC-503 will show a green supernatant and a white pellet (Figure 3B). On the contrary, the CC-125 control cells that were incubated in TAP -N display a clear supernatant and a dark green pellet like the untreated cells (Figure 3D). This is because the control cells are still protected by their cell wall, while the CC-503 strain is highly susceptible to lysis due to the absence of the cell wall and releases its chlorophyll content in the supernatant after detergent treatment. If the preparation works, the CC-125 cells treated with autolysin and Triton X-100 will show a similar result to the cell wall–less strain. Depending on the quality of the autolysin preparation, part of the pellet can still be green; in this case, serial treatment of cells to be transformed can be necessary. If no white pellet is observed, discard the frozen autolysin tube.
Note: Sometimes, the pellet from autolysin-treated cells will appear partly white even without detergent treatment because vortexing is harsh enough to lyse cell wall–less cells.
To ensure the best quality of autolysin, cell lysis efficiency can also be quantified by measuring chlorophyll release in the supernatant. The best result will be reached when the ratio of OD662nm after lysis over OD662nm before lysis is the closest to the value obtained with the cell wall–less strain. In Table 1, we provide an example of efficiency of chlorophyll release obtained from cell wall–less cells (CC-503) and from untreated cell-walled cells (CC-125), showing the difference observed between lysed and intact cells.
Table 1. Quantification of chlorophyll release by detergent treatment. Example of quantification of chlorophyll release in the supernatant from cell wall–less (CC-503) and cell-walled (CC-125) cells treated with Triton X-100.
OD662nm of the
cell suspension (OD1)
OD662nm of the
supernatant (OD2)
Efficiency (OD1/OD2, %) Average efficiency (%)
CC-503 0.27 0.183 67.7 67.7
0.261 0.187 71.6
0.276 0.176 63.8
CC-125 control 0.21 0.033 15.7 15.9
0.208 0.032 15.4
0.212 0.035 16.5
Validation of protocol
Autolysin prepared using this protocol has been used to generate targeted mutants by CRISPR/Cas9 in the TPT2 and TPT3 genes of Chlamydomonas reinhardtii (Huang et al., 2023). This protocol was also used for preparation of CC-124 cells prior to insertional mutagenesis and phenotypic screen for starch catabolism mutants (Tunçay et al., 2013).
Recipes
0.1% Triton X-100 (for 100 mL)
100 μL of Triton X-100
Complete with distilled water
Store at room temperature
Stocks solutions
Salts +N solution (for 1 L, 20×)
150 mM NH4Cl
16.6 mM MgSO4·7H2O
6.8 mM CaCl2·2H2O
Complete with distilled water
Store at room temperature
Salts -N solution (for 1 L, 20×)
150 mM NaCl
16.6 mM MgSO4·7H2O
6.8 mM CaCl2·2H2O
Complete with distilled water
Store at room temperature
2 M Tris base (for 1 L, 100×)
2 M Tris base
Complete with distilled water
Store at room temperature
Phosphate buffer (for 1 L, 200×)
687 mM KH2PO4
362 mM K2HPO4
Complete with distilled water
Store at room temperature
Hutner’s trace elements (for 1 L, 200×)
800 mL of distilled water
27 mM Na2EDTA·2H2O
Adjust pH to 6.5 with NaOH
3.6 mM FeSO4·7H2O
15 mM ZnSO4·7H2O
37 mM H3BO3
5.2 mM MnCl2·4H2O
1.3 mM CuCl2·2H2O
1.1 mM Na2MoO4·2H2O
1.3 mM CoCl2·6H2O
Complete with distilled water
Autoclave for 30 min at 120 °C
The solution’s color is light green after mixing all the powders. Let the solution oxidize for a week in the dark. It will turn orange, brown, and then purple
Store at room temperature in obscurity
TAP medium (for 1 L)
800 mL of distilled water
50 mL of +N Salt solution
10 mL of tris base
5 mL of phosphate buffer
5 mL of Hutner’s trace elements
1 mL of glacial acetic acid
Check pH range 7–7.2
Complete to 1 L with distilled water
Autoclave for 30 min at 120 °C
TAP 1.5% agar medium (for 1 L)
800 mL of distilled water
50 mL of salt +N solution
10 mL of Tris Base
5 mL of phosphate buffer
5 mL of Hutner’s trace elements
1 mL of glacial acetic acid
Check pH range 7–7.2
Complete to 1 L with distilled water
15 g of agar
Autoclave for 30 min at 120 °C
TAP -N medium (for 1 L)
800 mL of distilled water
50 mL of salt -N solution
10 mL of tris base
5 mL of phosphate buffer
5 mL of Hutner’s trace elements
1 mL of glacial acetic acid
Check pH range 7–7.2
Complete with distilled water
Autoclave for 30 min at 120 °C
TAP sucrose (for 1 L)
40 mM sucrose
Complete with TAP liquid medium
Filter sterilize
Acknowledgments
I am thankful to Neda Fakhimi, Sai Madireddi, and Carolyne Stoffel for feedback on this protocol. I acknowledge David Dauvillée from whom I adapted this protocol. This work was supported by the Carnegie Institution for Science, Department of Plant Biology.
Competing interests
I declare no competing interests.
References
Huang, W., Krishnan, A., Plett, A., Meagher, M., Linka, N., Wang, Y., Ren, B., Findinier, J., Redekop, P., Fakhimi, N., et al. (2023). Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light-sensitive. Plant Cell. doi: 10.1093/plcell/koad095.
Hwang, H. J., Kim, Y. T., Kang, N. S. and Han, J. W. (2018). A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase). J Mol Microbiol Biotechnol 28(4): 169-178.
Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87(3): 1228-1232.
Matsuda, Y. and Kubo, T. (2004). Gametolysin. In: Handbook of Proteolytic Enzymes (2nd Edition). Academic Press, 592-595.
Shin, S. E., Lim, J. M., Koh, H. G., Kim, E. K., Kang, N. K., Jeon, S., Kwon, S., Shin, W. S., Lee, B., Hwangbo, K., et al. (2016). CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep 6: 27810.
Tunçay, H., Findinier, J., Duchêne, T., Cogez, V., Cousin, C., Peltier, G., Ball, S. G. and Dauvillée, D. (2013). A forward genetic approach in Chlamydomonas reinhardtii as a strategy for exploring starch catabolism. PLoS One 8(9): e74763.
Zhang, N., Pazouki, L., Nguyen, H., Jacobshagen, S., Bigge, B. M., Xia, M., Mattoon, E. M., Klebanovych, A., Sorkin, M., Nusinow, D. A., et al. (2022). Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background). Plants 11(5): 585.
Article Information
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© 2023 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 molecular biology > DNA
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Isolation and purification
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Have you considered using agitation during gametogenesis and also using a higher concentration of cells?
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4,706 | https://bio-protocol.org/en/bpdetail?id=4706&type=0 | # Bio-Protocol Content
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Qualitative and Quantitative Methods to Measure Antibacterial Activity Resulting from Bacterial Competition
BT Boris Taillefer
MG Marie M. Grandjean
JH Julien Herrou
DR Donovan Robert
TM Tâm Mignot
CS Corinne Sebban-Kreuzer
EC Eric Cascales
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4706 Views: 1639
Reviewed by: Cristina Alvarez MartinezAksiniya Asenova Anonymous reviewer(s)
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Abstract
In the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors. Current laboratory techniques to evaluate antibacterial activity are usually labor intensive, low throughput, costly, and time consuming. Typical assays rely on the outgrowth of colonies of prey cells on selective solid media after competition. Here, we present fast, inexpensive, and complementary optimized protocols to qualitatively and quantitively measure antibacterial activity. The first method is based on the degradation of a cell-impermeable chromogenic substrate of the β-galactosidase, a cytoplasmic enzyme released during lysis of the attacked reporter strain. The second method relies on the lag time required for the attacked cells to reach a defined optical density after the competition, which is directly dependent on the initial number of surviving cells.
Key features
• First method utilizes the release of β-galactosidase as a proxy for bacterial lysis.
• Second method is based on the growth timing of surviving cells.
• Combination of two methods discriminates between cell death and lysis, cell death without lysis, or survival to quasi-lysis.
• Methods optimized to various bacterial species such as Escherichia coli, Pseudomonas aeruginosa, and Myxococcus xanthus.
Graphical overview
Keywords: Antibacterial activity Predation Competition β-galactosidase activity CPRG Bacterial growth Bacterial viability
Background
In the environment, bacteria do not live alone but rather build complex microbial communities in which several species coexist (Luo et al., 2022). These microbial communities, while dynamic, represent ecosystems that are usually stable and resilient by resisting changing conditions and disturbances. To ensure this stability, bacteria establish relationships with other bacterial species and microbes for mutual benefits. The most studied cooperation mechanisms are quorum sensing (i.e., a communication mechanism based on release and sensing of signaling molecules allowing the synchronized expression of specific genes), exchange of metabolic molecules, and division of labor (subpopulations that perform different tasks simultaneously) (West et al., 2006). Conversely, antagonist interactions, collectively known as competition, correspond to mechanisms evolved by microorganisms to destroy rivals—or prevent their growth—in order to ensure a privileged access to the niche or to limited resources. Competition can lead to the increase of one species at the expense of one or several others, and usually promotes formation of spatial segregation patterns inside the community. Competition comprises indirect (exploitative) or direct (interference) mechanisms, in which one species consumes the resources or eliminate rivals, respectively (Ghoul and Mitri, 2016). An attacker cell competing for nutrients or space is referred as a competitor. By opposition, an attacker using competition to directly eliminate and feed from a target cell is referred as a predator. At the molecular level, many mechanisms involved in direct competition have been revealed and described in detail (Ghoul and Mitri, 2016; Stubbendieck and Straight, 2016; Granato et al., 2019). All these mechanisms rely on common themes but need distinct specific machineries. They all require the delivery of a potent toxin that is usually co-produced with a cognate immunity protein conferring protection to the attacker cell (Chassaing and Cascales, 2018). Toxin-mediated competition mechanisms are categorized as contact-independent or contact-dependent. The best-known example of contact-independent mechanisms is represented by bacteriocins, such as colicins. These are peptide or protein antibiotics that are released by a quasi-lysis mechanism and bind to and penetrate into target cells by hijacking essential components of the cell envelope (Cascales et al., 2007; Kleanthous, 2010). By contrast, contact-dependent mechanisms rely on specific secretion machineries required for cell surface exposure or the delivery of toxic effectors. These are either directly injected inside the target cell or use a penetration mechanism similar to that of bacteriocins. Several dedicated secretion systems, such as Type IV, Type V, Type VI, and Type VII secretion systems and Type IV and Tad pili, have been demonstrated to play key roles in bacterial competition (Ruhe et al., 2013; Willett et al., 2015; Cao et al., 2016; Chassaing and Cascales, 2018; Granato et al., 2019; Sgro et al., 2019; Seef et al., 2021; Tassinari et al., 2022; Thiery et al., 2022).
Evaluating bacterial competition in laboratory conditions often requires mixing attacker and recipient cells and, after co-incubation, counting attacker and surviving recipient cell colony-forming units on selective solid growth medium (MacIntyre et al., 2010; Flaugnatti et al., 2016; Alcoforado Diniz et al., 2017). However, this method is labor intensive, low throughput, costly, and time consuming. There is, therefore, a need to overcome these limitations, particularly when a large number of strains needs to be tested or in the case of a natural antibiotic resistant model that does not allow recovering prey cells in selective medium (Hazan et al., 2012; Lin and Lai, 2020). Recently, a new procedure based on the cell-impermeable β-galactosidase chromogenic substrate chlorophenol-red β-D-galactopyranoside (CPRG) was proposed (Vettiger and Basler, 2016). Because of its inability to cross the cell envelope, the CPRG (yellow) is only hydrolyzed in chlorophenol red (CPR, red) when the cytoplasmic β-galactosidase is released from a lacZ+ reporter cell after lysis. In addition to the visual evaluation of the antibacterial activity, the colorimetric properties of CPR can be measured by absorbance at λ = 572 nm (A572) or fluorescence (excitation at λ = 580 nm and emission at λ = 620 nm, F620nm) for a semi-quantitative value (Sicard et al., 2014; Vettiger and Basler, 2016; Figure 1A).
Here, this colorimetric method, called LAGA for Lysis-associated β-galactosidase Assay, was optimized to evaluate the activity of various antibacterial mechanisms. In addition, we developed a quantitative and complementary method based on growth recovery of surviving cells, called SGK for Survivors Growth Kinetics, to measure the number of surviving reporter cells after a competition assay, which is directly linked to the antibacterial activity of the attacker cell (Figure 1B). This method measures the time necessary for a subculture of recipient cells in selective medium to reach a defined optical density at λ = 600 nm (A600) after a competition assay, which is dependent on the initial number of surviving cells. By using standard curves from different known reporter inoculums, the initial number of surviving recipients can be estimated. Here, we describe the application of these methods in different bacterial models using various antibacterial strategies.
Figure 1. Schematic representations of the LAGA and SGK assays. (A) Lysis-associated β-galactosidase assay (LAGA). (i) Attacker and lacZ+ reporter cells are mixed. (ii) The antibacterial activity of the attacker results in reporter cell lysis and release of β-galactosidase to the medium. (iii) The hydrolysis of the yellow-colored chlorophenol-red β-D-galactopyranoside (CPRG) into the purple product chlorophenol red (CPR) by the released β-galactosidase can be measured spectrophotometrically (iv). The intensity of the coloration is correlated with CPR concentration and thus directly proportional to the number of lysed reporter cells and to the attacker antibacterial activity. (B) Survivors growth kinetics (SGK). (i) After mixing attacker and reporter cells and incubation, antibiotic-resistant reporter cells are selected on liquid medium containing the antibiotic. The growth curve is compared to standard growth curves of a serially diluted reporter culture (grey lines and dots). A high-density sample (no competition, blue line) will thus reach a defined A600 (horizontal green line) faster than the low-density sample (active competition, red line). The time of emergence [Te, time necessary to reach the defined A600; Te(-), no competition; Te(+), active competition] can be plotted to the linear regression curve of slope α, obtained with the serially-diluted standards (ii), allowing the evaluation of the initial number of surviving cells in the sample.
Materials and reagents
Chlorophenol red β-D-galactopyranoside (CPRG), sodium salt (Roche, catalog number: 10884308001; Sigma-Aldrich, catalog number: 220588; or equivalent). Store powder and soluble preparation (1.2 mg/mL in water) at -20 °C for up to one year or at 4 °C for up to one week
Non-treated, clear, flat-bottom 96-well microtiter plates (Greiner Cellstar®, catalog number: M0812; Nunc, catalog number: 266120; or equivalent)
Equipment
Spectrophotometer for reading absorbance at λ = 572 and 600 nm
Microplate reader (Tecan, Infinite 200; or comparable microplate reader)
Incubators to grow bacterial cultures and to incubate plates.
1.5- and 2-mL plastic tubes
Procedure
A schematic diagram depicting the whole experimental procedure is shown in Figure 2.
Figure 2. Lysis-associated β-galactosidase assay (LAGA) and survivors growth kinetics (SGK) protocols at a glance. Attacker/predator and recipient/reporter/prey are cultured (A). Cell culture ODs are adjusted, and attacker and recipient mixed at the appropriate ratio (B). Cell mixtures are spotted on agar plates (C). Cell spots are either treated with chlorophenol-red β-D-galactopyranoside (CPRG) (D, LAGA assay) or resuspended and serially diluted into selective medium (E, SGK assay). After aliquoting into a 96-well plate (F), cell growth is spectrophotometrically monitored (G) and growth curves are plotted and compared to standard curves to measure the time of emergence (H).
LAGA protocol
Preparation of attacker (predator) and recipient (prey) cultures
Isolate the attacker(s) and the recipient(s) on specific medium agar plates, with any appropriate antibiotics if necessary.
Grow the strains in the adequate medium until the stationary phase.
Note: It is recommended to grow three independent colonies, for triplicate experiments.
Dilute the precultures in fresh medium to an optical density (A600) of 0.05–0.1 and incubate with shaking to the desired A600 (Figure 7A).
Notes:
To increase the expression of the lacZ gene, the recipient cell culture can be supplemented with 0.1 mM of IPTG.
lacZ expression could be significantly different depending on the recipient strain, growth medium, and growth conditions. We recommend using the same recipient culture with the different attackers for comparison.
Preparation of the attacker:recipient suspension and cell–cell contact
Prepare attacker and recipient cell suspensions at equal OD and mix them to the appropriate ratio (Figure 2B).
Note: For each attacker/recipient combination, the OD of the cell suspension and the attacker:recipient ratio should be optimized. Resuspension at 1–10 OD and 1:1–1:20 attacker:recipient ratio are typically used.
Spot 10 μL of the attacker:recipient cell suspension onto appropriate solid growth medium (medium allowing the growth of both attacker and recipient strains) and let dry (Figure 2C).
Notes:
It is recommended to spot each suspension three or four times for technical replicates (if three independent cultures and attacker:recipient have been made, this represents 9–12 spots for each attacker:recipient combination).
Control experiments should be performed by spotting the attacker only and the recipient only.
In some cases of predation that require flagellar motility (e.g., Bdellovibrio), the attacker:recipient cell mixture should be incubated in liquid medium.
For easy recovery of cells or in the case of sticky cells, the mixtures can be spotted on 0.45 μm membrane filters.
Incubate at the desired temperature for the optimized time.
Note: The optimal temperature depends on the attacker:recipient combination. The incubation time (usually between 1 and 24 h of contact) should be optimized.
CPRG hydrolysis and antibacterial activity evaluation
Add 10 μL of 1 mM of CPRG on top of the bacterial spot. Spot staining usually occurs in 10 min to 2 h (Figure 2D).
Note: CPRG hydrolysis could be measured with the supernatant. In this case, scrape the cells and resuspend the bacterial spot into 1 mL of PBS, centrifuge at 2,400× g for 5 min at 20 °C to pellet cells, and add 10 μL of 1 mM of CPRG into the supernatant. Measure the absorbance at λ = 572 and/or fluorescence (excitation at λ = 580 nm and emission at λ = 620 nm) after 10 min to 2 h. β-galactosidase activity could also be calculated by the slope obtained by measuring absorbance at λ = 572 every minute during 10 min, and expressed in μM/min using ϵM(CPR) = 45,000 M-1cm-1.
SGK protocol
Attacker and recipient cultures, preparation of attacker:recipient suspensions, and cell–cell contact
Follow sections A and B of LAGA protocol.
Serial dilutions and growth measures
Aliquot 100 μL of selective medium in the adequate number of wells of a 96-well microplate. Fill in extra wells with 200 μL of medium for blank measurements.
After incubation, scrape and resuspend the bacterial competition spots in 1 mL of selective medium in a 2 mL tube and vortex vigorously (Figure 2E).
Prepare serial dilutions of the suspensions (usually 1/2, 1/5, or 1/10 serial dilutions) (Figure 2E). At the same time, prepare 10-1 to 10-6 serial dilutions for the recipient alone to obtain standard curves.
Note: Serial dilutions to be done depend on the level of antibacterial activity and need to be optimized.
Add 100 μL of each serial dilution to the 96-well plates (Figure 2F).
Run a Tecan microplate reader program for measuring bacterial cell growth (A600) for 15 h with 5 min intervals at the optimal temperature with shaking (Figure 2G).
Note: To calculate a correlation between the initial number of surviving cells and the Te, plate 100 μL of recipient standard dilutions on selective agar plates. After incubation, count the number of colonies.
Plot the growth of the recipient standards and attacker:recipient suspensions over time and calculate the Te (Figure 2H). The Te could be represented on a graph alone (see Figures 6C and 7B) or with the standard curve (see Figure 5C).
Validation of protocol
CPR intensity is proportional to the number of lysed cells
To define whether the intensity of CPR correlates with the amount of released β-galactosidase, we first performed spectrophotometry analyses with lysed cells. LacZ+ W3110 E. coli K-12 cells were cultured at 37 °C to a A600nm of 1 in the presence of 0.1 mM IPTG and then lysed by sonication. The cleared supernatant was serially diluted and mixed with CPRG for 30 min; after quenching with 300 mM sodium carbonate, CPR concentration was spectrophotometrically measured using a microplate reader. Figure 3 shows that the CPR absorbance and fluorescence are function of the number of lysed cells, with a linear correlation ranging from 0.01 to 0.5 OD (R2 = 0.90 for absorbance and R2 = 0.95 for fluorescence).
Figure 3. β-galactosidase activity in serially diluted cell lysed extracts. Absorbance (A572) and fluorescence (F620) of chlorophenol red (CPR) after 30 min of incubation of serial dilutions of E. coli W3110 cell lysate in the presence of chlorophenol-red β-D-galactopyranoside (CPRG). Values are the means of three biological replicates with standard deviations.
Application of LAGA to estimate colicin activity
We first tested the applicability of the LAGA method to study colicin activity. Colicins are protein antibiotics released by E. coli and related species that penetrate and kill target cells through binding onto specific outer membrane receptors and translocation by exploiting transenvelope complexes such as the Tol (group A colicins) or TonB system (group B colicins) (Lazdunski et al., 1998; Cao and Klebba, 2002; Cascales et al., 2007; Housden and Kleanthous, 2012; Duché and Houot, 2019). 8 × 108 wild-type (WT) susceptible (W3110) or group B–resistant (W3110 ∆tonB; KP1344) E. coli cells were mixed with purified group B colicin D with a multiplicity of infection of ~100 and CPRG. The amount of CPR was then measured spectrophotometrically over time using a microplate reader. Figure 4 shows that no lysis of wild-type or ∆tonB cells is observed in the absence of colicin D. In contrast, wild-type cells treated with colicin D released β-galactosidase, whereas ∆tonB cells did not. Similar results were obtained with group A colicins, but in agreement with the role of the Tol-Pal system in maintaining outer membrane integrity (Lazzaroni et al., 1999; Lloubès et al., 2001; Szczepaniak et al., 2020), tol mutants released a significant amount of β-galactosidase even in the absence of colicin, and hence cannot be employed as controls. However, the LAGA approach can be used to measure Tol-dependent phenotypes in addition to SDS sensitivity, blebbing, and RNase I leakage (Fognini-Lefebvre et al., 1987; Bernadac et al., 1998; Cascales et al., 2002). Taken together, these results demonstrate that the LAGA method could be used to monitor the antibacterial activity of colicins. The SGK method is not suitable for studying sensitivity to colicins or other contact-independent antibacterial toxins, as they will remain in the supernatant during the growth recovery, unless cells are washed before measuring cell growth.
Figure 4. Colicin activity. Chlorophenol red (CPR) absorbance (A572, solid line) and fluorescence (F620, dashed line) of E. coli W3110 and ∆tonB cells treated (blue and purple) or not (red and green) with colicin D. Measurements were made every 15 min for 90 min after the addition of colicin and of chlorophenol-red β-D-galactopyranoside (CPRG). The mean of three biological replicates is indicated. The vertical bars represent standard deviations.
Application of LAGA and SGK methods to estimate type VI secretion-mediated activity
The type VI secretion system (T6SS) is a multiprotein machine widespread in Gram-negative bacteria, that uses a contractile mechanism to inject effectors into target cells, including bacteria (Cianfanelli et al., 2016; Brackmann et al., 2017; Cherrak et al., 2019; Coulthurst, 2019; Wang et al., 2020). T6SSs secrete a broad range of toxin effectors that usually target essential macromolecules such as nucleic acids, proteins, lipids, or peptidoglycan (Hernandez et al., 2020; Jurėnas and Journet, 2021). T6SSs can be distinguished based on their behavior: offensive T6SSs, such as in enteroaggregative E. coli and Serratia marcescens, assemble independently of target cell and fire any cell upon contact (Brunet et al., 2013; Gerc et al., 2015). By contrast, defensive T6SSs, such as the Pseudomonas aeruginosa H1 or Acinetobacter baumannii T6SSs, respond to cell–cell contacts or to cell envelope defects including those induced by a T6SS attack (Mougous et al., 2007; LeRoux et al., 2012; Basler et al., 2013; LeRoux et al., 2015; Lin et al., 2022). We thus tested whether the LAGA and SGK methods can be applied to the study of offensive and defensive T6SSs.
Enteroaggregative E. coli T6SS
The enteroaggregative E. coli (EAEC) strain 17-2 has been previously shown to eliminate E. coli K12 cells using its Sci1 T6SS (Flaugnatti et al., 2016). We tested the ability of a series of 17-2 strain isogenic derivatives to eliminate E. coli W3110 cells carrying a plasmid conferring ampicillin resistance and mCherry fluorescence. The 17-2 strain derivatives were chosen to provide antibacterial activities ranging from no activity (∆sci1; deletion of the entire sci1 T6SS gene cluster; Brunet et al., 2014), intermediate activities (∆tagA and ∆clpV retaining ~12% and ~4% of T6SS activity, respectively; Santin et al., 2018; Douzi et al., 2016), to maximal activity (WT strain). Reporter and attacker cell suspensions were adjusted to an A600 of 0.8 and mixed in a 1:4 ratio and 10 μL drops were spotted on synthetic minimal medium (inducing sci1 T6SS gene expression; Brunet et al., 2011) agar plates and incubated for 4 h at 37 °C. As shown in Figure 5A and 5B, the LAGA assay recapitulated previously published data, with a significant decrease of CPRG hydrolysis when ∆sci1 cells are used as attackers compared to wild-type 17-2 cells, and intermediate antibacterial activities of ∆tagA and ∆clpV cells.
Figure 5. T6SS activity in enteroaggregative E. coli (EAEC) cells. (A) Chlorophenol red (CPR) absorbance (A572, bars) and fluorescence (F620, red dots) of E. coli W3110 reporter cells after 4 h of incubation with the indicated EAEC attacker cells on solid synthetic minimal medium (SIM) agar. The mean of three biological replicates is indicated. The error bars represent standard deviation. (B) Qualitative evaluation of interbacterial activity. E. coli W3110 reporter cells producing mCherry were incubated (+) or not (-) with the indicated EAEC attacker cells on SIM agar for 4 h, before addition of 1 mM of chlorophenol-red β-D-galactopyranoside (CPRG) on spots. Antibacterial activity can be evaluated by the coloration of the spot due to CPRG hydrolysis or by the extinction of reporter cell mCherry fluorescence (f). (C) Time of emergence (Te, in hours) of the reporter strain. After 4 h of incubation with the indicated EAEC attacker cells, the reporter strain is selectively outgrown in lysogeny broth (LB) supplemented with ampicillin (means of three biological replicates, color diamonds). The linear regression of Te values of the standards (dilutions of reporter cells grown alone) is shown in dotted line (grey dots, means of three biological replicates; vertical bars, standard deviations).
Surviving recipient cells were then cultured in selective medium to measure the Te. Here, we used antibiotic for selective recovery, but any specific culture conditions excluding the attacker could be applied. To serve as standards, a recipient cell culture was serially diluted, and Te was measured. Figure 5C shows a linear regression of the Te values plotted as a function of the dilution factors (grey data points). Here, again, the data with the various T6SS mutant strains used as attackers were consistent with previous reports showing an absence of antibacterial activity of ∆sci1 cells (i.e., comparable to the recipient incubated in absence of attacker) and intermediate activities of ∆tagA and ∆clpV cells.
Pseudomonas aeruginosa H1-T6SS
P. aeruginosa strains usually carry several copies of T6SS gene clusters (Chen et al., 2015). Expression of the H1-T6SS, which is induced in the retS mutant background (Mougous et al., 2006), confers antibacterial activity (Hood et al., 2010; Russell et al., 2011). The H1-T6SS belongs to the family of the defensive T6SSs, as it has been established that it is activated by signals from lysed kin cells and by cell envelope damages such as those resulting from a T6SS attack by competitor cells (Basler et al., 2013; Ho et al., 2013; LeRoux et al., 2015). In agreement with these observations, the non-aggressive T6SS- strains DH5α pCR2.1, MG1655, and W3110 were lysed by P. aeruginosa PAK ∆retS cells with very low efficiency, while the aggressive T6SS+ EAEC 17-2 strain was efficiently lysed in a H1-T6SS-dependent manner when mixed in 1:1 ratio and incubated for 4 h (Figure 6A). Strains alone, as well as recipient strains mixed with the PAK∆retS∆H1-T6SS strain, remain yellow, indicating the absence of E. coli lysis.
We then tested different attacker strains of P. aeruginosa PAK: PAK∆retS∆tse1-tsi1 lacking the lytic amidase Tse1 and its immunity Tsi1, and PAK∆retS∆pppA lacking the PppA phosphatase that quenches the post-translation activation cascade (Figure 6B, lower panels). The results show that PAK lytic ability was strongly affected by the absence of the Tse1 effector. In contrast, the absence of PppA increased the antibacterial efficiency of the H1-T6SS apparatus, as previously shown (Mougous et al., 2007; Basler et al., 2013). To quantify the antibacterial activity of these strains, we measured the CPR absorbance after the addition of CPRG to the supernatants of the attacker/recipient mixtures (Figure 6B, upper panels). The spectrophotometric values correlated with the differences in coloration observed on agar plates. The LAGA protocol is thus sensitive enough to discriminate different levels of T6SS activity in P. aeruginosa.
We then performed the SGK protocol by monitoring recipient growth recovery. Here, again, the results shown in Figure 6C demonstrate that, with the exception of strain lacking Tse1, the times of emergence correlated well with the results of the LAGA assay. While the SGK assay measures cell survival, the LAGA assay measures β-galactosidase leakage and thus cell lysis. Because cell death is not necessarily associated to cell lysis, the difference for the PAK∆retS∆tse1-tsi1 can be explained by the fact that the Tse1 amidase causes the death of the recipient bacterium but does not lead to its lysis, as previously described (Russell et al., 2011; Chou et al., 2012). These data demonstrate that the two methods described here are relevant and complementary to better understand competition mechanisms.
Figure 6. H1-T6SS activity in P. aeruginosa PAK. (A) Selection of the best E. coli prey for antibacterial assay with P. aeruginosa PAK. Qualitative evaluation of P. aeruginosa PAK antibacterial activity using the lysis-associated β-galactosidase assay (LAGA) assay. The indicated attacker and recipient reporter strains were mixed and incubated for 4 h before addition of 1 mM of chlorophenol-red β-D-galactopyranoside (CPRG) on spots. The antibacterial activity can be evaluated by the coloration of the spot due to CPRG hydrolysis. (B and C) Antibacterial activity of P. aeruginosa PAK mutant strains. (B) Competition assays with the indicated P. aeruginosa PAK strains as attackers and enteroaggregative E. coli (EAEC) strain 17-2 pBBR1MCS-5 as recipient. P. aeruginosa and E. coli cells were grown in lysogeny broth (LB) medium to an A600 of 2 and in synthetic minimal medium (SIM) to an A600 of 0.8, respectively. Attackers and recipients were mixed in a 1:1 (v:v) ratio, and 10 μL of each mixture were spotted on dried LB agar plates and incubated for 4 h at 37 °C. Cells alone were spotted as controls. The lower panels show the spot assay with (+) or without (-) the recipient. The upper graph shows chlorophenol red (CPR) absorbance in the supernatants. (C) Time of emergence (Te, in hours) of the reporter EAEC 17-2 pBBR1MCS-5 strain after incubation with the indicated P. aeruginosa PAK attacker strains. The three biological replicate values (dots) and means (horizontal bars) are shown.
Application of LAGA and SGK methods to measure Myxococcus predation
M. xanthus is a predatory bacterium that hunts, attacks, and kills other microorganisms using a cell–cell contact mechanism involving the Kil Tad–like apparatus (Thiery and Kaimer, 2020; Zhang et al., 2020; Seef et al., 2021; Thiery et al., 2022). Mutation of genes encoding Tad subunits results in a partial or total loss of M. xanthus predation efficiency (Seef et al., 2021; Thiery et al., 2022). The LAGA colorimetric assay was optimized to evaluate predation by M. xanthus using kil mutants presenting intermediate (∆kilK and ∆kilKL) or null (∆kilC and ∆kilACF) predation efficiency (Seef et al., 2021).
The predation assay was performed in clone fruiting (CF) liquid medium for 24 h with M. xanthus as predator and lacZ+ E. coli MG1655 as prey, in a 1:20 ratio, by mixing 100 μL of E. coli at A600 of 10 and 100 μL of M. xanthus at A600 of 0.5. Measures of the A572 after addition of CPRG in culture supernatant revealed that predation levels of the ∆kilK and ∆kilKL strains were 1.5–2 times lower than the wild type, while β-galactosidase activities were comparable to the prey in absence of predator for the ∆kilC or ∆kilACF strains (Figure 7A). This result suggests that the LAGA CPRG colorimetric assay is an appropriate method to evaluate M. xanthus predation efficiency and is sensitive and robust enough to reveal subtle differences in its capacity to lyse a prey.
To test the SGK assay, a suspension of kanamycin-resistant E. coli cells was mixed with the different M. xanthus strain suspensions adjusted to an A600 of 5 in a predator:prey ratio of 1:8, and 10 μL were spotted on a CF agar plate. After 7 h of predation, cells were harvested, and the Te was measured to determine the relative number of E. coli cells that survived to predation. Figure 7B shows that all kil mutants presented an increase in E. coli survival in comparison to WT. In terms of predation, no intermediate phenotypes could be observed for ∆kilK and ∆kilKL. This method is therefore suitable to quantify the killing activity of M. xanthus. However, the difference between the LAGA and SGK for the kilKL and kilK mutants suggests that the SGK assay is not sensitive enough to reveal intermediate efficiencies, or that these mutants cause quasi-lysis, as shown for colicinogenic bacteria (Pugsley, 1983), an increase of cell permeability with limited impact on survival.
Figure 7. M. xanthus predatory activity. (A) Chlorophenol red (CPR) absorbance (A572) of the supernatants of co-incubation between the indicated Myxococcus strains as predators and E. coli MG1655 as prey for 24 h. The mean and standard deviations of three independent experiments are shown. The blue dotted horizontal bar represents the lysis of the prey alone. (B) Quantitative measure of M. xanthus predation over E. coli after 7 h of co-incubation using the survivors growth kinetics (SGK) method. Time of emergence (Te, in hours) of the MG1655 prey strain after incubation with the indicated M. xanthus predator strains. The five biological replicate values (dots) and means (horizontal black bars) are shown.
Conclusive remarks
We describe here the adaptation of a CPRG-based approach to estimate antibacterial competition, LAGA, and a quantitative and complementary approach, SGK. The first procedure measures the amount of CPR (red) due to the hydrolysis of CPRG (yellow) by the β-galactosidase released by bacterial lysis. The second procedure measures the amount of surviving attacker/recipient cells by following their growth in a selective medium. The two procedures are complementary: LAGA allows to measure lysis while SGK measures survival. The importance of using the two approaches has been exemplified here. As shown for P. aeruginosa Tse1 mutant, cell death is not systematically associated with cell lysis. By contrast, the M. xanthus kilK and kilKL examples showed that, in some conditions, bacteria can survive quasi-lysis. Hence, the systematic use of the two methods may help to better understand the molecular mechanisms underlying interbacterial competition by discriminating processes responsible for cell death and lysis, cell death without lysis, or survival to quasi-lysis.
Although these approaches are based on the use of recipient cells with specificities (producing the β-galactosidase for LAGA, presenting a character permitting selection for SGK), the two methods have significant advantages: they are simple to set up and optimize in any laboratory, fast, inexpensive, and not laborious. In addition, the two approaches can be adapted to different attacker bacterial species.
Acknowledgments
The LAGA protocol was adapted from previous studies (Vettiger and Basler, 2016; Sgro et al., 2018; Bayer-Santos et al., 2019; Schneider et al., 2019; Seef et al., 2021). We thank the members of the Cascales, Mignot, and Bordi laboratories for discussions and support, Kathleen Postle (Penn state University, USA) and Denis Duché (LISM, Marseille) for providing strain KP1344 and purified colicin D, respectively, Moly Ba, Annick Brun, and Audrey Gozzi for technical support, and the four anonymous referees for their very constructive comments and suggestions. This work was supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and by grants from the Agence Nationale de la Recherche (ANR-20-CE11-0017), the Fondation pour la Recherche Médicale (FRM, DEQ20180339165) and the Excellence Initiative of Aix-Marseille University - A*MIDEX, a French “Investissements d’Avenir” program (A-M-AAP-ID-17-33-170301-07.22) to E.C.
Competing interests
The authors declare that they have no competing interests.
Ethical considerations
Ethics approval was not required for this study.
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4,707 | https://bio-protocol.org/en/bpdetail?id=4707&type=0 | # Bio-Protocol Content
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Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction
GD Gloria Delfanti
PD Paolo Dellabona
GC Giulia Casorati
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4707 Views: 654
Reviewed by: Luis Alberto Sánchez VargasXiaokang Wu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Science Immunology Aug 2022
Abstract
Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow–derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.
Keywords: Primary iNKT cells Retrovirus transduction iNKT cell purification iNKT cell engineering iNKT cell expansion TCR transfer
Background
Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant αβ T-cell receptor (TCR), which is formed in mice by an invariant Vα14-Jα18 chain paired with a limited set of diverse Vβ chains (Bendelac et al., 2007). iNKT cells react to CD1d-restricted self and non self-lipid antigens in stress conditions, and are strongly implicated in tumor immunosurveillance (Godfrey et al., 2018). iNKT cells are evolutionary conserved; their investigation benefits from the availability of mouse models and the unequivocal detection, in mice and humans, by antigen-loaded CD1d tetramers and monoclonal antibodies specific for the semi-invariant TCR.
Several studies have shown that iNKT cells are an active component of the tumor microenvironment (TME) and control tumor progression by restraining cancer-supporting myeloid populations, such as modulating myeloid-derived suppressor cells and killing tumor-associated macrophages (De Santo et al., 2008 and 2010; Song et al., 2009; Liu et al., 2012; Gorini et al., 2017; Janakiram et al., 2017; Cortesi et al., 2018). Retargeting iNKT cells against cancer cells, by transducing tumor-specific TCR genes, generates enhanced effectors able to concurrently kill malignant cells and modulate detrimental myeloid cells in TME (Delfanti et al., 2022). This enhanced anti-tumor activity is peculiar of TCR-engineered iNKT, in which both the endogenous invariant TCR and the exogenous tumor-specific TCR exert anti-tumor effects—unlike conventional T cells, in which the endogenous TCRs are irrelevant for the therapeutic action, or even detrimental for possible off-target effects (Wolf et al., 2018). Therefore, iNKT cells are an attractive adoptive cell therapy (ACT) platform for engineering with anti-tumor TCRs or CAR (Heczey et al, 2020; Rotolo et al, 2018; Landoni et al, 2020; Delfanti et al., 2022), providing an appealing alternative to conventional T cells for the treatment of solid and hematological malignancies.
Because they are rare, iNKT cell harnessing for ACT requires two obligatory steps, consisting of ex vivo pre-selection followed by substantial in vitro expansion. We previously set up an optimized protocol for splenic iNKT cell purification and expansion (Delfanti et al., 2021) that can be used as the starting point for iNKT cell engineering. iNKT cells are enriched from the spleen of iVα14-Jα18 transgenic mice (iVα14 tg) (Griewank et al., 2007), in which iNKT cells are 30 times more frequent than in wildtype mice, significantly reducing the number of mice needed for the procedure. The setup of the cell culture requires only one day, instead of one week required by another widespread published protocol in which iNKT cells are stimulated with bone marrow–derived dendritic cells pulsed with the strong iNKT cell agonist α-GalCer (Chiba et al., 2009). In our protocol, iNKT cells are activated by anti-CD3/CD28 beads, and this activation is also useful for the retroviral (RV) transduction, which is done 48 h after. This procedure allows the generation of millions of ready-to-use, highly pure, and stably transduced iNKT cells within 15 days, so that they can be easily employed for functional studies (Figure 1). Indeed, by this method, we could engineer iNKT cells to express a second anti-tumor TCR (OT-I TCR), while maintaining the expression of the endogenous one, and we proved their efficacy both in vitro and in vivo (Delfanti et al., 2022). This protocol is also suitable for iNKT cell engineering with other effector molecules such as CARs or cytokines. A reporter gene, such as GFP, can also be introduced in the RV sequence to track iNKT cell transduction by flow cytometry over time.
Figure 1. Timeline for engineered invariant natural killer T (iNKT) cell production. Schematic representation of the protocol passages that lead to the generation of engineered iNKT cells.
Materials and reagents
Millex-HV 0.45 μm filter (Merck Millipore, catalog number: SLHVM33RS)
Polypropylene centrifuge tubes (Beckman Coulter, catalog number: 326823)
Microplate 96-well shape (Greiner Bio One, catalog number: 650101)
75 cm2 flask (Corning, catalog number: 430720U)
100 mm × 20 mm tissue culture dish (Falcon, catalog number: 353003)
5 mL polystyrene round-bottom tube (Falcon, catalog number: 352052)
70 μm cell strainer (Falcon, catalog number: 352350)
15 mL polypropylene conical tube (Euroclone, catalog number: ET5015B)
50 mL polypropylene conical tube (Euroclone, catalog number: ET5050B)
iVα14-Jα18 tg mice, strain name: C57BL/6-Tg(Cd4-TcraDN32D3)1Aben/J, purchased from the Jackson Laboratory (USA)
IMDM, with HEPES and L-Gln (Lonza, catalog number: 12-277F)
Hyclone FetalClone I (Cytiva, catalog number: SH30080.03)
Penicillin/streptomycin solution (Gibco, catalog number: 15140-122)
Phoenix-eco packaging cell line (ECO) cells, purchased from ATCC
Chloroquine (Sigma-Aldrich, catalog number: C6628)
dH2O, tissue-culture grade (Sigma-Aldrich, catalog number: W-4502)
TE buffer (Invitrogen, catalog number: 8019005)
Vector DNA plasmid, purified using PureLink, HiPure Plasmid Filter Maxiprep kit (Invitrogen, catalog number: K210017)
Dulbecco’s phosphate buffered saline (PBS) (Euroclone, catalog number: ECB4004L)
Fetal bovine serum (FBS), ultra-low endotoxin (Euroclone, catalog number: ECS0186L)
Purified rat anti-mouse CD16/CD32 (mouse BD Fc block), clone: 2.4G2 (BD Pharmingen, catalog number: 553142)
CD19-FITC, clone: 6D5 (BioLegend, catalog number: 115506)
H2(IAb)-FITC, clone: AF6-120.1 (BioLegend, catalog number: 114406)
TCRβ-APC, clone: H57-597 (BioLegend, catalog number: 109212)
Vβ5.2 PE/Cy7, clone MR9-4 (BioLegend, catalog number: 139508)
Mouse PBS57-CD1d-tetramer, provided by NHI tetramer core facility
DAPI (Santa Cruz, catalog number: SC-3598)
Live/Dead Zombie Violet Fixable Viability kit (BioLegend, catalog number: 423114)
Bovine serum albumin (BSA) fraction V (Roche, catalog number: 10735094001)
EDTA stock solution (Cayman Chemical Company, catalog number: 600215)
Anti-FITC microbeads (Miltenyi Biotec, catalog number: 130-048-801)
Anti-PE microbeads (Miltenyi Biotec, catalog number: 130-048-701)
LD column (Miltenyi Biotec, catalog number: 130-042-901)
Pre-separation filter (Miltenyi Biotec, catalog number: 130-041-407)
LS column (Miltenyi Biotec, catalog number: 130-042-401)
MS column (Miltenyi Biotec, catalog number: 130-042-201)
Dynabeads mouse T-Activator CD3/CD28 (Gibco, catalog number: 11452D)
RPMI 1640-GlutaMAX (Gibco, catalog number: 61870-010)
Non-essential amino acids (Gibco, catalog number: 11140-035)
Sodium pyruvate (Gibco, catalog number: 11360-039)
β-mercaptoethanol (Gibco, catalog number: 31350010)
Polybrene hexadimethrine bromide (Sigma-Aldrich, catalog number: H9268)
(hr)IL-2 (Chiron Corp)
Recombinant mouse IL-7 (R&D System, catalog number: 407-ML-025)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)
CaCl2 (Sigma-Aldrich, catalog number: C9702)
NH4Cl (Sigma-Aldrich, catalog number: A0171)
KHCO3 (Sigma-Aldrich, catalog number: 237205)
N2EDTA (BDH, catalog number: 280254-D)
NaCl (Sigma-Aldrich, catalog number: S5886)
HEPES (Sigma-Aldrich, catalog number: H4034)
Na2HPO4 (Sigma-Aldrich, catalog number: S3264)
IMDM + 10% Hyclone (see Recipes)
TE 0.1-H2O (see Recipes)
2× HEPES buffered saline (HBS) (see Recipes)
ACK (ammonium-chloride-potassium) lysing buffer (see Recipes)
MACS separation buffer (see Recipes)
Complete RPMI (see Recipes)
Equipment
MACS® MultiStand (Miltenyi Biotec, catalog number: 130-042-303)
QuadroMACSTM Separator (Miltenyi Biotec, catalog number: 130-090-976)
DynaMagTM-15 Magnet (Invitrogen, catalog number: 12301D)
Polyallomer centrifuge tubes (Beckman Coulter, catalog number: 326823)
Eppendorf centrifuge (Eppendorf, model: 5810 R)
Ultracentrifuge (Beckman Coulter, model: Optima XPN-90)
Rotor (Beckman Coulter, model: SW32Ti)
S@FEGROW 188 incubator (Euroclone)
FACS Canto II (BD Biosciences)
Software
FlowJo_V10 (BD Biosciences)
Procedure
Retrovirus production
Note: Universal precautions must be taken while handling RV supernatants and RV-transduced samples, and all experiments must be carried out in at least class II biological safety cabinets and using appropriate protection equipment. RV must be handled and disposed of in accordance with your institutional biohazard control regulations.
Maintain Phoenix-ECO cells in 10 mL of IMDM + 10% Hyclone (see Recipe 1) in a T75 flask in a 37 °C incubator supplied with 5% CO2.
Seed 2 × 106 Phoenix-ECO cells into a 100 mm × 20 mm tissue culture dish with 9 mL of IMDM + 10% Hyclone.
Transfect Phoenix-ECO cells at ~70%–80% confluency, normally ~20–24 h after seeding.
Ten minutes before transfection, add 25 μL of 10 mM chloroquine to the cell and incubate at 37 °C.
Prepare the plasmid mix:
125 μL of 1 M CaCl2
20 μg of vector plasmid DNA (20 μL if DNA concentration is 1 μg/μL)
and the proper volume of TE 0.1-H2O (see Recipe 2) to reach a final volume of 500 μL
While vortexing the plasmid mix, add 500 μL of 2× HBS (see Recipe 3) (use the pipette gun and a 2 mL pipette).
Immediately add (with the same pipette) the precipitate to the Phoenix-ECO cells drop by drop.
Return cells to the incubator.
After 14–16 h, completely replace the medium and add 7 mL of fresh prewarmed IMDM + 10% Hyclone.
Twenty-four hours after step A9, collect culture media and replace with 7 mL of fresh prewarmed IMDM + 10% Hyclone.
Centrifuge the collected media at 300× g for 5 min at 4 °C and then pass through a 0.45 μm filter into a clean 50 mL tube. Store filtered media at 4 °C.
Twenty-four hours after step A11, repeats steps A10 and A11 and combine filtered media into 50 mL tubes.
Make 32 mL aliquots of retroviral supernatant in ultracentrifuge tubes (Polyallomer centrifuge tubes) carefully balancing the volumes (add medium or PBS if necessary).
Ultracentrifuge at 69,000× g for 2 h at 4 °C (rotor SW32Ti).
Discard supernatant and pay attention to dry all drops that may remain on the tube walls.
Add 70 μL of PBS per tube and wait 15 min at room temperature (RT).
Resuspend the samples at least 10 times setting the pipette volume at 60 μL, without creating bubbles, and pool all the samples in a 1.5 mL Eppendorf tube.
Parafilm the Eppendorf tube and rotate on a wheel for 30 min at 4 °C.
Make 20 μL aliquots and store at -80 °C. Under these conditions, RV may be stored up to two years after production.
Primary iNKT cell purification
Note: All the procedures must be performed under sterile conditions.
Spleen processing
Euthanize iVα14-Jα18 mice according to institutional policy, by inhalation of CO2.
Note: iVα14-Jα18 mice must be eight weeks old or older.
To dissect the spleen, place the animal on a clean dissection board and rinse with 70% reagent alcohol. Incise the abdominal cavity and collect the spleen using scissors and tweezers. The spleen is located to the left side of the abdomen, inferior to the stomach (Dowling et al., 2020).
Place a 70 μm cell strainer on a 50 mL tube and prepare the strainer by rinsing with 3 mL of PBS + 2% FBS (hereafter PBS-FBS).
Place the spleen on the strainer and homogenize the tissue via grinding in 10–20 mL final volume of PBS-FBS.
Centrifuge at 450× g for 5 min at 4 °C.
Remove the supernatant by inversion and resuspend the cell pellet with 1 mL of sterile ACK lysing buffer (see Recipe 4); for red blood cell lysis, incubate for 3 min at room temperature and block with 5 mL of PBS-FBS.
Centrifuge at 450× g for 5 min at 4 °C.
Remove the supernatant by inversion and resuspend the cell pellet in 3 mL of PBS-FBS. Remove fat residues by pipetting and determine cell number.
Note: Expected recovery from the spleen of one iVα14-Jα18 mouse is 2 × 106 iNKT cells; if more iNKT cells are needed, increase the number of mice accordingly. Cells coming from different mice can be pooled at this point prior to the determination of cell number.
T-cell enrichment
Note: For the enrichment steps, work fast, keep the cells cold, and use solutions pre-cooled at 4 °C overnight and then kept on ice.
Resuspend the total spleen cells in the appropriate volume of PBS-FBS (500 μL for 107 cells) + Fc blocker (5 μL × 107 cells) and incubate for 15 min at RT.
Wash with 1–2 mL of MACS separation buffer (MB) (see Recipe 5) per 107 total cells and centrifuge at 450× g for 10 min at 4 °C.
Remove the supernatant by inversion and stain the cell with CD19-FITC and H2(IAb)-FITC (use 5 μL × 107 cells in 100 μL of MB); mix well and incubate for 15 min in the dark at 4–8 °C.
Wash cells by adding 1–2 mL of MB per 107 cells and centrifuge at 450× g for 10 min at 4 °C.
Pipette off the supernatant completely and resuspend the cell pellet in 90 μL of MB per 107 total cells.
Add 10 μL of anti-FITC microbeads per 107 total cells. Mix well and incubate for 15 min in the dark at 4–8 °C.
Wash the cells by adding 1–2 mL of MB per 107 cells and centrifuge at 450× g for 10 min at 4 °C.
Pipette off the supernatant completely and resuspend up to 1.25 × 108 cells in 500 μL of MB.
Place a LD column in the magnetic field of MACS Separator to proceed with the depletion. To avoid clogging, apply a pre-separation filter on the LD column and rinse with 2 mL of MB. When the column reservoir is empty, apply the cell suspension onto the filter. This step depletes B and myeloid antigen-presenting cells from the spleen cell suspension.
Collect the unlabeled cells that pass through the column.
Wash three times with 1 mL of MB, only when the column reservoir is empty.
Collect the total effluent, which will be enriched in T cells, and count the cells.
iNKT cell enrichment
Centrifuge the T cell–enriched suspension at 450× g for 5 min at 4 °C and remove the supernatant by inversion.
Stain the cells with CD1d-tetramer-PE (mouse PBS57-CD1d-tetramer), according to the antibody titration, in 50 μL of MB per 106 cells. Mix well and incubate for 30 min in the dark on ice.
Wash the cells by adding 1–2 mL of MB per 107 cells and centrifuge at 450× g for 10 min at 4 °C.
Pipette off the supernatant completely and resuspend the cell pellet in 80 μL of MB per 107 total cells.
Add 20 μL of anti-PE microbeads per 107 total cells. Mix well and incubate for 15 min in the dark at 4–8 °C.
Wash cells by adding 1–2 mL of MB per 107 cells and centrifuge at 450× g for 10 min at 4 °C.
Pipette off the supernatant completely and resuspend up to 108 cells in 500 μL of MB; otherwise, if cells exceed 108, adjust the volume accordingly.
According to cell count, place a LS (up to 108) or MS (up to 107) column in the magnetic field of MACS Separator. Rinse the column with MB (3 mL for LS, 500 μL for MS).
Apply the cell suspension onto the column. Collect unlabeled cells that pass through.
Wash column three times by adding the appropriate volume of MB (3 × 3 mL for LS column, 3 × 500 μL for MS column), only when the column reservoir is empty. The total effluent is the negative fraction.
Remove column from the magnetic field and place it on a new collection tube.
Add MB onto the column (5 mL for LS column or 1 mL for MS column), push the provided plunger into the column, and flush out the positive fraction (enriched in iNKT cells).
To further increase the iNKT cell recovery, centrifuge the negative fraction at 450× g for 10 min at 4 °C and repeat steps h–l with a new LS or MS column.
Pool the positive fractions and determine cell count.
Check the purification steps by FACS analysis. Samples are spleen ex vivo, T cell–enriched fraction, iNKT-positive fraction, and iNKT-negative fraction. Stain the cells with CD19-FITC, IAb-FITC, CD1d-tetramer-PE, TCRβ-APC, and DAPI (Figure 2 and Figure 3).
Figure 2. Primary invariant natural killer T (iNKT) cell purification. A. Schematic representation of the purification protocol. B. Flow cytometry analysis of each enrichment step. Upper plots show the percentage of T-cell frequencies, gated on viable lymphocytes (see data analysis section for details). Lower plot show the percentage of iNKT cells along each step, gated on viable CD19- IAb- TCRβ+ lymphocytes. Adapted from Delfanti et al. (2021).
Figure 3. Gating strategy for analysis of invariant natural killer T (iNKT) cells purification steps. Representative dot plots of gating strategy for analysis of ex vivo splenic iNKT cells used to evaluate each purification step.
iNKT cell activation and transduction
Activate purified iNKT cells with mouse T activator anti-CD3/CD28 magnetic beads in 1:1 ratio.
Centrifuge the iNKT cell positive fraction at 450× g for 5 min at 4 °C.
Meanwhile, transfer the appropriate volume of mouse T activator anti-CD3/CD28 magnetics beads to a 15 mL tube, add an equal volume of PBS, vortex for 5 s, place the tube on the magnet (DynaMagTM-15 Magnet) for 1 min, and discard the supernatant.
Remove the tube from the magnet and resuspend the washed magnetic beads in the proper volume of complete RPMI (see Recipe 6) to have 5 × 105 iNKT cells in 1 mL. Use this suspension to resuspend the iNKT cell pellet.
Plate 1 mL of the cell suspension (5 × 105 iNKT cells) + anti-CD3/CD28 magnetic beads in a 48-well plate with 20 U/mL human recombinant (hr)IL-2 and incubate at 37 °C.
Transduce iNKT cells after 48 h in culture.
Note: All handling of retroviral production and transduction is carried out in at least class II biosafety cabinets.
Centrifuge the 48-well plate at 450× g for 7 min at RT.
Carefully remove the cell media by pipetting, starting from the top of the well to avoid disturbing the cells.
Add 200 μL of complete RPMI, plus the appropriate volume of concentrated RV (Section A), and 8 μg/mL of polybrene to the wells.
Note: Always leave one well for the non-transduced (NT) control by adding complete RPMI and 8 μg/mL polybrene only.
Centrifuge the plate at 800× g for 2 h at 30 °C.
Add 800 μL of prewarmed media to each well with original culture conditions [complete RPMI and 20 U/mL of (hr)IL-2] and incubate at 37 °C.
Analyze iNKT cell transduction five days post-infection as described below (Section E).
iNKT cell expansion
Split the cells 1:2 when they reach 80%–90% confluency (usually starting from day 3–4 after activation)
Maintain the cell in complete RPMI and 20 U/mL of (hr)IL-2 until day 5 in culture.
After five days in culture, add 10 ng/mL of mouse IL-7.
In these culture conditions [complete RPMI, 20 U/mL of (hr)IL-2, 10 ng/mL of mouse IL-7], iNKT cells can be expanded for up to 15 days, when in vivo or in vitro assays can be performed.
Note: For iNKT adoptive cell transfer, beads have to be removed from the cell culture by loading the cell suspension on the magnet (incubate for 3 min). Cells are washed three times with PBS before injection.
Analysis of iNKT cell transduction
At five days post-infection, iNKT cell transduction can be assessed by flow cytometry.
Note: Work under a class II biosafety cabinet, since at this time point some RV particles can still be present in cell supernatant.
Collect 200 μL of cell suspension and transfer to a U-bottom 96-well plate for staining.
Centrifuge at 450× g for 5 min at RT.
Remove the supernatant by pipetting (change the tips and bleach them).
Add 50 μL of PBS + 0.5 μL of Fc blocker per sample and incubate for 15 min at RT.
Centrifuge at 450× g for 5 min at 4 °C.
Remove the supernatant, add 50 μL of PBS + 0.1 μL of Live/Dead Fixable Violet per sample, and incubate for 20 min at RT.
Centrifuge at 450× g for 5 min at 4 °C.
Remove the supernatant and stain the cells with CD1d-tetramer-PE, according to the antibody titration, in 50 μL of PBS-FBS per sample. Mix well and incubate for 30 min in the dark on ice.
Centrifuge at 450× g for 5 min at 4 °C.
Meanwhile, prepare the flow cytometry mix for surface staining:
100 μL of PBS-FBS.
1 μL of TCRβ-APC and 1 μL of Vβ5.2-PE/Cy7 (for OT-I TCR staining) per sample.
Note: If the RV contains a GFP reporter, avoid the use of FITC-conjugated antibodies for the cell staining.
Remove the supernatant and add 100 μL of flow cytometry mix. Mix well and incubate for 15 min in the dark on ice.
Centrifuge at 450× g for 5 min at 4 °C.
Remove the supernatant and add 200 μL of 2% (w/v) PFA in PBS. Incubate for 10 min in the dark on ice.
Centrifuge at 600× g for 7 min at 4 °C.
Remove the supernatant and wash twice with PBS: add 200 μL of PBS, centrifuge at 450× g for 5 min at 4 °C, and repeat.
Resuspend the cells in 200 μL of PBS and transfer to flow cytometry tubes for FACS analysis.
Use gating strategy in Figure 4 to identify transduced iNKT cells.
iNKT cell transduction can also be assessed later on over cell culture. Starting from day 7 after infection, fixation with PFA is no longer required.
Data analysis
Use gating strategy lymphocytes/single cell/viable cells/ CD19- IAb- TCRβ+/TCRβ+ CD1d tetramer+ for analysis of ex vivo splenic iNKT cells (Figure 3).
Use gating strategy lymphocytes/single cell/viable cells for analysis of transduced iNKT cells (Figure 4). Here, iNKT cells were transduced to express the OT-I TCR, and transduction efficiency can be evaluated by the expression of the Vβ5.2, which is the specific Vβ chain of the OT-I TCR. The co-expression of the endogenous invariant TCR can be evaluated by staining iNKT cells also with the CD1-tetramer. In case the RV also encodes a GFP reporter, the transduction efficiency can simply be evaluated by the GFP expression.
Figure 4. Gating strategy for analysis of transduced invariant natural killer T (iNKT) cells. Representative dot plots of gating strategy for analysis of control non-transduced (NT), OT-I TCR, and OT-I TCR GFP-transduced iNKT cells. The expression of the transgene is assessed on viable cells by staining with anti-Vβ5.2 monoclonal antibody (mAb). Dotted grey lines in the histograms represent the NT cells. When the RV also expresses a GFP reporter (bottom line), iNKT cells transduction can also be evaluated by the GFP expression.
Notes
iVα14 tg mice are commercially available; however, their availability could be a limitation for the reproducibility of the current protocol. In absence of these mice, we envisage the possibility of using a large number of C57BL/6 mice, but the protocol needs to be set up accordingly due to the paucity of iNKT cells in C57BL/6 mice [iNKT cell frequencies in C57BL/6 and iVα14 tg are shown in Supplementary Figure 1 of Delfanti et al. (2022)].
For unequivocal detection of iNKT cells in the present protocol, we used mouse PBS-57-CD1d-tetramers provided by NIH. PBS-57 is an analog of the prototypical antigen recognized by iNKT cells αGal-Cer, with improved solubility (Liu et al., 2006). The NIH Tetramer Facility provides PBS-57 ligand complexed to CD1d tetramers. However, we envisage the possibility to adjust the protocol for the use of commercially available CD1d dimers/tetramers/dextramers that can be loaded with lipid antigens as αGal-Cer.
For optimal iNKT cell transduction, it is recommended to determine the RV titer. To do so, actively proliferating mouse hybridoma cells (i.e., 58α-β- cells) can be transduced as described in Section C steps 5–6, with serial dilution of the RV (from 1:50 up to 1:100,000). Five days after transduction, the transgene expression can be assessed by flow cytometry (fix cells with 2% PFA), and RV titer (expressed as transducing units/milliliter, TU/mL) is calculated according to the formula [N × (F/100) × D]/0.2, where N is the number of the transduced cells (usually 2.5 × 105), F is the percentage of cells expressing the transgene, and D is the fold dilution of virus used in transduction. The RV titer allows the calculation of the multiplicity of infection (MOI) with the following formula: MOI = V(mL) × TU/mL/N.
Transduction efficacy can vary depending on the construct used. In the case of the TCR transfer (Delfanti et al., 2022), we obtained a good (60%) transduction using MOI 4 of our RV. However, there are two possible strategies to increase iNKT cell transduction efficacy: by increasing the MOI employed, or by transducing iNKT cells 48 h after activation and repeating the transduction also 72 h after the activation.
Fresh, non concentrated RV supernatants can also be employed for iNKT cell transduction. In this case, the maximum volume of RV that can be added to the 48-well plate is 1.5 mL. We recommend always adding 8 μg/mL of polybrene and performing spin inoculation as described above.
Recipes
IMDM + 10% Hyclone
IMDM with L-Glutamine
10% Hyclone
1× Penicillin/streptomycin solution
TE 0.1-H2O
Dilute TE buffer 1:10 in dH2O to obtain TE 0.1; then, mix two volumes of TE 0.1 and one volume of dH2O.
2× HEPES buffered saline (HBS)
281 mM NaCl
100 mM HEPES
1.5 mM Na2HPO4
Dissolve in dH2O, pH 7.14, and 0.22 μm filter.
Store at -20 °C and avoid re-freezing.
ACK (ammonium-chloride-potassium) lysing buffer
0.15 M NH4Cl
10 mM KHCO3
0.1 mM N2EDTA
Dissolve in dH2O, pH 7.2–7.4, and 0.22 μm filter.
Store at 4 °C. ACK is also commercially available.
MACS separation buffer
0.5% BSA
2 mM EDTA stock solution
In PBS, pH 7.2, 0.22 μm filtered
Store at 4 °C. MACS buffer is also commercially available.
Complete RPMI
RPMI 1640-GlutaMAX
10% heat-inactivated FBS
1× Penicillin/streptomycin solution
1× non-essential amino acids
1 mM sodium pyruvate
50 μM 2-mercaptoethanol
Acknowledgments
This work was funded by Associazione Italiana Ricerca sul Cancro (AIRC) project grant IG2017-ID.20081 (to G.C.), AIRC “under-5-per-Mille” 2019-ID.22737 (to P.D.), Italian Healthy Ministry project on CAR T RCR-2019-23669115 (to P.D.), Worldwide Cancer Research project grant 19-0133 (to G.C.). G.D. was supported by FIRC-AIRC Fellowship number 2019-22604 and by the “young researchers mobility programme” fellowship by Associazione Giovanna Tosi per la lotta contro i tumori. The authors acknowledge the NIH tetramer facility for providing the mouse CD1d tetramers. The protocol presented here was adapted from previously published works (Delfanti et al., 2021 and 2022).
Competing interests
The authors declare no conflict of interest.
Ethical considerations
All procedures were reviewed and approved by the San Raffaele Scientific Institute Institutional Animal Care and Use Committee (678 and 1067) and by the Italian Ministry of Health (Rome, Italy) and were conducted in compliance with national laws and policies.
References
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Cortesi, F., Delfanti, G., Grilli, A., Calcinotto, A., Gorini, F., Pucci, F., Lucianò, R., Grioni, M., Recchia, A., Benigni, F., et al. (2018). Bimodal CD40/Fas-Dependent Crosstalk between iNKT Cells and Tumor-Associated Macrophages Impairs Prostate Cancer Progression. Cell Rep 22(11): 3006-3020.
Delfanti, G., Cortesi, F., Perini, A., Antonini, G., Azzimonti, L., de Lalla, C., Garavaglia, C., Squadrito, M. L., Fedeli, M., Consonni, M., et al. (2022). TCR-engineered iNKT cells induce robust antitumor response by dual targeting cancer and suppressive myeloid cells. Sci Immunol 7(74): eabn6563.
Delfanti, G., Perini, A., Zappa, E. and Fedeli, M. (2021). Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies. J Vis Exp (168). doi: 10.3791/62214.
De Santo, C., Arscott, R., Booth, S., Karydis, I., Jones, M., Asher, R., Salio, M., Middleton, M. and Cerundolo, V. (2010). Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A. Nat Immunol 11(11): 1039-1046.
De Santo, C., Salio, M., Masri, S. H., Lee, L. Y., Dong, T., Speak, A. O., Porubsky, S., Booth, S., Veerapen, N., Besra, G. S., et al. (2008). Invariant NKT cells reduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans. J Clin Invest 118(12): 4036-4048.
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Heczey, A., Courtney, A. N., Montalbano, A., Robinson, S., Liu, K., Li, M., Ghatwai, N., Dakhova, O., Liu, B. and Raveh-Sadka, T., et al. (2020). Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis. Nat Med 26(11): 1686–1690.
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4,708 | https://bio-protocol.org/en/bpdetail?id=4708&type=0 | # Bio-Protocol Content
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3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM)
NF Nuria Ferrandiz
SR Stephen J. Royle
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4708 Views: 674
Reviewed by: Nafisa M. Jadavji Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in The Journal of Cell Biology Apr 2022
Abstract
Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.
Keywords: Chromosome missegregation Mitosis Endoplasmic reticulum Volume EM Microscopy Scanning electron microscopy
Background
Cell division is essential for living organisms, as it is important for growth, repair, development, and reproduction. Accurate chromosome segregation during mitosis is essential to maintain genomic stability in cell division. Entry into mitosis involves a dramatic, large scale cellular reorganization. After the nuclear envelope breaks down, the chromosomes congress at the cell equator. The mitotic spindle, a complex subcellular machine, coordinates this congression and then the accurate segregation of sister chromatids to the two daughter cells. The combination of light and electron microscopy (EM), together with computer-based 3D reconstruction, shows that the mitotic spindle is localized in an exclusion zone, which is free of endomembranes: nuclear envelope and Golgi remnants, endoplasmic reticulum (ER), and vesicles (Puhka et al., 2007; Lu et al., 2009; Puhka et al., 2012; Champion et al., 2017; Nixon et al., 2017). Beyond the exclusion zone, the endomembranes are densely packed and mitochondria are randomly distributed. Recently, we described how chromosomes that do not congress to the cell equator can lie beyond the exclusion zone and become ensheathed by endomembranes, constituting a risk factor for chromosome instability (Ferrandiz et al., 2022). Our aim here is to provide a detailed step-by-step protocol to visualize, at the ultrastructural level, these chromosomes that become ensheathed by endomembranes. We use light microscopy to locate the cell of interest and then semi-automated large volume EM method (serial block-face scanning electron microscopy, SBF-EM) and 3D reconstruction to visualize the mitotic cell. The protocol will be useful for other applications where the total endomembrane content of mitotic cells, or indeed non-dividing cells, needs to be visualized.
Materials and reagents
Cell culture
DMEM/F-12 Ham (Merck Life Science, catalog number: D6421-6)
Fetal bovine serum (FBS) (Sigma, catalog number: F7524)
Trypsin-EDTA solution (Sigma, catalog number: T3924)
l-glutamine (Sigma, catalog number: G7513)
Penicillin/streptomycin (Gibco, catalog number: 15140-122)
Sodium bicarbonate (NaHCO3) (Sigma, catalog number: D8662)
MatTek gridded glass-bottom culture dishes (MatTek Corporation, catalog number: P35G-1.5-14-CGRD)
Fugene HD transfection reagent (Promega, catalog number: E2312)
OptiMEM (Gibco, catalog number: 10149832)
GSK923295 (Selleckchem, catalog number: s7090)
Thymidine (Sigma, catalog number: T1895)
RO-3306 (Sigma-Aldrich, catalog number: SML0569)
SiR-DNA (Spirochrome, catalog number: SC007)
Poly-L-Lysine (Sigma-Aldrich, catalog number: P8920)
Concanavalin A (Sigma-Aldrich, catalog number: C2010)
Sample processing
Sodium phosphate dibasic (Na2HPO4) (Sigma, catalog number: S0876)
Sodium phosphate monobasic (Na HPO4) (Sigma, catalog number: S0751)
Potassium hydroxide (KOH) (Sigma, catalog number: 221473)
Glutaraldehyde 25% (Agar, catalog number: R1011)
Paraformaldehyde 16% (Thermo Fisher, catalog number: 28908)
Tannic acid, low molecular weight (Electronic Microscopy Science, catalog number: 21710)
OsO4 (TABB, catalog number: O014)
Potassium ferrocyanide (Sigma, catalog number: 14459-95-1)
Thiocarbohydrazide (TABB, catalog number: T009)
Uranyl acetate (UA) (TAAB, catalog number: U008)
Methanol anhydrous (Sigma, catalog number: 322415)
Lead nitrate (TAAB, catalog number: L019)
l-Aspartic acid (Sigma, catalog number: A9256)
Ethanol molecular grade (Sigma, catalog number: 51976)
Agar 100 premix kit-Hard (Agar Scientific, catalog number: R1140)
Silver conductive paste (TAAB, catalog number: S384)
Gold/Palladium (80/20%) 60 mm × 0.1 mm (Quorum technologies, catalog number: SC500-314B)
0.1 M phosphate buffered (PB) saline (see Recipes)
Fixation buffer (see Recipes)
2% reduced osmium (see Recipes)
1% thiocarbohydrazide (TCH) solution (see Recipes)
1% uranyl acetate (UA) (see Recipes)
Lead aspartate solution (see Recipes)
Agar 100 premix kit (see Recipes)
Equipment
Nikon CSU-W1 spinning disc confocal system with SoRa upgrade (Yokogawa). Used with a Nikon, 20×/0.50 objective (Nikon) with optional 2.3× intermediate magnification or 60×, 1.40 NA, oil, Plan Apo VC objective (Nikon), and 95B Prime camera (Photometrics). The system has CSU-W1 (Yokogawa) spinning disk unit with 50 μm and SoRa disks (SoRa disk used), Nikon Perfect Focus autofocus, Okolab microscope incubator, Nikon motorized XY stage, and Nikon 200 μm z-piezo. Excitation was via 405, 488, 561, and 638 nm lasers with 405/488/561/640 nm dichroic and Blue, 446/60; Green, 525/50; Red, 600/52; FRed, 708/75 emission filters
Ultramicrotome (Leica Microsystems, EM UC7)
Gatan 3 View system installed on an FEI Quant 250 ESEM, with digital micrograph software (Gatan)
Software
NiS Elements (Nikon)
Digital micrograph software (Gatan)
Open-source programs for analyzing images such as:
Fiji (https://fiji.sc) (version 2.9.0)
Microscopy Image Browser (http://mib.helsinki.fi) (version 2.60)
IMOD (https://bio3d.colorado.edu/imod/) (version 4.10.49)
Procedure
Cell culture (Timing: 2–4 d)
This protocol can be adapted to most mammalian cell lines; we have imaged HeLa, RPE1, and HCT116 cell lines using this protocol. Here, RPE1 GFP-Sec61β cells were maintained in DMEM/F-12 Ham supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin/streptomycin, and 0.26% NaHCO3 in a humidified incubator at 37 °C and 5% CO2. Seed the cells 24 h before starting the experiment (see Note 1). Seed approximately 90,000 RPE-1 GFP-Sec6β cells onto MatTek gridded glass-bottom culture dishes (see Note 2) once they are detached using trypsin-EDTA solution. The alphanumeric photoetched grid provides a reference for tracking the same cell throughout the workflow. The degree of confluency, ideally 60%, on the day of imaging, is important to achieve the aim. We require as many cells as possible at the correct mitotic stage determined by the fluorescent tags. However, too many cells can obscure the photoetched grid and make correlation difficult.
Note 1: The specific media and additives mentioned in this section are for RPE1 cells. If a different cell line is being used, please use the appropriate media and additives for that cell line.
Note 2: If transfection is required, approximately 25,000 RPE1 GFP-Sec61b cells need to be seeded 48 h before starting the experiment. RPE1 cells are transfected using Fugene (3:1); this ratio is a guide, depending on the plasmid size and cell type. Add 3 μL of Fugene to 100 μL of OptiMEM, incubate for 5 min, and add a total of 1 μg of purified plasmid DNA to the mixture of OptiMEM-Fugene. Incubate for 20 min at room temperature (RT), then add to the cells in the MatTek dish containing whole DMEM/F-12 Ham, and leave for 12–16 h. Typically, this results in around 20%–30% transfection efficiency. The following morning, wash off the transfection media and replace with full DMEM/F-12 Ham. Cells are then imaged by light microscopy 48 h later.
Antimitotic drug treatment to induce chromosome misalignment (see Note 3): Our protocol uses 150 nM GSK923295, a CENP-E inhibitor, for 3 h. Wash off the drug 1 h before imaging to allow cells to enter mitosis. Typically, around 30% of cells are at metaphase in this protocol.
Note 3: OPTIONAL: Coating the glass coverslip with poly-l-lysine or concanavalin reduces the risk of mitotic cells washing away during imaging and sample processing.
Note 4: OPTIONAL: Mitosis is a rare event, occurring for 1%–8% of the cell cycle; if a specific stage needs to be visualized, this reduces the chance that a cell of interest can be readily found. The way to increase the chance to capture the desired cell is to synchronize them or use drugs to arrest them in early mitosis. Our current protocol for chemical synchronization uses a 2.5 mM thymidine block for 16 h, release for 6 h, and incubation in 9 μM RO-3306 for 16 h. After that time, RO-3306 is washed off for 45 min before imaging. This results in 30% of the cells being at metaphase.
To dye chromosomes in live-cell imaging, incubate cells with 0.5 μM SiR-DNA for 30 min before imaging commences.
Light microscopy
Keep cells in full DMEM/F-12 Ham media during imaging.
The microscope used in our experiments is a Nikon CSU-W1 spinning disc confocal system with SoRa upgrade (Yokogawa). Sora disk is used with Nikon 20×/0.5 with optional 2.3× intermediate magnification or 60×, 1.40 NA, oil, Plan Apo VC objective, and 95B Prime camera (Photometrics). The system has the Okolab microscope incubator used for imaging at 37 °C and 5% CO2. Acquisition and image capture was via NiS Elements (Nikon). A widefield microscope would also be sufficient to visualize the cells.
Select mitotic cells of interest by light microscopy (desired stage of mitosis, moderate fluorescent protein expression, etc.). For our experiment, a single misaligned chromosome should be identified to be ensheathed by endomembranes (marked by GFP-Sec61β). Acquire live images at 20× with 2.3× intermediate magnification or 60×, 1.40 NA, oil, Plan Apo VC objective; brightfield images are taken at 20× magnification to record the position of the cell on the grid (Figure 1).
Note: Correlating the selected cell imaged by light microscopy to SBF-SEM is a manual process.
Following imaging, fix cells in fixation buffer for 1 h. Fixation after imaging must be immediate in order to accurately correlate the light and electron microscopy images. To do this rapidly and to minimize the possibility of mitotic cells detaching from the coverslip, we find that adding the fixative solution directly to the dish while it is still in the microscope is the optimal method. Replace this media-fixative solution with a fresh fixative solution after 1 min.
Figure 1. Correlating a cell of interest from light microscopy to electron microscopy. A. Light microscopy imaging of a cell of interest. A mitotic cell expressing GFP-Sec61β is selected at the desired stage of mitosis and with a misaligned chromosome. Scale bar, 10 μm. B. Layout of photoetched grids on MatTek dishes. C. Low magnification image of the position of the cell on the grid (coord 6G can be seen). Scale bar, 100 μm. D. Resulting serial block-face scanning electron microscopy (SBF-SEM) image following correlation. Scale bar, 2 μm.
Sample processing
Perform all processing steps with the cells still in the MatTek dish used for imaging. At all stages, the surface of the dish must be kept covered in order to prevent the sample from drying out. All solution exchanges must be gentle to prevent mitotic cells from detaching from the coverslip.
Following fixation, wash cells three times in PB saline for 5 min each to remove residual aldehydes. Pause point: Samples can stay in PB for up to 12 h at 4 °C. This can be useful to collect all the samples before progressing to the next stage.
Postfix, incubate the cells in fresh 2% reduced osmium (equal volumes of 4% aqueous OsO4 and 3% potassium ferrocyanide) for 1 h at RT.
During this incubation, prepare a 1% TCH solution for the next step. Always make this solution fresh for each experiment. Add 0.1 g of thiocarbohydrazide to 10 mL of prewarmed dH2O and incubate at 60 °C for 1 h. Gently swirl every 5–10 min to facilitate dissolving. It is recommended to filter the solution through a 0.22 μm syringe filter prior to use. TCH acts as a mordant to facilitate heavy metal staining.
Then, wash cells three times for 5 min in PB, removing the excess osmium, at RT.
Incubate cells in prepared TCH for 15 min at RT.
Wash cells three times for 5 min in PB, removing the TCH solution, at RT.
Incubate the cells in an aqueous 2% OsO4 solution (not reduced osmium) for 30 min at RT.
Wash the cells three times for 5 min in PB, removing the TCH solution, at RT.
Incubate cells in 1% UA solution in water at 4 °C for 16 h in the dark. Prepare the UA solution from a filtered 20% stock in methanol. Spin down the stock before diluting to remove any precipitate.
Next morning, prepare Walton’s lead aspartate for the next step. To make 10 mL lead aspartate, add 66 mg of lead nitrate to 9 mL of filtered 0.03 M aspartic acid solution and adjust the pH to 4.5 with KOH. Adjust the final volume to 10 mL and the final pH to 5.5. Incubate the lead aspartate at 60 °C for 30 min in a glass vial. The pH adjustment step is tricky and can result in precipitation if an error is made. We adjust the pH in two steps during the preparation of the lead aspartate solution to facilitate dissolution: first to 4.5 with KOH, and then to a final pH of 5.5. The solution is ready to use later if precipitate is not formed. This volume is suitable for 10 samples.
Wash the samples from UA solution following three washes in dH2O for 5 min each.
Incubate cells in the previously prepared lead aspartate solution for 30 min at RT.
Remove lead aspartate by washing cells three times in dH2O for 5 min each.
Next, dehydrate the samples in a graded series of molecular grade ethanol (30%, 50%, 70%, 90%, and 100%) for 5 min at each concentration.
Now, infiltrate the samples with resin. A hard resin is used when embedding samples for SBF-SEM to minimize beam-induced damage. We use Agar 100 premix kit-Hard. Incubate cells with a mixture of resin:ethanol: first, in a 1:2 proportion for 30 min; second, in a 2:1 proportion for 30 min; and third, full resin for a further 30 min.
Add fresh full resin to fill the MatTek dish (approximately 2 mm).
Polymerize resin to hardness by incubating for 24 h at 60 °C following the manufacturer’s instructions.
Once the resin is cooled to RT, remove the glass coverslip on the MatTek dish by briefly plunging it into liquid nitrogen and using a razor blade to prize the edges of the glass coverslip away from the plastic dish until it detaches, leaving the resin surface and imprinted grid coordinates exposed. The cell of interest is then relocated using the previously acquired brightfield images, and its position is highlighted using a marker pen to aid excision.
Excise the resin block containing the cell of interest using a hacksaw. Trim away excess resin around the grid coordinate using a glass knife on ultramicrotome (EM UC7, Leica Microsystems) to produce a 200 × 200 μm block face. To aid in conductivity around the block face and reduce charging from the electron beam, the block is first painted with silver conductive paste (TAAB) and gold/palladium (80/20%) 60 mm × 0.1 mm thick is subsequently evaporated onto it (Quorum technologies).
SBF-SEM
SBF-SEM imaging is done using a Gatan 3View system on a FEI quanta 250 ESEM. Load the pin with resin block into the SBF-SEM and relocate the cell of interest using the light microscopy images. Imaging is automated, such that a diamond knife cuts a section, an image is taken, and then the process is repeated. Typically, coarse sections are taken until the cell is in view and then the block is centralized, and higher magnification imaging and fine sectioning is performed. The exact conditions depend on the experiment, but for RPE-1 mitotic cells, imaging at 8.8 nm in x and y and 60 nm in z, in a 4,000 × 4,000 pixel window is sufficient to capture the entire cell and all of the features in which we are interested.
Image analysis
Large volume EM datasets obtained from SBF-SEM are used for segmentation and subsequent 3D rendering or analysis. The aim is to model the cellular features of the mitotic cell of interest. There are a number of software tools available for segmentation, including Microscopy Image Browser (MIB), AMIRA, ImageJ/Fiji, 3DMOD/IMOD, and Vaa3D. We use a combination of Fiji, MIB, and 3DMOD/IMOD to reconstruct the key features of mitotic cells such as endomembranes and chromosomes.
FIJI
Import the image sequence into Fiji, using BioFormats to convert the DM3 file to an image stack, or by reading in a series of TIFF images exported by Gatan Digital Micrograph (Schindelin et al., 2012).
Ensure that the voxel size is read correctly. If not, correct it using Image > Properties.
It is recommended to convert the 16 bit images into 8 bit to speed up their processing and reduce the final size of the data downstream.
SBF-SEM data is inherently in-register because the block is fixed relative to the detector. However, image registration may be required if imaging is interrupted, and the block remounted. Our favorite method is SIFT, to ensure that the stack is in register (Plugins > Registration).
Normalize the contrast throughout the stack by selecting Process > Enhance Contrast. Change the saturated pixel to 1% and select Normalize and Process All.
OPTIONAL: Pre-process the stack. The goal here is to aid segmentation. Subtraction of a gaussian-blurred version of the stack can reduce uneven density across the image. Additional filters may be used at this stage.
Save the adjusted image as a TIFF series using File > Save as Image sequence.
MIB
MIB can be opened through MATLAB or as a standalone program (Belevich et al., 2016). For more information on how to do either, see the MIB website (http://mib.helsinki.fi/).
Import the TIFF image stack using Directory Contents.
Highlight all the images to be analyzed, right-click, and select Combine Selected Datasets.
Change the spatial parameters by going to Dataset > Parameters.
If required, it is possible to align or correct the stack for drift by using Dataset > Alignment, or by choosing Mode > Algorithm > Drift correction (correlate with, color channel, and background). In Options, it is possible to align a selected area.
Next, start the segmentation process by clicking Create in the Segmentation panel and select 63 or 255 models, depending on how many objects will be segmented, to start the segmentation.
Add a material to the Segmentation panel by clicking the plus button; change the name of the material and it will appear in the column.
During segmentation in MIB, pixels can only be selected and assigned to one material at a time. In our case, we determine four materials: chromosomes, mitochondria, ER, and plasma membrane.
Each material can be named by double-clicking and selecting the desired color.
A variety of tools can then be used for the segmentation of the cellular structures of the mitotic cells.
Below the material panel is the segmentation tool drop-down list. The default when you start the program will be the Brush tool, a manual segmentation tool.
Use the Brush tool to trace the outline of the object and repeat this over each slice. Press Shift and F to fill the object throughout all the slices.
Hold the Ctrl button to turn the brush tool into an eraser and remove any errors.
The selected pixels will appear green on the image.
Assign the selected pixels to the corrected material in the Add to box by choosing the material and pressing Shift and A.
MIB offers tools for semiautomatic segmentation:
i. Interpolation using the brush tool manually: draw on every nth slice and then click I or go to Selection > Interpolation. Check and correct any errors that have occurred.
ii. Thresholding takes a user inputted contrast range to select pixels. There are two types: B/W thresholding and the Magic Wand tool.
1) Select B/W Thresholding from the menu. Alter the range by adjusting the two sliders until a correct selection is made over the entire image.
2) Click All for B/W thresholding to be applied to the stack of images.
3) Choose the Magic Wand-Region growing tool and change the variation and radius of the selection tool.
4) Click on a single pixel in the object—pixels within the range and radius will be selected—and alter the variation and radius until the desired selection is made.
5) Select 3D in the Selection panel to apply the thresholding to the stack.
6) Check for any errors and correct them using the brush tool.
The chromosomes and plasma membrane of the mitotic cells are segmented using the interpolation tool. Mitochondria were segmented mainly using the thresholding tool. In both cases, the timing was relatively fast for >50 images. However, the ER is segmented manually and is extremely time consuming. We typically render a sub stack using this approach to reduce the time required for manual segmentation. Automated segmentation methods are likely to supplant the manual steps in the next year or so.
Save File once the segmentation is finished by Model > Save Model. We export the models for using IMOD software for 3D reconstruction. Therefore, we export them as Contours (*.mod) and a density factor for points in the XY plane is available depending on the resolution required. In addition, we also need the volume file (*.mrc) through File > Save Image as > MRC format for IMOD, confirming dataset parameters.
3DMOD/IMOD
IMOD is a suite of programs to visualize 3D biological image data (Kremer et al., 1996). Instructions to download and launch 3DMOD/IMOD can be found at https://bio3d.colorado.edu/imod/.
At the launch window, select Image File (.mrc) and specify the model file (.mod).
Load data into the Zap window; all the material and objects are segmented.
To visualize the model, select Image > Model View. This window shows the 3D view of our model (Figure 2A).
To mesh the surface follow Edit > Object > Meshing > Mesh All. Adjust the settings (brightness, size, color) if they are required (Figure 2B).
Save as TIFF by File > Snap TIFF as. Additionally, a movie/montage can be created.
Figure 2. 3D model of cellular features. In 3DMOD/IMOD, the 3D model can be viewed in various ways, analyzed, or exported to make figures for publication. Scale bar, 2 μm.
General notes and troubleshooting
Cell density: The density of cells is critical for the accurate reading of grid coordinates. High density can lead to a lack of correlation between cell position and grid coordinates, while low density can result in a reduced number of mitotic cells at the right stage, making the experiment more challenging. A desirable number on the imaging day is around 60% confluency.
Imaging mitotic cells: To locate mitotic cells of interest expressing fluorescently tagged proteins, brightfield images are taken at 20× magnification to record the position of the cell relative to the grid coordinates for future reference. The same cell is relocated, and live-cell fluorescence microscopy is performed at 60× magnification (oil objective). Be cautious not to miss the cell of interest when changing objectives. Using 20× with 2.3× intermediate magnification can provide the necessary information depending on the protein of interest and fluorescent protein used.
Fixation: Fixation before imaging can be an option, but it should be done with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h. After fixation, wash the samples three times for five minutes each in PB and leave them in PB for imaging. Once imaging is complete, the samples require a second fixation step in 0.5% glutaraldehyde and 0.1% tannic acid in 0.1 M phosphate buffer, pH 7.4, for 1 h before processing with osmium staining. Avoid using glutaraldehyde before imaging, because it causes high autofluorescence, reacting with proteins and peptides to generate visible to near-infrared (IR) emitters.
Osmium staining: Make sure the reduced osmium is dissolved correctly before staining the cells. Any precipitates in the dish will not be acceptable for imaging. If the problem persists, it is recommended to filter the solution through a 0.22 μm syringe filter before use. This solution should always be fresh and never reused from previous staining. It is also important to wash the sample properly after fixation before adding the osmium-reduced solution. A hint of aldehyde fixative left in the dish can cause precipitation once the osmium solution is added. When using correlative dishes, ensure all solutions are removed from the coverslip and at least three washes are performed between the fixation and osmium staining steps.
Resin polymerization: This is a crucial step because once the coverslip comes off, if a small amount of resin stays on the glass, it is challenging to get the reference grid to identify the correct area. The cells may not have been embedded in the resin properly and slightly stand out of the resin. Additionally, it would make it more difficult to trim the resin as it is either very brittle or still gel-like, causing it to crumble away when trimming is done. The problem may be that the coverslip still contains some solvent and did not mix well enough with the fresh resin to polymerize properly. To avoid these issues, ensure the previous solutions are completely removed before starting the step to embed the samples with resin. Follow the manufacturer’s instructions provided for each resin to determine the time and temperature incubations required for proper polymerization.
Analysis tools: There are very helpful online tools, such as tutorials and a mailing list.
MIB Tutorial: http://mib.helsinki.fi/tutorials.html
IMOD Tutorial: https://www.andrewnoske.com/wiki/IMOD_-_tutorial
https://www.youtube.com/watch?v=Nu7TzloKfWU
https://www.youtube.com/watch?v=enMEEjPDRE0
Recipes
0.1 M phosphate buffered (PB) saline
Dissolve 1.41 g of Na2HPO4 in 50 mL of H2O (0.2 M solution)
Dissolve 1.2 g of NaH2PO4 in 50 mL of H2O (0.2 M solution)
Add 40.5 mL of Na2HPO4 solution to 9.5 mL of NaH2PO4
Adjust pH to 7.4 with 10 N KOH solution
Add 50 mL of H2O to make 0.1 M solution
Storage: 4 °C
Fixation buffer
2.5% glutaraldehyde
2% paraformaldehyde
0.1% tannic acid
0.1 M phosphate buffer, pH 7.4
Storage: Immediate use only
2% reduced osmium
4% aqueous OsO4
Dissolve 0.2 g of potassium ferrocyanide power in 10 mL of 0.1 M PB (for 3% solution)
To make 2% reduced osmium, mix equal volumes of 4% OsO4 and 3% potassium ferrocyanide
Storage: Immediate use only
1% thiocarbohydrazide (TCH) solution
Measure 0.1 g of TCH
10 mL of prewarmed dH2O
Mix the TCH and water and incubate at 60 °C for 1 h
Gently swirl every 5–10 min to dissolve
Storage: Immediate use only
Note: It is recommended to filter the solution through a 0.22 μm syringe filter prior to use.
1% uranyl acetate (UA)
Prepared from a filtered 20% stock in methanol
Measure 0.5 g of UA
2.5 mL of methanol
Mix the UA and the methanol. Dissolve by shaking
Filter and spin down the 20% stock solution
To make 1%, take 0.25 mL from 20% in 5 mL of H2O
Storage: 4 °C for up to a month in foil and parafilm lid
Note: UA precipitates under light.
Lead aspartate solution
Make up 0.03 M aspartic acid stock
Heat at 60 °C for 60 min to dissolve
Filter and store at RT
Add 0.066 g of lead nitrate to 9 mL of 0.03 M aspartic acid stock
Adjust the pH to 4.5 with 10 N KOH
Make up to 10 mL of aspartic acid stock
Adjust the pH to 5.5 with 10 N KOH
Incubate in a glass vial for 30 min at 60 °C
Agar 100 premix kit
One component is in a bottle of 50 mL capacity; second component is in a bottle of 100 mL capacity
Add the contents of the smaller bottle to the larger bottle
Add the contents of one of the ampoules of accelerator
Mix thoroughly and approximately 100 g of resin is ready to use
Storage: at 4 °C for a few days
Note: If the unused mixed resin is stored at a reduced temperature before use, the resin must be allowed to reach RT before opening and use.
Acknowledgments
This protocol is derived from Ferrandiz et al. (2022). DOI: 10.1083/jcb.202203021. We thank Claire Mitchell and Laura Cooper from the Computing and Advanced Microscopy Unit (CAMDU) for their help and support. Alison Beckett and Ian Prior at the Liverpool Biomedical EM Unit provided SBF-SEM imaging. Previous lab members Faye Nixon, Nick Clarke, and Daniel Booth, contributed to this imaging pipeline. This work was supported by a Pioneer Award and Programme Award from Cancer Research UK (C25425/A24167 and C25425/A27718).
Competing interests
The authors declare no conflicting interests.
References
Belevich, I., Joensuu, M., Kumar, D., Vihinen, H. and Jokitalo, E. (2016). Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets. PLoS Biol 14(1): e1002340.
Champion, L., Linder, M. I. and Kutay, U. (2017). Cellular Reorganization during Mitotic Entry. Trends Cell Biol 27(1): 26-41.
Ferrandiz, N., Downie, L., Starling, G. P. and Royle, S. J. (2022). Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes. J Cell Biol 221(6): e202203021.
Kremer, J. R., Mastronarde, D. N. and McIntosh, J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1): 71-76.
Lu, L., Ladinsky, M. S. and Kirchhausen, T. (2009). Cisternal organization of the endoplasmic reticulum during mitosis. Mol Biol Cell 20(15): 3471-3480.
Nixon, F. M., Honnor, T. R., Clarke, N. I., Starling, G. P., Beckett, A. J., Johansen, A. M., Brettschneider, J. A., Prior, I. A., and Royle, S. J. (2017). Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis. J Cell Sci 130(10): 1845-1855.
Puhka, M., Joensuu, M., Vihinen, H., Belevich, I., and Jokitalo, E. (2012). Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Mol Biol Cell 23(13): 2424-2432.
Puhka, M., Vihinen, H., Joensuu, M. and Jokitalo, E. (2007). Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells. J Cell Biol 179(5): 895-909.
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: 676-682.
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4,709 | https://bio-protocol.org/en/bpdetail?id=4709&type=0 | # Bio-Protocol Content
Improve Research Reproducibility
A Bio-protocol resource
Peer-reviewed
easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria
EB Edna Bode
DA Daniela Assmann *
PH Petra Happel *
EM Elmar Meyer *
KM Karin Münch *
NR Nicole Rössel *
HB Helge B. Bode
(*contributed equally to this work)
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4709 Views: 566
Reviewed by: Dennis J NürnbergSelcuk Hazir Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Scientific Reports Jun 2022
Abstract
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
Keywords: easyPACId Biosynthetic gene cluster activation NRPS PKS Natural product Promoter exchange Bioactivity testing Bioactive compounds isolation Xenorhabdus Photorhabdus Pseudomonas
Background
Entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus live in symbiosis with soil dwelling nematodes of the genera Steinernema or Heterorhabditis, respectively (Goodrich-Blair and Clarke, 2007; Chaston et al., 2011). They are able to propagate without their nematode host and can be easily cultivated in the laboratory using standard media. Their genome harbors a rich diversity for natural products (NPs) with potential activities as antibiotics, antifungals, and cytotoxic or signaling functions. These NPs are often produced by non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and mixed NRPS-PKS hybrids, encoded by biosynthetic gene clusters (BGCs) (H. B. Bode, 2009; Shi and Bode, 2018; Shi et al., 2022).
The access to this promising source of bioactive compounds is possible via different approaches: either by optimizing the culture conditions for enhanced NP production, by heterologous expression or promoter activation of the BGC of interest, or chemical synthesis of the NP derived thereof (H. B. Bode et al., 2002; Biggins et al., 2011; Biggins et al., 2014; E. Bode et al., 2017). All these methods have their limitations: chemical synthesis might be impossible, difficult, or too time consuming, requiring profound knowledge of the NP structure as a starting point. Heterologous production of the desired NP is dependent on building blocks that might only be present in the natural host, on the size of the BGC to be cloned into the heterologous host, on its toxicity, or on special transport systems or modifying enzymes (Brachmann et al., 2012; Nollmann et al., 2015). Isolation of natural products from crude extracts from wildtype (WT) bacteria might be hindered by low production titers, regulatory elements that silence the expression of BGCs, and/or sophisticated culture conditions that have to be established. A promoter activation of desired gene clusters in WT strains might result in NP overproduction that facilitates the isolation of the compound for bioactivity testing and structure elucidation, but still requires NP isolation from crude extracts. The easyPACId (easy Promoter Activation and Compound Identification) approach circumvents all these limitations.
easyPACId describes a simple and fast enrichment and preparation of selected NPs for bioactivity screening just from the crude extract of a bacterial culture of an induced ∆hfq_pCEP-gene_xyz easyPACId mutant. It avoids laborious steps often encountered during NP isolation from WT crude extracts and is not hindered by limitations concerning heterologous expression or chemical synthesis. The targeted activation via the PBAD promoter (Guzman et al., 1995) in a clean ∆hfq background serves as an excellent tool to enable production of only desired NPs or NP classes derived from single BGCs (Tobias et al., 2017; Valverde, 2017; E. Bode et al., 2019). The RNA chaperone Hfq is widespread in proteobacteria. It has a pleiotropic effect on gene expression by mediating the interaction of small non-coding RNAs (sRNA) and mRNAs, and thereby can activate or suppress translation (Vogel and Luisi, 2011). For NP-producing bacteria like Xenorhabdus, Photorhabdus, or Pseudomonas, a ∆hfq mutant has lost the ability to produce all or most NPs or shows a severely reduced NP amount. Consequently, the chromatogram of crude extracts from a ∆hfq mutant analyzed via HPLC/MS shows no NP-derived signals anymore (clean background) compared to HPLC/MS chromatograms of the corresponding WT where all NPs are produced, and their corresponding signals show a typical pattern for the strain (Figure 1a).
Briefly, the experiment starts via detection of potential BGCs encoding NRPSs, PKS, hybrids thereof, or other BGC classes, via antiSMASH analysis (or based on other bioinformatic tools) (Medema et al., 2011) in the genome of selected NP producers, here exemplified for the genera Photorhabdus and Xenorhabdus. By deletion of the gene hfq, encoding the so-called RNA-chaperone Hfq, a mutant is generated, which is deficient in NP production (Tobias et al., 2017). By exchange of the natural promoter against the L-arabinose inducible promoter PBAD (Guzman et al., 1995), encoded on the conjugatable vector pCEP via homologous integration (E. Bode et al., 2015), the selective activation of the targeted BGC in a ∆hfq background is now possible (Figure 1). Cell-free culture supernatants or even whole cultures from respective ∆hfq_pCEP-gene_xyz can be freeze dried and dissolved in water for subsequent testing on various target organisms (E. Bode et al., 2015 and 2019; Gulsen et al., 2022). Often, the selected compounds are overproduced and can be easily isolated for further investigations like structure elucidation.
This simple and beneficial method of gaining potentially bioactive compounds can be used to activate natural product biosynthesis gene clusters, not only in Xenorhabdus and Photorhabdus bacteria. It has been applied already to Pseudomonas and is likely to work in any other proteobacteria that encode Hfq, like Serratia or Vibrio (Thoma and Schobert, 2009; Hmelo et al., 2015). Besides Hfq itself, other regulative components of the hfq-sRNA regulon like ArcZ (Neubacher et al., 2020) or even other global regulators might also be useful to generate NP non-producing phenotypes in the described or other Gram-negative bacteria.
Figure 1. Schematic overview of the easyPACId workflow
Materials and reagents
∆hfq strain from Xenorhabdus spp. (E. Bode et al., 2019)
Sterile tips for micropipettes [Starlab, catalog numbers: S1110-3700-c (10–20 μL), S1111-1706-c (200 μL), S1111-6000-c (1,000 μL)]
Sterile reaction tubes [Sarstedt, catalog numbers: 72.706 (1.5 mL), 72.695.500 (2.0 mL)]
Black reaction tubes (Carl Roth, catalog number: AA80.1)
PCR tubes (Starlab, catalog number: 1402-3700)
50 mL tubes (Sarstedt, catalog number: 62.547.254)
Petri dishes [Sarstedt, catalog numbers: 82.1473.001 (92 mm × 16 mm), 82.1184.500 (150 mm × 20 mm)]
Electroporation cuvettes (Bio-Rad, catalog number: 1652089)
Single-use syringe Omnifix LuerLock Solo (Braun, catalog number: 4617207V)
Filtropur S 0.2 (Sarstedt, catalog number: 83.1826.001)
Steritop 45 mm neck size Millipore Express Plus 0.22 μm PES (Millipore, catalog number: S2GPT05RE)
Tryptone (BD, catalog number: 211699)
Yeast extract (BD, catalog number: 288620)
Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)
Bacto-agar (BD, catalog number: 214030)
LE agarose (Biozym, catalog number: 840004)
L-Arabinose (Carl Roth, catalog number: 5118.5)
Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771.1)
Kanamycin monosulfate (km) (Sigma, catalog number: K4000)
5-Aminolevulinic acid hydrochloride (5-ALA) (Sigma, catalog number: 8339)
E. coli ST18 competent cells (Thoma and Schobert, 2009) (DSMZ, Braunschweig, Germany)
Conjugatable cloning vector pCEP-kmR with R6K ori, PBAD promoter, selection marker kanamycin (kmR) (E. Bode et al., 2015 and 2019)
Phusion High-Fidelity DNA Polymerase (New England Biolabs, catalog number: M0530L)
dNTP-Set (Carl Roth, catalog number: K039.2)
PstI/PstI HF (New England Biolabs, catalog numbers: R0140L/R3140L)
NdeI (New England Biolabs, catalog number: R0111L)
Monarch Genomic DNA Purification kit (New England Biolabs, catalog number: T3010L)
100 bp DNA ladder (New England Biolabs, catalog number: N3231L); 1 kb DNA ladder (New England Biolabs, catalog number: N3232L)
Midori Green Advance (Biozym, catalog number: 617004)
QIAprep Spin Miniprep kit (Qiagen, catalog number: 27106)
Wizard® SV Gel and PCR Clean-Up System (Promega, catalog number: A9285)
NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, catalog number: E2621L)
Methanol optima LC/MS grade (Fisher Scientific, catalog number: A456-212)
Ethanol (Carl Roth, catalog number: 9065.4)
Glycerol (Carl Roth, catalog number: 3783.2)
Acetic acid (Carl Roth, catalog number: 3738.5)
Trizma-Base (Sigma, catalog number: T6066)
Na2-EDTA (Carl Roth, catalog number: 8043.2)
Luria-Bertani medium (LB) (see Recipes)
5-Aminolevulinic acid hydrochloride (5-ALA) (50 mg/mL) (see Recipes)
L-Arabinose (20% w/v) (see Recipes)
Kanamycin (50 mg/mL) (see Recipes)
TAE-buffer, pH 8.0 (see Recipes)
0.5 M EDTA pH 8.0 (see Recipes)
Equipment
Super Duty Erlenmeyer flasks, 50 mL (Omnilab, catalog number: 5425046)
500 mL Erlenmeyer flasks (Omnilab, catalog number: 5010476)
Cellulose plug F 17.0–18.5 mm (VWR International, catalog number: 391-0161)
Caps for flasks with neck Ø38 mm (Carl Roth, catalog number: K396.1)
Sterile glass pipettes
Gel chambers for agarose gel electrophoresis
Inoculation loop, Platin-Iridium (neoLab, catalog number: 1-2112)
Drigalski spatula (Carl Roth, catalog number: T724.2)
Sterile toothpicks
Conical centrifuge tube 50 mL (Sarstedt, catalog number: 62.547.254)
ChemiDoc MP imaging systems with Lab Touch Software (Bio-Rad, catalog number: 12003154) and Blot/UV/Stain-Free tray (Bio-Rad, catalog number: 12003028)
Transilluminator (Intas Science Imaging Instruments)
Micropipettes (Gilson, Pipetman G P2G, catalog number: F144054M; Pipetman G P10G, catalog number: F144055M; Pipetman G P20G, catalog number: F144056M; Pipetman G P200G, catalog number: F144058M; Pipetman G P1000G, M, catalog number: F144059M)
Centrifuge Pico 17 microcentrifuge (Fisher Scientific, catalog number: 75002410)
Centrifuge Eppendorf 5810 R (V8.8), rotor S-4-104 with adapter (Eppendorf, catalog numbers: 5825734009 and 5825733002)
ProFlexTM PCR system, 96-well (Applied BiosystemsTM, catalog number: 4484075)
Eppendorf ThermoMixer C (Eppendorf, catalog number: 5382000015)
Infors HT-Multitron Standard shaker
Gene Pulser Xcell (Bio-Rad)
NanoDrop One WiFi (Life Technologies, catalog number: ND-ONE-W)
UV-vis-spectrophotometer Biochrom Libra S60 (Omnilab, catalog number: 80-7000-12)
Lyovapor L-300 (Büchi)
HPLC vials X1000 Microflaschen ND9 (Fisher Scientific, catalog number: 11707597)
Lid (for HPLC vials) X1000 (Fisher Scientific, catalog number: 11787567)
HPLC/MS Thermo Scientific UltiMate 3000 coupled to an amaZon speed ion trap (Bruker)
Vortex Genie 2 (Fisher Scientific, catalog number: 15547335)
Software
Geneious Prime® 2022.1.1 or earlier versions (https://www.geneious.com)
DataAnalysis (Bruker Daltonics GmbH)
antiSMASH (https://antismash.secondarymetabolites.org/#!/start) (Medema et al., 2011)
Procedure
Plasmid planning and construction
Choose the ∆hfq strain of interest and scan the genome for natural product BGCs using antiSMASH (Medema et al., 2011). This protocol will focus on Xenorhabdus_∆hfq as model organism for Xenorhabdus and Photorhabdus. For generating a ∆hfq strain in other proteobacteria, please refer to suitable methods for strain generation.
Streak the ∆hfq strain on a LB agar plate using a sterile inoculation loop in order to obtain single colonies. Incubate the inoculated plate at 30 °C for at least 72 h; look daily for growth and examine the strain for antibiotic resistance, pigmentation, swarming, or other phenotypical characteristics.
Note: For swarming strains, increase agar concentration of solid medium up to 30 g/L.
Store the grown plates at 18 °C for up to two weeks. For long-term storage, prepare glycerol stocks from fresh overnight cultures [720 μL of glycerol (50% v/v) and 1,280 μL of culture].
Inoculate 5 mL of LB medium in a 50 mL Erlenmeyer flask with a single colony from the plate and incubate the culture overnight at 30 °C rotating at 200 rpm. Purify chromosomal DNA from 1 mL (~2 × 109 cells) of bacterial culture with Monarch Genomic DNA Purification kit. Dissolve the DNA pellet in 100 μL of elution buffer or dH2O. Store dissolved DNA at 4 °C during the experimental period or freeze it at -20 °C for long-term storage.
Streak E. coli ST18_pCEP-kmR (Cluster Expression Plasmid) on LB agar containing 5-ALA (50 μg/mL) and kanamycin (50 μg/mL). Incubate overnight at 37 °C. Store the plates at 4 °C for up to two months. For long-term storage, follow step A3.
Note: The plasmid is also available as pCEP-cmR (E. Bode et al., 2015). This protocol will focus on pCEP-kmR.
Inoculate 10 mL of LB medium supplemented with 5-ALA (50 μg/mL) and kanamycin (50 μg/mL) with a single colony and cultivate the cells overnight at 37 °C shaking at 200 rpm.
Note: Always add 5-ALA (50 μg/mL) for cultivation of E. coli ST18 to the media, except in step C9.
Isolate pCEP plasmid DNA (Figure 2) (low copy vector) from 10 mL of culture (E. Bode et al., 2015), following the manual instruction of Qiagen Plasmid Mini Prep kit.
Note: For higher yield of the low copy vector pCEP, extract the vector from E. coli EC100 λpir cells, cultivate in SOC medium, or use Qiagen Midi Prep kit and extract plasmid DNA from 100 mL of overnight culture.
The cloning strategy is based on the HiFi DNA assembly method and carried out with the NEBuilder HiFi DNA Assembly Master Mix following the instruction manual.
Use Geneious Prime® and design a primer pair of 20–25 bp targeting the first 300–600 bp (homologous region) of the gene of interest (Figure 1b) with homologous arms (overlaps) (Table 1) to the pCEP vector (Figure 2c). For later validation of the promoter exchange in Xenorhabdus_∆hfq, design an individual verification primer Vxyz-rv binding approximately 20–100 bp downstream of the homologous region (Figure 3). Its melting temperature (Tm) should match the Tm of the verification primer VpCEP-fw that binds to the pCEP vector (Table 2, Figure 3).
Perform PCR on the genomic DNA, extracted in step A4, with Phusion polymerase. Amplify the first 300–600 bp of the target gene (Figure 1b). Run PCR on an agarose gel (1% agarose in TAE-buffer, stain with Midori Green Advance 0.5 µL/100 mL). Use the 100 bp DNA ladder for determination of the right fragment size and cut the appropriate DNA fragment out of the gel. Use Wizard® SV Gel and PCR Clean-Up System and extract the fragment from the gel slice.
Note: Other polymerases can be used for amplification, especially for GC-rich DNA. If no fragment is obtained via PCR, sometimes a 1:10 dilution of genomic DNA with deionized H2O is helpful. An optimization of the PCR reaction mixture by adding more DMSO or additional MgCl2 might be beneficial. Adjust the PCR protocol and cycle to the respective polymerase.
Figure 2. Schematic overview of the multiple cloning site of pCEP-km and pCEP_xabA. Vector map of pCEP-kmR (a) and pCEP_xabA (b), highlighting the empty multiple cloning site (MCS) and the restriction sites for the selected enzymes NdeI and PstI (c) and the inserted gene fragment xabA (d).
Figure 3. Schematic picture of “easyPACId mutant” Xenorhabdus_∆hfq_pCEP_xyz. The general verification primer VpCEP-fw (depicted in blue) binds to the integrated pCEP backbone, and the individual verification primer Vxyz-rv (depicted in red) binds in the genome, downstream of the homologous region.
Table 1. Sequences for oligonucleotide-overlaps to the pCEP plasmid required for cloning/amplification of the gene of interest
Oligo Sequence 5′-3′
overlap-P-fw TTTGGGCTAACAGGAGGCTAGCAT_binding primer
overlap-P-rv CCGTTTAAACATTTAAATCTGCAG_binding primer
PCR protocol
1 μL
10 μL
1.5 μL
0.4 μL
0.5 μL
0.5 μL
1 μL
35.1 μL
50 μL
gDNA
5× HF-buffer
DMSO
dNTPs (25 mM)
Primer forward (100 pmol/μL)
Primer reverse (100 pmol/μL)
Phusion-polymerase
H2O
volume
PCR cycle
98 °C 2 min
98 °C
10 s
65 °C 30 s 35×
72 °C 30 s
72 °C 10 min
20 °C ∞
Perform a restriction of pCEP with enzymes NdeI and PstI (Figure 2). The restriction sites are located in the multiple cloning site of pCEP and enable the ligation of the prepared gene fragment via its homologous arms in front of the PBAD promoter, which is located upstream of the NdeI restriction site (Figure 2).
Run the restricted plasmid on an agarose gel (1% agarose in TAE buffer). Use 1 kb DNA ladder and determine the linear 4.84 kb plasmid backbone. Cut the fragment out of the gel and extract it from the gel slice with Wizard® SV Gel and PCR Clean-Up System.
Determine the DNA concentration of the plasmid backbone and insert fragments using a NanoDrop. Ligate the fragments using NEBuilder HiFi DNA Assembly Master Mix for 1 h at 50 °C, following the manual’s instructions. Additionally, set up a ligation mix that contains the restricted pCEP vector only and replace the volume of insert with dH2O as a control for restriction efficiency. After ligation, construct samples can be stored at -20 °C.
Note: Perform ligation in a PCR tube in a PCR thermocycler. If no clones are obtained, variate the vector/insert ratio.
Transformation and validation of plasmid constructs
Thaw the competent E. coli ST18 cells on ice. Transform 1–10 μL of the ligated construct (depending on the transformation method) into the donor cells via heat shock (a) or electroporation (b) (Figure 1b).
Thaw 50 μL of chemically competent cells on ice for 10 min. Add 5–10 μL of pCEP_xyz ligation mixture and incubate on ice for 30 min. Heat shock the cells for 45 s at 42 °C and subsequently incubate on ice for 2 min. Add 200 μL of LB with 5-ALA (50 μg/mL) medium. Let cells regenerate at 37 °C and 800 rpm in a ThermoMixer for 1 h.
Add 1–4 μL of pCEP_xyz ligation mixture to 50 μL of electro competent cells and mix well by pipetting up and down. Pipette the mixture onto the bottom of a pre-chilled electroporation cuvette (1 mm). Use Gene Pulser Xcell and pulse at 1.8 kV, 200 Ω, and 25 μF. Immediately add 950 μL of pre-warmed LB medium + 5-ALA (50 μg/mL), transfer the cells into a 1.5 mL reaction tube, and let cells recover at 37 °C shaking at 800 rpm in a ThermoMixer for 1 h.
Plate the transformed cells (Figure 1c) on LB agar containing 5-ALA and kanamycin. Incubate the agar plates overnight at 37 °C. Usually, colonies will appear the next day.
For cells transformed via heat shock: plate 100–200 μL of the transformed E. coli pCEP_xyz. Spin down the remaining culture at 17,000× g for 15 s, decant the supernatant, dissolve cells in the remaining liquid, and plate them on a second LB agar plate.
For electroporated cells: plate 100 μL on a first plate, then spin down the remaining culture at 17,000× g for 15 s, decant the supernatant, dissolve cells in the remaining liquid, and plate them on a second LB agar plate.
Screen for the accurate E. coli_pCEP-xyz donor by checking at least 10 single colonies by colony PCR: pick colonies with a sterile toothpick or sterile tip and suspend the cells in the colony PCR reaction mix. Set up a master plate with the corresponding selective antibiotic and 5-ALA by streaking each respective clone on the plate to save it for later applications.
Use verification primers VpCEP-fw and VpCEP-rv (Table 2) (Figure 2a and 2b) for amplification of the ligated fragment. A 234 bp fragment is the result for an empty vector.
Note: Alternatively, lyse colony material in 50 μL of dH2O and take 1 μL from the lysate for PCR. If the colony PCR gives no result, check your plasmid via restriction. Therefore, set up a small overnight culture from the respective mutant (5 mL in 50 mL flasks) in LB medium + antibiotic. Extract the plasmid from 5 mL of overnight culture and digest it with NdeI and PstI to obtain the ligated fragment size or use a single restriction enzyme that cleaves your plasmid twice.
Table 2. Oligonucleotide sequences for verification of the correct E. coli_pCEP_xyz clone binding upstream and downstream of the ligated fragment on the pCEP backbone (Figure 2)
Oligo Sequence 5′-3′ Tm
VpCEP-fw GCTATGCCATAGCATTTTTATCCATAAG 64 °C
VpCEP-rv ACATGTGGAATTGTGAGCGG 64 °C
Colony PCR
1 μL
4 μL
0.6 μL
0.16 μL
0.2 μL
0.2 μL
0.5 μL
13.34 μL
20 μL
Cell material or 1 μL DNA lysate
5× HF-buffer
DMSO
dNTPs (25 mM)
Primer VpCEPforward (100 pmol/μL)
Verification primer (100 pmol/μL)
Phusion polymerase
dH2O
volume
PCR cycle
98°C 3 min
98 °C 10 s
65 °C 30 s 35×
72 °C 30 s
72 °C 10 min
20 °C ∞
Mating procedure
Inoculate 5 mL of LB in a 50 mL flask with a single acceptor strain colony Xenorhabdus_∆hfq. Cultivate at 30 °C and 200 rpm overnight.
Note: For experiments with Xenorhabdus and Photorhabdus, the colonies should not be older than 10 days.
In parallel, inoculate the desired E. coli_pCEP-xyz donor in 5 mL of LB medium supplemented with 5-ALA and kanamycin in a 50 mL flask. Cultivate at 37 °C and 200 rpm overnight.
On the next day, inoculate the acceptor strain in 5 mL of fresh LB medium in two different ratios from the acceptor overnight culture—1:25 and 1:50—and incubate until OD600 = 0.6–1.0 at 30 °C while shaking at 200 rpm. Measure all OD600 with UV-vis-spectrophotometer Biochrom Libra S60.
In parallel, inoculate the donor E. coli_pCEP-xyz strain in 5 mL of LB medium + 5-ALA in two different ratios from the donor E. coli_pCEP-xyz overnight culture—1:50 and 1:100—and incubate until OD600 = 0.6–1.0 at 37 °C while shaking at 200 rpm. The OD is reached after 3–5 h.
Note: Prepare glycerol stocks from the overnight culture of the E. coli mutant for long-term storage. If the donor cells reach the appropriate OD earlier than the acceptor, store them on ice until the acceptor is ready.
When the OD600 is appropriate, pipette 1 mL from the acceptor strain into a reaction tube. Spin down the cells at 17,000× g for 1 min. Decant the supernatant.
Spin down 1 mL of the E. coli_pCEP_xyz donor at 17,000× g for 1 min.
Wash the E. coli_pCEP_xyz donor cells twice with 1 mL of LB medium to eliminate 5-ALA. Discard the supernatant.
Dissolve the pelleted cells in 400 μL of LB medium.
Combine donor and acceptor cells in a drop on an LB agar plate (do NOT add 5-ALA or antibiotics in this step) in the recommended acceptor:donor ratios: 1:3 (25 μL:75 μL) and 3:1 (75 μL:25 μL) (Figure 4a). Mix the cells in the drop by gently agitating the plate; do not spread the cells over the whole plate!
Incubate the plates for 3 h at 37 °C and subsequently switch to 30 °C for 21 h. Bacterial conjugation happens via the rolling circle mechanism. pCEP-xyz integrates via its homologous region “-xyz” into the genome of the acceptor strain, replacing the natural promoter by the inducible PBAD promoter (Figure 1d, Figure 3), generating the promoter exchange mutant ∆hfq_pCEP_xyz (Figure 1e, Figure 3).
Note: Do not exceed the incubation time at 37 °C as Xenorhabdus and Photorhabdus will not survive. If time is a problem, just incubate at 30 °C for 24 h, which also works well in most cases.
Selection for promoter exchange mutants
Scrape the cell plaque (Figure 4b) from the agar carefully with a sterile inoculation loop and suspend the cells in 1 mL of LB medium by pipetting up and down.
Inoculate the selection plate for ∆hfq_pCEP_xyz promoter exchange mutants by spreading 300 μL of the cell suspension on a large Petri dish containing LB agar + kanamycin (without 5-ALA!) by using a sterile Drigalski spatula or a glass pipette. Incubate the plates at 30 °C for at least 72 h and check for colonies daily (Figure 4c).
Note: Depending on the target gene, colonies may appear early, after 24 h of incubation, or late, after 72 h, if bacterial growth is severely affected by the silencing of the gene by promoter exchange.
Streak 1–10 single ∆hfq_pCEP_xyz colonies on an LB agar plate + selective antibiotic (master plate) (Figure 4d).
Perform colony PCR from selected cells: pick colony material from the plate and suspend the cells directly in the PCR samples, or dissolve material from the colony in 50 μL of dH2O or 25 μL of NaOH (0.02 M) in a PCR tube, and lyse cells at 99 °C for 5 min in the thermocycler to obtain genomic DNA. Take 1 μL for PCR and use VpCEP-fw (Table 2) and the corresponding individual verification primer Vxyz-rv (Figure 5) binding downstream of the homologous region in the genome, designed in step A9.
Note: If no fragment is obtained by colony PCR, extract genomic DNA described in step A for PCR.
Figure 4. Different steps of conjugation. Fresh (a) and 24 h incubated (b) mating plaque of Xenorhabdus_∆hfq + E. coli ST18-pCEP_xyz, which will be scraped off and dissolved in LB (according to the protocol step D1). (c) Promoter exchange mutants Xenorhabdus_∆hfq_pCEP_xyz after plating on LB-km agar and 48 h of incubation at 30 °C. (d) Master plate on LB-km agar of selected mutants from plate depicted in (c).
Production cultures and HPLC/MS analysis
Inoculate 5 mL of LB + selective antibiotic in a 50 mL flask with a single ∆hfq_pCEP_xyz colony. Prepare respective cultures from the ∆hfq acceptor strain and the corresponding WT strain as controls.
Next day: wash the cells twice with LB medium to eliminate antibiotics.
Inoculate 100 mL of LB medium in 500 mL flasks with washed cells from the pre-cultures 0.1% (v/v) and induce all cultures with L-arabinose (final concentration 0.2%). Cultivate for 48–72 h at 30 °C and 140 rpm on a rotary shaker.
Transfer the cultures in 50 mL plastic tubes with a threaded cover and spin down the cells at 3,220× g for 20 min.
Note: Extend centrifugation time if the cells do not form a solid pellet.
Transfer the cell-free supernatant into new plastic tubes.
For HPLC/MS analysis: Pipette 50 μL of the supernatant in a reaction tube and extract it with 500 μL of methanol by mixing vigorously for 20 s on a vortex. Centrifuge the samples at 17,000× g for 20 min.
Pipette a sample of 50–120 μL into a HPLC vial for HPLC/MS analysis (Figure 5). Use DataAnalysis to analyze the HPLC/MS data.
Freeze the remaining cell-free supernatant at -80 °C.
Subsequently freeze dry the supernatant in a lyophilisator (Lyovapor L-300).
The lyophilizate can be stored at -20 °C for further investigations.
Dissolve the lyophilizate in 10 mL of deionized water and use the 10-fold concentrate for further analysis.
Note: Adjust solvents to specific testing conditions. To consider compounds that are located in the cell membrane, freeze dry the whole culture.
Data analysis
Exemplary data
Figure 5. Extracted ion chromatograms (EICs) for xenoamicin (m/z 658.2 [M+2H]2+). Depicted are chromatograms from cultures in LB medium from X. doucetiae WT (green), X. doucetiae∆hfq (blue), and easyPACId mutant X. doucetiae∆hfq_pCEP_xabA (red) after induction with 0.2% L-arabinose. Cell-free supernatant was extracted with methanol according to the protocol step E6.
Recipes
Luria-Bertani medium (LB) (1 L)
Tryptone 10 g
Yeast extract 5 g
NaCl 5 g
dH2O 1 L
For solid medium, add
Bacto-agar 15 g
Sterilize by autoclaving for 20 min at 121 °C.
Note: Add antibiotics, 5-ALA, or L-arabinose after autoclaving.
5-Aminolevulinic acid hydrochloride (5-ALA) (50 mg/mL)
Prepare a stock solution with 50 mg/mL in dH2O, filter sterilize, and store at -20 °C in black reaction tubes to avoid photochemical degradation. Working concentration 50 μg/mL.
L-Arabinose (20% w/v)
Prepare a stock solution of 200 g/L in dH2O. Sterilize it by filtration via Steritop 45 mm neck size Millipore Express Plus.
Store at 4 °C or -20 °C for long-term storage.
Kanamycin (50 mg/mL)
Prepare a 50 mg/mL stock solution in dH2O, filter sterilize, and store at -20 °C.
TAE buffer, pH 8.0 (1 L)
Tris-Base 2.0 M 242 g
Acetic acid 2.0 M 57.1 mL
10% (v/v) EDTA 0.5 M pH 8.0 100 mL
0.5 M EDTA pH 8.0 (1 L)
Na2-EDTA·2H2O 182.1 g
Dissolve in ddH2O
Add 15 g of NaOH pellets and adjust pH with 5 M NaOH
Sterilize by autoclaving at 121 °C for 15 min
Acknowledgments
Part of this work was supported by an ERC Advanced Grant (835108) to H.B.B. The authors are grateful for support of the Max-Planck-Society. This protocol is derived from earlier versions described in the publications (E. Bode et al., 2015 and 2019).
Competing interests
The authors state that there are no competing interests.
References
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Biggins, J. B., Liu, X., Feng, Z. and Brady, S. F. (2011). Metabolites from the induced expression of cryptic single operons found in the genome of Burkholderia pseudomallei. J Am Chem Soc 133(6): 1638-1641.
Bode, E., Brachmann, A. O., Kegler, C., Simsek, R., Dauth, C., Zhou, Q., Kaiser, M., Klemmt, P. and Bode, H. B. (2015). Simple “on-demand” production of bioactive natural products. Chembiochem 16(7): 1115-1119.
Bode, E., He, Y., Vo, T. D., Schultz, R., Kaiser, M. and Bode, H. B. (2017). Biosynthesis and function of simple amides in Xenorhabdus doucetiae. Environ Microbiol 19(11): 4564-4575.
Bode, E., Heinrich, A. K., Hirschmann, M., Abebew, D., Shi, Y. N., Vo, T. D., Wesche, F., Shi, Y. M., Grün, P., Simonyi, S., et al. (2019). Promoter Activation in Deltahfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing. Angew Chem Int Ed Engl 58(52): 18957-18963.
Bode, H. B. (2009). Entomopathogenic bacteria as a source of secondary metabolites. Curr Opin Chem Biol 13(2): 224-230.
Bode, H. B., Bethe, B., Höfs, R. and Zeeck, A. (2002). Big effects from small changes: possible ways to explore nature's chemical diversity. Chembiochem 3(7): 619-627.
Brachmann, A. O., Kirchner, F., Kegler, C., Kinski, S. C., Schmitt, I. and Bode, H. B. (2012). Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens. J Biotechnol 157(1): 96-99.
Chaston, J. M., Suen, G., Tucker, S. L., Andersen, A. W., Bhasin, A., Bode, E., Bode, H. B., Brachmann, A. O., Cowles, C. E., Cowles, K. N., et al. (2011). The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes. PLoS One 6(11): e27909.
Goodrich-Blair, H. and Clarke, D. J. (2007). Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination. Mol Microbiol 64(2): 260-268.
Gulsen, S. H., Tileklioglu, E., Bode, E., Cimen, H., Ertabaklar, H., Ulug, D., Ertug, S., Wenski, S. L., Touray, M., Hazir, C., et al. (2022). Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach. Sci Rep 12(1): 10779.
Guzman, L. M., Belin, D., Carson, M. J. and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177(14): 4121-4130.
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Nollmann, F. I., Dauth, C., Mulley, G., Kegler, C., Kaiser, M., Waterfield, N. R. and Bode, H. B. (2015). Insect-specific production of new GameXPeptides in photorhabdusluminescens TTO1, widespread natural products in entomopathogenic bacteria. Chembiochem 16(2): 205-208.
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Visualization of Actin Cytoskeleton in Cellular Protrusions in Medaka Embryos
TK Toru Kawanishi
AH Ann Kathrin Heilig
AS Atsuko Shimada
HT Hiroyuki Takeda
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4710 Views: 474
Reviewed by: Chiara AmbrogioIstvan StadlerIndranil Malik
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Original Research Article:
The authors used this protocol in eLIFE May 2022
Abstract
Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell–cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy.
Keywords: Cellular protrusion Actin Confocal imaging Microinjection Medaka Developmental biology
Background
Cellular protrusions are fundamental structures that play pivotal roles in various aspects of developmental biology such as cell migration, cell-cell communication, and signal reception (Sanders et al., 2013; Roy et al., 2014; Sagar et al., 2015; Genuth et al., 2018; Mattes et al., 2018; Dalle Nogare et al., 2020; Bischoff et al., 2021; Heilig et al., 2022). These protrusions can be morphologically categorized into several types, including filamentous projections called filopodia, sheet-like structures called lamellipodia, and round-shaped blebs (Ridley, 2011). The distinct morphologies of cellular protrusions are manifested by the spatial distribution of cytoskeletal actin: in filopodia, for example, actin filaments elongate radially and change their length over time, while actin in lamellipodia constitutes a planar meshwork and exhibits treadmilling motions (Rottner et al., 2017). Thus, visualizing actin distribution is a good proxy to describe the morphology and dynamics of cellular protrusions. Actin distribution in living cells has been visualized with fluorescent probes, including actin-binding peptides/proteins such as LifeAct and Utrophin, and GFP-labeled actin itself (Melak et al., 2017). These actin probes are genetically encodable and therefore can be expressed in diverse types of cells and tissues via transgenesis or mRNA injection (Blaser et al., 2006; Riedl et al., 2010; Dalle Nogare et al., 2020). More recently, a new fluorescent actin probe called Actin-Chromobody was developed based on a single monomeric antibody (nanobody) against actin (Rocchetti et al., 2014; Panza et al., 2015). Use of this new probe could potentially minimize interference with the endogenous actin dynamics, which is sometimes observed for the traditional actin probes (Spracklen et al., 2014; Flores et al., 2019; Xu and Du, 2021).
Medaka (Oryzias latipes) is a freshwater fish with beneficial features for developmental biology, similar to zebrafish; daily spawning and external development of transparent embryos as well as a small genome size enable investigation of cellular events taking place during embryogenesis (Wittbrodt et al., 2002; Takeda and Shimada, 2010). Furthermore, there is a series of spontaneous and mutagenesis-mediated medaka mutants, some of which turned out to have unique mutations and phenotypes that have not been identified in zebrafish (Omran et al., 2008; Kawanishi et al., 2013; Porazinski et al., 2015), demonstrating that medaka is a model genetic organism complementary to zebrafish. Medaka thus offers an expanding opportunity for analyses of unprecedented cellular mechanisms underlying embryonic development.
Live imaging of cellular protrusions in an embryo requires its complete immobilization. In medaka, contractile movements of the yolk surface and the periderm occur between gastrulation and mid-somitogenesis stages (Yamamoto, 1975; Robertson, 1979; Fluck et al., 1983; Iwamatsu, 2004), which can be inhibited by administration of n-heptanol (Rembold and Wittbrodt, 2004). Moreover, as with zebrafish, trunk twitching caused by muscle contraction takes place later until the hatching stage (Iwamatsu, 2004). The twitching can also be blocked by tricaine treatment, although a long exposure to the drug leads to mild developmental defects (Swinburne et al., 2015). α-bungarotoxin, which blocks acetylcholine receptors, has been recently shown to efficiently inhibit muscle twitching in zebrafish and medaka embryos without perturbing embryonic development (Swinburne et al., 2015; Lischik et al., 2019).
Here, we describe a detailed protocol to visualize actin dynamics in cellular protrusions of medaka embryos step by step. We label actin filaments in some of the cells by introducing mRNA encoding Actin-Chromobody-TagGFP2 into a cell at the 4-cell stage. We also fully immobilize the medaka embryo while imaging at the subcellular level under a confocal microscope by injecting α-bungarotoxin mRNA as well. Since the expression of the fluorescent probe lasts up to 5–6 days, our method exploiting mRNA injection is a convenient way to visualize actin distribution during embryogenesis without the need to create transgenic lines. Our protocol should be applicable for a wide range of functional investigation of cell protrusions in living medaka embryos, as well as in zebrafish and other fish embryos that are amenable to microinjection and dechorionation.
Materials and reagents
Medaka (Oryzias latipes) d-rR strain. Can be ordered from NBRP, Japan
(https://shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true&strainId=5666). Adult medaka should be kept at 26–28 °C in a room with a 14:10 h light/dark cycle to promote spawning
pMTB-AC-TagGFP2 (Heilig et al., 2022). Available from the authors upon request
pMTB-α-bungarotoxin (Addgene Plasmid #69542) (Swinburne et al., 2015)
AvaI (New England Biolab, catalog number: R0152S). 10× CutSmart buffer is included in the product
EcoRV (New England Biolab, catalog number: R0195S). 10× CutSmart buffer is included in the product
0.5% phenol red in DPBS (Sigma-Aldrich, catalog number: P0290)
Penicillin-streptomycin solution (Thermo Fisher Scientific, catalog number: 15140122)
Hatching enzyme (store at -80 °C). Can be ordered from NBRP, Japan (https://shigen.nig.ac.jp/medaka/strain/hatchingEnzyme.jsp). You can also make hatching enzyme by yourself [see pp. 254–255 in Kinoshita et al. (2009)]
Agarose (Funakoshi, catalog number: GA-001)
Agarose, low melting point (LMP) (Promega, catalog number: V2831)
Wizard SV gel and PCR clean-up system (Promega, catalog number: A9285)
mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher Scientific, catalog number: AM1340). 2× NTP/CAP, reaction buffer, and enzyme mix are included in the kit
RNeasy mini kit (Qiagen, catalog number: 74104)
Fish-scooping net with fine mesh
9 cm plastic dish, Asnol (As One, catalog number: 1-8549-04)
Multidish, 4 wells, Nunc (Thermo Fisher Scientific, catalog number: 176740)
Glass bottom dish (Iwaki, catalog number: 3911-035)
Injection mold. You need to make the mold by yourself; to design it, see pp. 279 in Kinoshita et al. (2009). You can order a plastic mold using the design through the Shapeways website (https://www.shapeways.com/); choose “Smoothest Fine Detail Plastic” for the material
Glass capillary (Harvard Apparatus, catalog number: GC100F-10)
Microcapillary tip (Eppendorf, catalog number: 5242956003)
Forceps (Dumont, catalog number: No.5-INOX)
Pasteur pipette (Asahi Glass, catalog number: IK-PAS-9P)
Yamamoto’s Ringer’s solution (store at 4 °C) (see Recipes). Dilute 50 mL of 10× Yamamoto’s Ringer’s solution in 450 mL of water, autoclave it, and add 5 mL of penicillin-streptomycin solution
Hatching buffer (store at room temperature) (see Recipes). Dilute 100× hatching buffer in water
Equipment
Heating dry bath incubator (Major Science, model: MD-MINI)
Heating chamber (e.g., Tokai Hit, model: INUC-KRi)
Microvolume spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 2000c)
Capillary puller (Narishige, model: PC-10)
Microinjector (Eppendorf, model: FemtoJet 4i)
Manipulator (Narishige, model: M-152)
Magnet stand for the manipulator (Narishige, model: GJ-8)
Iron plate for the magnet stand (Narishige, model: IP)
Incubator (Mitsubishi Electric Engineering, model: CN-25C)
Stereomicroscope (Leica, model: M165 FC)
External light source for fluorescence excitation (Leica, model: EL6000)
Inverted confocal microscope (Zeiss, model: LSM 710)
25× water immersion objective with a long working distance (Zeiss, catalog number: 420852-9871-000)
40× water immersion objective with a long working distance (Zeiss, catalog number: 421867-9970-000)
Software
Zen (Zeiss, https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html)
Fiji (https://imagej.net/software/fiji/) (Schindelin et al., 2012)
Procedure
mRNA synthesis
mRNA is synthesized in vitro from a plasmid containing both a promoter sequence for SP6/T7/T3 RNA polymerase and an SV40-derived polyA signal. Here, we use the pMTB vector, which was originally designed for zebrafish transgenesis as well as in vitro mRNA synthesis (Wagner et al., 2018). pCS2+ is another vector that is commonly used for in vitro mRNA synthesis.
Linearize the pMTB-AC-TagGFP2 and pMTB-α-bungarotoxin plasmids with restriction enzymes. To do so, mix the following solutions and incubate at 37 °C for 2 h:
Plasmid (pMTB-AC-TagGFP2 or pMTB-α-bungarotoxin), 2 μg
10× CutSmart buffer, 2 μL
Restriction enzyme (EcoRV for pMTB-AC-TagGFP2, AvaI for pMTB-α-bungarotoxin), 1 μL
Water up to 20 μL
Run electrophoresis with 1 μL of the reaction solution to verify that the plasmids have been fully linearized.
Purify the linearized templates with Wizard SV gel and PCR clean-up system kit as per the manufacturer’s instructions. Elute the DNA in 25 μL of water.
Assess the concentration of the eluents with a NanoDrop spectrometer. The concentration should be approximately 100 ng/μL.
Synthesize mRNA using the mMESSAGE mMACHINE SP6 transcription kit. After thawing the kit solutions at room temperature (except the RNA polymerase enzyme mix), mix the solutions listed below (from top to bottom) at room temperature and incubate at 37 °C for 2 h:
Nuclease-free water up to 10 μL (in total)
2× NTP/CAP, 5 μL
10× reaction buffer, 2 μL
Template, 0.5–1 μg
Enzyme mix, 1 μL
While handling RNA, be sure to avoid RNase contamination by wearing gloves.
Digest the DNA templates. Add 1 μL of TURBO DNase (included in the kit) and incubate at 37 °C for 15 min.
Purify the mRNA with RNeasy mini kit following the manufacturer’s instructions and elute the mRNA in 30 μL of nuclease-free water.
Assess the concentration of the mRNA eluents with a NanoDrop spectrometer. The concentration should be 100–500 ng/μL. The A280/A260 ratio of purified mRNA is typically approximately 2.0. The quality of mRNA can be further validated by agarose gel electrophoresis, whereby a single band with little smear will appear unless mRNA is degraded or unsuccessfully synthesized. The solution can be stored at -80 °C for months.
Preparation of injection tools
Injection gel
Pour 20 mL of melted 1% agarose dissolved in the hatching buffer into a 9 cm plastic dish and apply the injection mold onto the agarose before it solidifies. Trapping the air bubbles under the mold should be prevented by slowly sliding the mold onto the agarose surface with an angle of 45° (Figure 1A and 1B).
Once the agarose is solidified, remove the mold and pour the hatching buffer on the gel to prevent drying out (Figure 1C). The agarose gel can be stored at 4 °C for a few months and repeatedly used.
Injection needles
Pull the glass capillaries using a Narishige capillary puller. Adjust the No. 2 heater adjustment parameter to 57.5 and set the mode selector knob to the step 1 mode. This configuration provides an ideal needle shape for microinjection into medaka embryos.
Store the needles in a container so that the fragile tips do not touch anything (Figure 1D).
Figure 1. Injection tools for medaka embryos. (A, B) Injection mold sliding onto melted agarose. Note that the mold is gently put onto the agarose with an angle of 45° to exclude air bubbles between the mold and agarose (A). (C) Injection gel with 0.9 mm wide wells. (D) Injection needles stored in a container.
Microinjection into medaka eggs (Video 1)
Video 1. Microinjection into medaka eggs. After adult medaka pairs are crossed, collect fertilized eggs and remove attaching filaments on the chorion with a fish-scooping net to separate each other. Transfer separated eggs onto the injection gel and align them within the grooves of the gel. At the 4-cell stage, inject the mRNA solution into one of the cells.
In the evening of the day before injection, pair adult medaka for mating in fish tanks. Separate females from males using transparent partitioners.
In the morning of injection day (typically within 2 h after the room light turns on), remove the partitioner to enable the pairs to start mating. Mating will happen typically within 5–10 min.
While waiting for the eggs (approximately 10–30 min), prepare the injection solution by mixing the following ingredients:
Actin-Chromobody-TagGFP2 mRNA, 1.5 μg
α-bungarotoxin mRNA, 0.25 μg
0.5% phenol red, 0.75 μL
Nuclease-free water up to 10 μL
Collect eggs from the female and transfer them into the hatching buffer.
Medaka eggs tend to stick together because of long attaching filaments on the chorion. To separate eggs, transfer them into a fish-scooping net and gently roll the eggs with the net between your fingers until the egg clumps disintegrate and all eggs are separated (Figure 2A–2C). During the process, some short attaching filaments are removed as well, and tiny holes are created on the chorion; these holes will allow hatching enzyme to enter the chorion and digest it (Procedure D).
Transfer the eggs onto the injection gel, which is covered with the hatching buffer, and gently align the eggs within the wells using forceps (Figure 2D).
Load the prepared glass needle with ~3 μL of the injection solution using a Microloader tip and attach the needle to the injector.
Initiate the injector and adjust the injection pressure (Pi) and compensation pressure (Pc) to appropriate levels. During the injection process, the needle should neither take up the embryo medium nor eject the injection solution too fast. Pressure values vary between individual needles, but we typically set Pi and Pc to ~500 and ~150 hPa, respectively.
Break the tip of the needle by gently scratching the surface of a chorion, until injection solution slowly leaks out of the tip.
When the embryos reach the 4-cell stage (Figure 2E), inject the solution into the cytoplasm of one cell of each embryo. This will lead to mosaic expression of Actin-Chromobody-TagGFP2, herewith highlighting actin dynamics in individual cells. The injection volume should be up to approximately half the size of the cell (corresponding to 2–3 nL).
Figure 2. Separation of eggs using a fish-scooping net. (A) Gently roll the eggs (arrow) with a net to separate them. (B) Eggs with intact attachment filaments. (C) Eggs after separation using a net. (D) Eggs aligned on an injection gel. (E) A 4-cell embryo. Scale bars = 1 cm.
Incubate the injected embryos in the hatching buffer at 23–32 °C.
Dechorionation
Dechorionation should be performed on the day of injection to maximize the chances of obtaining healthy embryos. Although dechorionation can be done later, digestion of the chorion is more likely to be incomplete, potentially leading to damaging the embryo with partially digested chorion pieces.
Dilute the hatching enzyme 1:5–1:20 with 1/2× Yamamoto’s Ringer’s solution. The dilution ratio should be determined based on the enzymatic activity of the enzyme lot; when you use the lot for the first time, we recommend that you begin with a 1:20 dilution and examine how long it takes to digest most of the chorion (see step D3).
Transfer embryos to a container with a small bottom area, e.g., a 4-well dish, and remove the culture medium (Figure 3A).
Apply the diluted hatching enzyme onto the embryos and incubate them at 28 °C until the chorion is mostly digested (Figure 3B–3D). The incubation duration depends on the enzymatic activity of the lot, but usually takes up to 2–3 h. The enzyme will degrade the hard layer of the chorion, while a thin membranous layer will remain undigested (Figure 3D). It is recommended to check the digestion status in regular intervals (30–60 min). If the chorion is not digested within a few hours, try a higher concentration for the enzyme solution.
Figure 3. Dechorionation with hatching enzyme. (A) Apply diluted hatching enzyme solution onto embryos in a 4-well dish. (B-D) Temporal change of the chorion during hatching enzyme treatment. (B) Intact chorion has a thick hard layer. (C) Partially digested chorion shows a rough surface inside. (D) Digested chorion retains a thin membranous layer that can be torn with forceps. Scale bar = 500 μm.
Carefully transfer the embryos to a glass dish filled with Yamamoto’s Ringer’s solution to rinse off the enzyme. Most of the embryos are still surrounded by the thin layer of the chorion, but some may be fully out of it. Transfer all embryos to a new glass dish filled with Yamamoto’s Ringer’s solution. Since physical contact with the bottom surface of a brand-new plastic dish could damage the embryo, keeping the embryos in a glass dish is preferred.
Incubate the injected embryos at 23–32 °C until they reach the desired developmental stage. For stage 28, it takes approximately three days at 26 °C (Iwamatsu, 2004).
Mounting embryos in agarose for imaging
In this protocol, we focus on embryos from stage 28 onward.
When the embryos reach the desired stage, screen for embryos exhibiting GFP fluorescence at the region of interest (ROI), such as somites or epidermis. For the screening, use a fluorescent stereomicroscope and transfer suitable embryos into a new dish filled with Yamamoto’s Ringer’s solution. Using forceps, gently remove the remaining chorion of the selected embryos.
Dissolve 1% LMP agarose in hot Yamamoto’s Ringer’s solution and keep it at ~40 °C using a heating incubator.
Pour a few drops of the LMP agarose solution to cover the entire glass part of a glass bottom dish and immediately transfer 2–3 embryos into the agarose. Mount the embryos before the agarose solidifies. Orient the embryos with forceps in a way that the ROI is in contact with the glass bottom (Figure 4). This allows the ROI to be within working distance of the confocal microscope. Once the agarose solidifies (within a minute), avoid moving the embryos. If the orientation of the embryos is not adequate, add Yamamoto’s Ringer’s solution to the dish, break the agarose with forceps to release the embryos, and repeat the mounting process.
Figure 4. Mounting of embryos in low-melting-point agarose for imaging. Embryos are mounted adjacently to the glass surface of the dish (arrows) for subsequent imaging with an inverted confocal microscope.
Imaging cellular protrusions using an inverted confocal microscope
Using an objective with a long working distance is critical for imaging cells located inside the embryo.
Start the confocal microscope and the microscope software Zen. Use the 25× water immersion objective and select the Locate tab on the software to allow observation through the eyepieces.
Place the dish with the mounted embryos on the microscope stage and find the ROI by visual inspection through the eyepieces. Screen the embryos by their GFP fluorescence at the ROI.
Once a good candidate is found, select the Acquisition tab on the software and start imaging the focal plane. Overall, tissue structures can be captured with this magnification.
To obtain subcellular resolution, switch to the 40× water immersion objective and acquire images (Figure 5). Timelapse imaging can also be performed by activating the Time series checkbox on the software (Figure 6). If timelapse imaging takes a few hours or more, install a heating chamber on the stage to maintain the incubation temperature.
Figure 5. Representative images of cellular protrusions in different cell types. Actin-Chromobody-TagGFP2 (green) highlights cellular protrusions in a dorsal-most dermomyotomal cell (A), a dermomyotomal cell located more ventrally (B), and epidermal cells (C) of medaka embryos at stage 28. Arrowheads and arrows indicate filopodia and lamellipodia, respectively. Scale bars = 5 μm.
Figure 6. Temporal dynamics of cellular protrusions. Actin-Chromobody-TagGFP2 labels actin dynamics in epidermal cells of a medaka embryo at stage 28 (A) and one minute later (B). The merged image is shown in (C). Arrow indicates growth of a lamellipodium. Scale bar = 5 μm.
Data analysis
Addition of a scale bar to the confocal image
Open the image in Fiji.
Go to Analyze > Tools > Scale bar… and choose from displayed options. A scale bar with the defined length will appear on the image.
Recipes
10× Yamamoto’s Ringer’s solution (store at room temperature)
Reagent Final concentration Amount
NaCl 1.3 M (7.5%) 75 g
KCl 27 mM (0.2%) 2.0 g
CaCl2·2H2O 18 mM (0.27%) 2.7 g
NaHCO3 24 mM (0.2%) 2 g
H2O n/a Up to 1,000 mL
Total n/a 1,000 mL
100× Hatching buffer (store at room temperature)
Reagent Final concentration Amount
NaCl 1.7 M (10%) 100 g
KCl 40 mM (0.3%) 3 g
CaCl2·2H2O 27 mM (0.4%) 4 g
MgSO4·7H2O 65 mM (1.6%) 16 g
H2O n/a Up to 1,000 mL
Total n/a 1,000 mL
Acknowledgments
We thank Ms. I. Fukuda, M. Sakamoto, Y. Yamagishi, and F. Hattori for fish husbandry. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP15H05859 (H.T.), JP21K15101 (T.K.) and Japan Science and Technology Agency CREST Grant Number JPMJCR13W3 (H.T.). This protocol was derived from Heilig et al. (2022).
Competing interests
The authors declare no competing interests.
Ethical considerations
All experimental procedures and animal care were carried out according to the animal ethics committee of the University of Tokyo.
References
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Cell Biology > Cell imaging > Live-cell imaging
Molecular Biology > RNA > mRNA translation
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Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis
HW Haoyu Wu *
PW Ping Wang *
ZL Ziying Liu
CL Chunyan Lu
WY Wenguang Yin
(*contributed equally to this work)
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4711 Views: 370
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Original Research Article:
The authors used this protocol in Nature Communications Jul 2018
Abstract
The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.
Keywords: Trachea Smooth muscle cell Cartilage Mesenchymal condensation Cell polarity
Background
Understanding tracheal formation is a fundamental goal in the field of pulmonary development and disease. The trachea consists of mesoderm-derived smooth muscle (SM), cartilage, and connective tissue, as well as endoderm-derived epithelium (Brand-Saberi and Schafer, 2014). SM is positioned dorsally to control tracheal contraction, whereas the cartilage rings are located ventrally to prevent airway collapse (Hines et al., 2013; Yin et al., 2018 and 2019).
In humans, defects in the formation of the tracheal tube have been reported to lead to tracheomalacia, tracheostenosis, or complete tracheal ring deformity, which are characterized by a deficiency of the supporting cartilage or narrowing of the tracheal lumen and may lead to respiratory distress and death (Landing and Dixon, 1979; Fraga et al., 2016; Sinner et al., 2019).
In mice, tracheal SM differentiation starts by embryonic day 11.5 (E11.5) with the appearance of α-smooth muscle actin (αSMA) positive cells at the dorsal side (Hines et al., 2013; Yin et al., 2018). Newly differentiated tracheal SM cells exhibit round shapes and are not well organized (Yin et al., 2018 and 2019). They progressively develop into spindle-shaped cells that circumferentially align the tube (Yin et al., 2018 and 2019). During tracheal elongation, differentiated SM cells proliferate with increased area of SM stripes from E11.5 to P0 (Yin et al., 2018).
Tracheal cartilage development initiates as early as E9 (Elluru and Whitsett, 2004). Uncondensed SOX9+ mesenchymal cells appear and are restricted to the ventral trachea as early as E10.5 (Hines et al., 2013). Both Sox9 mRNA levels and the number of SOX9+ mesenchymal cells increase at E12.5 (Hines et al., 2013). By E13.5, SOX9+ mesenchymal cells condense to resemble cartilaginous rings with the number of 11–13. The number of C-shaped rings appears to be unchanged, whereas both the distance between rings and ring width increase during tracheal elongation (Yin et al., 2019). By E15.5, SOX9+ mesenchymal cells start differentiating into chondrocytes characterized by positive alcian blue staining, as well as aggrecan and type II collagen expression (Park et al., 2010; Yin et al., 2019).
Analysis of tracheal formation has mostly focused on cell condensation, differentiation, proliferation, and apoptosis by using tissue sections (Park et al., 2010; Hines et al., 2013; Lin et al., 2014). These studies have provided information on cell behavior in two-dimensional views, allowing for a better understanding of the cellular and molecular mechanisms underlying tracheal tubulogenesis. The method we present here for whole-mount immunostaining and imaging overcomes some of their limitations; for example, the precise sites of the three-dimensional trachea are not properly located. Thus, it is presently unclear how cells are organized in the whole trachea. Another not fully investigated question is the characteristics of SM cells. Although SM cell differentiation and proliferation have been tested (Hines et al., 2013; Gerhardt et al., 2018), cell alignment, polarization, and changes in cell shape in tracheal development remain largely unknown. To analyze tissue structure and cell organization, we provide a detailed protocol for examining SM cell organization and changes in cell shape as well as mesenchymal cell condensation, an important process during tracheal cartilage formation (Yin et al., 2019).
Materials and reagents
Coverslips (Thermo Fisher Scientific, catalog number: 11961988)
Adhesion slides (Thermo Fisher Scientific, catalog number: 10219280)
10 cm Petri dish (Corning, catalog number: 430167)
Pipette (Sigma-Aldrich, catalog number: Z331759-1PAK)
Cy3-conjugated anti-mouse αSMA (Sigma-Aldrich, catalog number: C6198)
Anti-rat CDH1 (E-cadherin) (Santa Cruz, catalog number: sc-59778)
Anti-rabbit SOX9 (SRY-box transcription factor 9) (Millipore, catalog number: AB5535)
Sheep anti-GM130/GOLGA2 (Golgin subfamily A member 2) (R&D systems, catalog number: AF8199)
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 30525-89-4)
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418)
Mowiol (Millipore, catalog number: 475904)
30% hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 7722-84-1)
Methanol (Sigma-Aldrich, catalog number: 34860)
Bovine serum albumin (BSA) (Thermo Fisher Scientific, catalog number: 30036578)
Fetal bovine serum (FBS) (Gibco, catalog number: 10270-106)
4',6-Diamidino-2-Phenylindole, dilactate (DAPI) (Thermo Fisher Scientific, catalog number: D3571)
Phosphate buffered saline (PBS) (Capricorn Scientific, catalog number: PBS-1A)
Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H6648)
Triton X-100 (Sigma-Aldrich, catalog number: 9036-19-5)
Glycerol (Sigma-Aldrich, catalog number: USA56-81-5)
Benzyl benzoate (Sigma-Aldrich, catalog number: B6630)
Benzyl alcohol (Sigma-Aldrich, catalog number: 305197)
Benzyl benzoate (Sigma-Aldrich, catalog number: B6630-1L)
Sucrose (Sigma-Aldrich, catalog number: 57-50-1)
Tissue-Tek OCT (SAKURA, catalog number: 4583)
Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa FluorTM 488 (Thermo Fisher Scientific, catalog number: A-11034)
Donkey anti-sheep IgG (H+L) secondary antibody, Alexa FluorTM 647 (Thermo Fisher Scientific, catalog number: A-21448)
Goat anti-rat IgG (H+L) secondary antibody, Alexa FluorTM 568 (Thermo Fisher Scientific, catalog number: A-11077)
Phosphate buffered saline (PBS) (see Recipes)
4% formaldehyde solution (see Recipes)
Permeabilization solution (see Recipes)
10% sucrose (see Recipes)
30% sucrose (see Recipes)
Mowiol mounting medium (see Recipes)
0.3% Triton X-100/PBS solution (see Recipes)
5% FBS/PBS/0.5% Triton X-100/3% BSA solution (see Recipes)
70% ethanol solution (see Recipes)
DMSO:methanol (1:4) solution (see Recipes)
H2O2/DMSO/methanol (1:1:4) solution (see Recipes)
80% methanol solution (see Recipes)
50% methanol solution (see Recipes)
Benzyl alcohol:benzyl benzoate (BABB) (1:2) solution (see Recipes)
5% FBS/PBS/3% BSA blocking solution (see Recipes)
Equipment
Scissors (FST, catalog number: 15011-12)
Forceps (FST, catalog number: 11252-20)
Stereoscopic dissecting microscope (Zeiss, model: Stemi 305)
Stereomicroscope (Nikon, model: SMZ25)
Upright laser scanning confocal microscope (Zeiss, model: LSM 880) or cryostat (Leica, model: CM1950)
Quantification of immunofluorescence intensity of lumen area, tube length, SM area, SM cell orientation, nuclear, aspect ratio (NAR), and Golgi-apparatus position relative to the nucleus was performed using ImageJ (Version 1.8.0.172) (Schneider et al., 2012)
Software
ImageJ, GraphPad Prism (Version 8.0.2)
Zeiss ZEN (Version 2.3)
Procedure
A schematic diagram of the protocol overview is presented in Figure 1.
Figure 1. Schematic diagram of the protocol overview
Mouse tracheal dissection
Euthanize pregnant mice at E11.5–E16.5 by CO2 exposure, consistent with institutional ethical guidelines and approved protocols. Place one mouse in a CO2 chamber and gradually fill CO2 at a flow rate of 20% of the chamber volume per minute. Keep the animal in the chamber for 4 min and maintain gas flow for an additional 2 min after apparent clinical death. Perform cervical dislocation following CO2 treatment (Figure 1).
Isolate embryos from euthanized animals. Clean and sterilize the abdomen with 70% ethanol. Perform laparotomy using surgical scissors and forceps. Transfer the uterus and keep it in ice-cold HBSS in a 10 cm Petri dish. Open the uterus and transfer embryos to a new dish containing ice-cold HBSS (Supplementary Figure 1).
Dissect the trachea and lungs from the embryo under the stereoscopic dissecting microscope. For embryonic mice, it is best to start dissection from the abdominal cavity so as not to damage the trachea and lungs in the thoracic cavity. Fix the head and forelimb, remove the skin, carefully open the abdominal cavity not to damage the trachea and lungs, and remove the heart using scissors and forceps. Separate the trachea and lungs from the embryo and trim away the esophagus using forceps. The trachea is cut above the larynx to ensure its integrity. The esophagus is dissected behind the trachea and down from the larynx with two tiny forceps. Transfer the trachea and lungs to a 24-well plate containing ice-cold PBS using a 1 mL pipette to ensure tissue integrity (Supplementary Figure 1 and Supplementary Figure 2).
Whole-mount immunostaining and imaging
The mouse embryonic trachea is tiny. To avoid sucking it away in washing procedures, we perform whole-mount immunostaining using the whole trachea and lungs and suck out washing buffer when tissues sink to the bottom of the tube. Fix E11.5–E16.5 tracheas and lungs in DMSO:methanol (1:4) (see Recipes) overnight at 4 °C. Incubate samples in H2O2/DMSO/methanol (1:1:4) (see Recipes) for 5 h at room temperature (RT). The purpose of soaking the tissue in a fixative containing hydrogen peroxide is to remove endogenous catalase.
Wash samples twice in 100% methanol for 1 h each, once in 80% methanol for 1 h, once in 50% methanol for 1 h, twice in PBS for 1 h each, and twice in 5% FBS/PBS/0.5% Triton X-100/3% BSA (see Recipes) for 1 h each.
Incubate tracheas and lungs in primary antibodies diluted in 5% FBS/PBS/0.5% Triton X-100/3% BSA (SOX9, 1:400; αSMA-Cy3, 1:200; GM130, 1:50) for 24 h at 4 °C. Wash samples five times in 5% FBS/PBS/0.5% Triton X-100/3% BSA for 1 h each at 4 °C.
Incubate samples in secondary antibodies (Alexa Fluor 488, 1:1,000; Alexa Fluor 647, 1:1,000; DAPI, 1:2,000) for 24 h at 4 °C. Wash samples five times in 5% FBS/PBS/0.5% Triton X-100/3% BSA for 1 h each at 4 °C.
Dehydrate samples in methanol for 6 h (incomplete dehydration may result in tissue disintegration), and then clear samples in BABB (1:2) (see Recipes) for 24 h.
Mount samples in BABB and seal the edges of the cover glass completely with nail polish.
After whole-mount immunostaining, tissues are transferred to the microscope for imaging. The position of the trachea is gently adjusted with a pipette as follows: throat and upper trachea in the anterior direction, lower trachea in the posterior direction, cartilage in the upward direction, and SM in the downward direction to obtain a dorsal and ventral view. Acquire images of tracheal SM and cartilage rings in an anterior–posterior direction with a laser scanning confocal microscope by using a 20× objective and a stereomicroscope with a fluorescence mode by using a 2.5× objective, respectively. Take 10–15 images for whole-mount immunostaining. Take one image for section immunostaining (Figure 1; Figure 2A, 2C, 2E, 2G; Figure 5A–5D).
Immunostaining of sections and imaging
Fix E11.5–E14.5 tracheas and lungs in 4% PFA overnight at 4 °C. Incubate samples in 10% and 30% sucrose for 24 h each at 4 °C.
Mount samples in OCT embedding compound. Make transverse sections of the trachea at 10 µm.
Fix sections in 4% PFA for 10 min at 4 °C. Incubate samples in permeabilization solution (0.25%Triton X-100/PBS) (see Recipes) for 15 min at RT and in blocking solution (5% FBS/PBS/3% BSA) for 1 h at RT.
Incubate samples in primary antibodies (αSMA-Cy3, 1:1,000; CDH1, 1:200, or SOX9, 1:400) overnight at 4 °C. Wash samples three times in 5% FBS/PBS/0.5% Triton X-100/3% BSA for 10 min each at RT.
Incubate samples in secondary antibodies [Alexa FluorTM 568 goat anti-rat IgG (H+L), 1:1,000 or Alexa FluorTM 488 goat anti-rabbit IgG (H+L), 1:1,000] for 2 h at RT. Wash samples three times in 5% FBS/PBS/0.5% Triton X-100/3% BSA for 10 min each at RT.
Perform DAPI counterstaining (1:2,000) for 5 min at RT. Wash samples three times in 5% FBS/PBS/0.5% Triton X-100/3% BSA for 10 min each at RT.
Mount samples with Mowiol mounting medium. Acquire images using a laser scanning confocal microscope (Figure 1; Figure 4A, 4C, 4E, and Figure 5E).
Quantification of SM cell alignment
Select the whole area of the imaged tracheal SM.
Draw a horizontal line and then a line along the extension of a SM cell using the angle tool in ImageJ. Select the menu Analyze and click the button Measure. Record the value of the angle for each SM cell.
For an individual trachea, measure the value of the angle for 50 SM cells. Quantify six tracheas. Count the number of SM cells in each defined angle value range (-90° to -30°, -30° to 0°, 0° to 30°, 30° to 90°). Calculate the ratio of SM cells (Figure 2).
Note: Distribution of SM cell alignment is reflected by the percentage of SM cells in the defined angle value range.
Quantification of SM cell polarity
Select the whole area of the imaged tracheal SM.
The Golgi apparatus for polarity analysis has been used in airway SM cells and vascular endothelial cells (Kwon et al., 2016; Yin et al., 2018). Draw a horizontal line to the middle of the SM cell nucleus and then a line to the GM130-labeled Golgi using the angle tool in ImageJ. Select the menu Analyze and click the button Measure. Record the value of angle for each SM cell.
For an individual trachea, measure the value of the angle for 50 SM cells. Quantify six tracheas. Count the number of SM cells in each defined angle value range (-135° to -45°, -45° to 45°, 45° to 135°, 135° to -135°) (Chen et al., 2015). Calculate the ratio of SM cells (Figure 3).
Note: Distribution of SM cell polarity is reflected by the percentage of SM cells in the defined angle value range.
Quantification of SM cell nuclear aspect ratio (NAR)
Select the whole area of the imaged tracheal SM.
Draw a line along the horizontal dimension of the SM cell nucleus using Straight Line in ImageJ and assign it as the length 2a. Select the menu Analyze and click the button Measure. Record the value of length (Supplementary Figure 3).
Draw a line perpendicularly across the middle of the length using Straight Line in ImageJ and assign it as the width 2b. Select the menu Analyze and click the button Measure. Record the value of length (Supplementary Figure 3).
Calculate the value of NAR using the equation:
NAR = a/b
For an individual trachea, calculate the value of NAR for 50 SM cells. Quantify six tracheas. Count the number of SM cells in each defined NAR range (0–1.0, 1.0–2.0, 2.0–3.0, 3.0–4.0). Calculate the ratio of SM cells.
Note: Distribution of SM cell shape is reflected by the percentage of SM cells in the defined NAR value range.
Data analysis
Results
SM cell alignment
SM is critical for tubulogenesis in the trachea, lungs, gut, and blood vessels (Knot and Nelson, 1998; Shyer et al., 2013; Kim et al., 2015; Yin et al., 2018 and 2019). To analyze SM organization during tracheal formation, we examined SM cell alignment using αSMA antibody staining. SM cells were aligned approximately perpendicular to the direction of tube elongation from E12.5 to P60 (Figure 2A–2H). SM exhibited no significant differences in cell alignment among these stages.
Figure 2. Tracheal smooth muscle (SM) cell alignment at several embryonic and postnatal stages. (A) Dorsal views of αSMA immunostaining (red) and DAPI staining (blue) of E12.5 tracheas. (B) Quantification of E12.5 tracheal SM cell orientation (n = 7). (C) Dorsal views of αSMA immunostaining (red) and DAPI staining (blue) of E13.5 tracheas. (D) Quantification of E13.5 tracheal SM cell orientation (n = 7). (E) Dorsal views of αSMA immunostaining (red) and DAPI staining (blue) of E14.5 tracheas. (F) Quantification of E14.5 tracheal SM cell orientation (n = 7). (G) Dorsal views of αSMA immunostaining (red) and DAPI staining (blue) of P60 tracheas. (H) Quantification of P60 tracheal SM cell orientation (n = 7). Scale bar = 20 μm (A, C, E, G).
SM cell polarity
Correct orientation of the cell body depends on the establishment of cell polarity (Li and Dudley, 2009). To examine the polarization of tracheal SM cells, we quantified the Golgi apparatus position relative to the nucleus by using the cis-Golgi matrix marker GM130 (Nobes and Hall, 1999; Kwon et al., 2016). GM130+ Golgi localized preferentially along the longitudinal edges of the nucleus in SM cells at E14.5 (Figure 3A and 3B).
Figure 3. Tracheal smooth muscle (SM) cell polarity at E14.5. (A) Immunostaining for αSMA (red) and GM130 (green) and DAPI staining (blue) in dorsal views of E14.5 tracheas. (B) Quantification of E14.5 Golgi apparatus (green) position relative to the nucleus (blue) (n = 8). Scale bar = 20 μm (A and B).
SM cell NAR
To examine changes in SM cell shape during tracheal formation, we examined NAR. SM cells exhibited approximately round shapes at E11.5–12.5 (Figure 4A–4D) and developed spindle shapes by E13.5 (Figure 4E and 4F), indicating that SM cell elongates significantly between E12.5 and E13.5.
Figure 4. Tracheal smooth muscle (SM) cell nuclear aspect ratio (NAR) at several embryonic stages. (A) Immunostaining for αSMA (red) and CDH1 (green), and DAPI staining (blue) of transverse sections of E11.5 tracheas. (B) Quantification of E11.5 tracheal SM cell NAR (n = 6). (C) Immunostaining for αSMA (red) and CDH1 (green), and DAPI staining (blue) of transverse sections of E12.5 tracheas. (D) Quantification of E12.5 tracheal SM cell NAR (n = 6). (E) Immunostaining for αSMA (red) and CDH1 (green), and DAPI staining (blue) of transverse sections of E13.5 tracheas. (F) Quantification of E13.5 tracheal SM cell NAR (n = 6). Scale bar = 20 μm (A, C, E).
Mesenchymal condensation
The condensation of mesenchymal cells into chondrogenic nodules drives chondrogenesis (Bi et al., 1999; Sala et al., 2011). To analyze the process of tracheal cartilage ring formation, we examined mesenchymal condensation in E12.5–E16.5 tracheas by using SOX9 antibody staining. An even distribution of SOX9+ mesenchymal cells was observed in E12.5 tracheas (Figure 5A). Starting at E13.5, a clear pattern of condensed SOX9+ mesenchymal cells resembling C-shaped cartilaginous rings was readily distinguished (Figure 5B), and these rings are becoming clearer at E14.5 and E16.5 (Figure 5C–5E), indicating that mesenchymal condensation begins between E12.5 and E13.5.
Figure 5. Tracheal mesenchymal condensation at several embryonic stages. (A) Immunostaining for SOX9 (green) in ventral views of E12.5 tracheas. (B) Immunostaining for SOX9 (green) in ventral views of E13.5 tracheas. (C) Immunostaining for SOX9 (green) in ventral views of E14.5 tracheas. (D) Immunostaining for SOX9 (green) in ventral views of E16.5 tracheas. (E) Immunostaining for SOX9 of transverse sections of E14.5 tracheas. Arrows point to tracheal rings. The arrowhead points to the ventral part of the trachea. Scale bar = 1 mm (A–D); 50 μm (E).
Discussion
In this protocol, we provided detailed descriptions about tissue preparation, immunostaining, clearing, and imaging as well as novel methods to measure tracheal SM cell alignment, polarity, and NAR. By using immunostaining in both whole-mount and sectioned tracheas at several embryonic stages in combination with confocal and stereomicroscopy imaging, we clearly observed the process of tracheal SM cell organization and changes in cell shape as well as mesenchymal condensation. Our methods enable researchers to visualize and analyze how tracheal mesenchyme forms and identify key timepoint(s) for cell behavior, organization, and shape changes (Hines et al., 2013; Lin et al., 2014; Park et al., 2010).
It is necessary to use the intact trachea for whole-mount immunostaining and quantification of SM cells and cartilage rings. Isolation of the tracheas from early embryos is not easy due to its fragility and tight connection with surrounding tissues. We used fine forceps to carefully remove tissues surrounding the trachea under stereomicroscopy.
SM lies beneath other mesenchymal cells in the dorsal part of the trachea. For fluorescence imaging of SM cells in the whole-mount immunostained trachea, it is critical to clear samples. We used BABB to transparentize the trachea to make deep structures visible without compromising immunofluorescence.
A limitation of this protocol is that fixation of the trachea will disable live-cell imaging. To track live cell behavior, we need to generate mouse lines expressing fluorescent reporters in mesenchymal cells such as SOX9CreERT2/+;ROSAmT/mG or Myh11CreERT2/+;ROSAmT/mG mice (same as SMMHCCreERT2/+;ROSAmT/mG mice) (Muzumdar et al., 2007; Wirth et al., 2008; Soeda et al., 2010), and perform time-lapse imaging.
SM cells surround epithelial and endothelial tubes in a number of organ systems, including the digestive tract, exocrine glands, lungs, kidneys, and vasculature (Hogan and Kolodziej, 2002; Iruela-Arispe and Beitel, 2013). Our protocol provides a useful technique to analyze SM cell organization, polarity, and nuclear shape in these organs.
Recipes
Phosphate-buffered saline (PBS)
8.0 g of NaCl
0.2 g of KCl
1.15 g of Na2HPO4·7H2O
0.2 g of KH2PO4
Adjust to pH 7.4
4% formaldehyde solution
40 g of PFA powder
Add 1× PBS up to 1 L
Permeabilization solution
1× PBS containing 0.25% Triton X-100
10% sucrose
5 g of sucrose
Add distilled deionized water up to 50 mL per tube
30% sucrose
15 g of sucrose
Add distilled deionized water up to 50 mL per tube
Mowiol mounting medium
200 mL of 1× PBS
50 g of Mowiol
25 mL of water-free glycerol
Add 50 g of Mowiol to 20 mL of 1× PBS, stir for 16 h at RT, add 25 mL of water-free glycerol, stir for 16 h at RT, centrifuge at 1,3525× g for 15 min at 4 , and store at -20 before use.
0.3%Triton X-100/PBS solution
0.3 mL of Triton X-100
Add 1× PBS up to 1 L
5% FBS/PBS/0.5% Triton X-100/3% BSA solution
50 mL of FBS
0.5 mL of Triton X-100
30 g of BSA
949.5 mL of 1× PBS
70% ethanol solution
737 mL of 95% ethanol
Add distilled deionized water up to 1 L
DMSO:methanol (1:4) solution
50 mL of DMSO
200 mL of methanol
H2O2/DMSO/methanol (1:1:4) solution
100 mL of 30% H2O2
100 mL of DMSO
400 mL of methanol
80% methanol solution
Diluting 400 mL of methanol
Add distilled deionized water up to 500 mL
50% methanol solution
Diluting 250 mL of methanol
Add distilled deionized water up to 500 mL
Benzyl alcohol:benzyl benzoate (BABB) (1:2) solution
50 mL of benzyl alcohol
100 mL of benzyl benzoate
5% FBS/PBS/3% BSA blocking solution
50 mL of FBS
30 g of BSA
950 mL of 1× PBS
Acknowledgments
The author would like to acknowledge State Key Laboratory of Respiratory Disease, Guangzhou, Max Planck Institute for Heart and Lung Research, Bad Nauheim for the assistance and previous work (Yin et al., 2018 and 2019). This work was supported by the National Natural Science Foundation of China (81970019) and R&D Program of Guangzhou Laboratory (SRPG22-016 and SRPG22-021). Open Project of the State Key Laboratory of Respiratory Disease (SKLRD-OP-202110). Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (No. SKLGE-2305).
Competing interests
The author has nothing to disclose.
Ethical considerations
C57BL/6J mice were used in the experiments. All mouse husbandry was performed under standard conditions in accordance with the Institutional Animal Care and Use Committees of Guangzhou Medical University, and institutional (Max Planck Society) and local ethics committee (Regierungspräsidium Darmstadt, Hessen, Germany). All animal experiments were performed in compliance with ethical guidelines and approved protocols.
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Supplementary information
The following supporting information can be downloaded here:
Supplementary Figure 1. Isolated embryos from euthanized animals
Supplementary Figure 2. Dissection of the trachea and lungs from the embryo
Supplementary Figure 3. Length and width measurements of the SM cell nucleus using the ImageJ software
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Tissue analysis > Tissue staining
Cell Biology > Cell imaging > Confocal microscopy
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Peer-reviewed
Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections
BK Bernhard A. Kramer *
JC Jacobo Sarabia del Castillo *
LP Lucas Pelkmans
GG Gabriele Gut
(*contributed equally to this work)
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4712 Views: 1577
Reviewed by: Chiara AmbrogioAnthony FlamierAkira Karasawa
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Original Research Article:
The authors used this protocol in Science Jul 2022
Abstract
Highly multiplexed protein measurements from multiple spatial scales using fluorescence microscopy recently emerged as a powerful way to investigate tumor microenvironments in biomedicine and the multivariate nature of complex systems’ interactions. A range of methods for this exist, which either rely on directly labeling the primary antibody with oligonucleotides/rare metals or employing methods to remove fluorescence for cyclic acquisition. Here, we describe a protocol that uses off-the-shelf primary and secondary antibodies without further need for modification and only commonly available chemical reagents. The method harnesses the observation that antibodies can crosslink to bound epitopes during light exposure, thus preventing elution. By utilizing a simple oxygen radical scavenging buffer during imaging and by blocking free sulfhydryl groups before antibody incubation, the presented method can employ comparably mild conditions to remove bound antibodies from epitopes, which preserves sample integrity. Thus, with the stated minor modifications, it allows for a standard immunofluorescence imaging protocol in cyclic fashion, currently permitting staining of up to ~80 unique epitopes.
Graphical overview
Keywords: Fluorescence microscopy Multiplexed imaging Antibody High-throughput imaging Histology Tissue imaging FFPE sections
Background
A multitude of research questions across various fields of biology can only be addressed by the ability to simultaneously visualize the abundance and subcellular distribution of proteins in single cells, within their tissue context. Multiplexed imaging enables the identification of multiple cell types in a single sample, which facilitates the characterization of tissue microenvironments and cellular ecosystems in biomedicine (Cole et al., 2022; Wahle et al., 2022). It further enables an in-depth description of the multivariate nature of cellular states, which are predictive of higher-level biological phenomena in basic research. Given the substantial research potential of multiplexed imaging methods, multiple methods have recently been developed, as outlined in Hickey et al. (2022). These methods rely either on directly conjugating the primary antibodies to oligonucleotides or metal-chelating moieties that can bind rare metals, which requires additional processing of the antibodies, or employ chemical conditions to elute the bound antibodies, which might affect sample integrity. For researchers who wish to use multiplexed imaging but are not specialists in these methods, or who require the detection of epitopes for which no modified antibodies exist, a protocol that uses off-the-shelf antibodies would be highly beneficial.
During fluorescence imaging, the generated oxygen radicals can crosslink antibodies to their bound epitopes, thereby hindering antibody removal. By employing a radical scavenging buffer during imaging, this crosslinking can be prevented, allowing antibody removal using relatively mild conditions. This allows for cyclic imaging using a conventional immunofluorescence staining protocol and off-the-shelf primary and secondary antibodies.
The ease of application of the described protocol, lack of need for specialized equipment, and the reliance on off-the-shelf antibodies and commonly available reagents allows researchers with various backgrounds to easily obtain multiplexed images of their sample of interest. While here we describe the protocol for a conventional 96-well imaging plate, using adherent cell lines and formalin-fixed paraffin-embedded (FFPE) sections, the procedure can readily be extended to a variety of samples and formats. For instance, it can readily be employed on coverslips, 384-well plates, and various other formats with minor adaptations. Combined with automated liquid-handling equipment, the approach can be scaled up.
Materials and reagents
For both tissue culture cells and FFPE tissue sections
L-Glycine-hydrochloride (Sigma-Aldrich, catalog number: G2879); storage: room temperature (RT)
Guanidium-hydrochloride (Sigma-Aldrich, catalog number: G3272); storage: RT
Urea (Sigma-Aldrich, catalog number: U5128); storage: RT
Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich, catalog number: C4706); storage: 4 °C
N-acetyl-cysteine (Sigma-Aldrich, catalog number: A7250); storage: 4 °C
Bovine serum albumin (Sigma-Aldrich, catalog number: A9418); storage: 4 °C
Maleimide (Sigma-Aldrich, catalog number: 129585); storage: 4 °C
Phosphate-buffered saline (PBS) (Thermo Fisher, catalog number: 10010023); storage: RT
1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher, catalog number: 15630080); storage: 4 °C
12 M hydrochloric acid (Sigma-Aldrich, catalog number: 320331); storage: RT
10 M sodium hydroxide (Sigma-Aldrich, catalog number: 72068); storage: RT
Primary antibodies (various manufacturers); storage: according to manufacturers’ recommendations
Secondary antibodies (Thermo Fisher, choose according to color requirements and species of primary antibodies. We recommend Donkey-anti-Primary antibodies); storage: according to manufacturers’ recommendations
4’,6-Diamidino-2-phenylindole (Thermo Fisher, catalog number: 62247); storage: -20 °C
Specific for tissue culture cells
96-well imaging plate (Greiner, catalog number: 655096); storage: RT
16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710); storage: RT
Triton-X (Sigma-Aldrich, catalog number: X100); storage: RT
Succinimidyl Ester Alexa Fluor 647 (Thermo Fisher, catalog number: A20006)
Specific for FFPE tissue sections
Glass bottom Nexterion (Schott Glass, catalog number: 1535661)
Grace Bio-Labs ProPlate MPTM microtiter plate superstructure (Sigma-Aldrich, catalog number: GBL204969-1EA)
National DiagnosticsTM Histo-ClearTM tissue clearing agent (Chemie Brunschwig AG, catalog number: D1620333)
100% EtOH (chemical under K20 chemical hood, was diluted in ddH2O)
Feather A35 1 × 50 Blades (Biosystems Switzerland AG, catalog number: 81-0358-00); storage: RT
30% w/v acrylamide/0.8% w/v bis-acrylamide (Sigma-Aldrich, catalog number: A3699-100ML); storage: 4 °C
TEMED (Thermo Fisher, catalog number: 17919); storage: 4 °C
Poly-L-lysine solution (Sigma-Aldrich, catalog number: P4832-50ML); storage: 4 °C
Glass basin length 21 cm × 15 cm × 6 cm
Superfrost plus microscope slides, 1 × 72 slides, 25 mm × 75 mm × 1 mm (Menzer-Glaseritem, catalog number: J1800AMNZ); storage: RT
PAP pen (Electron Microscopy Science, catalog number: 71310); storage: RT
Water-resistant, ethanol-soluble pen, 1 × 72 (Staedtler Lumocolor® permanent pen 318)
Histoclear clearing agent (National Diagnostic, catalog number: HS-200)
Solutions
40 mL elution buffer (EB) (see Recipes)
6 mL 4i blocking buffer (sBB) (see Recipes)
10 mL conventional blocking buffer (cBB) (see Recipes)
20 mL 4i imaging buffer (IB) (see Recipes)
20 mL fixation solution (FS) (see Recipes)
10 mL permeabilization solution (PS) (see Recipes)
5 mL primary antibody staining solution (1° AS) (see Recipes)
5 mL secondary antibody staining solution (2° AS) (see Recipes)
10 mL DNA stain solution (DSS) (see Recipes)
10 mL whole protein staining solution (WPSS) (see Recipes)
Embedding solution (ES) (see Recipes)
Equipment
Microm HM355S (Thermo Scientific, catalog number: 90 520 0STS)
Steamer (Betty Bossi, catalog number: 10003614)
(Optional: to ease liquid handling steps) Washer dispenser (Agilent, catalog number: EL406)
Procedure
The following procedure is based on using a standard 96-well plate containing 100 μL per well. Every volume and step are indicated on a per well basis. However, the protocol works for any container and adherent cell type if the volumes are adjusted accordingly. Numbers in bold indicate the final dilution.
For every step described in section C, the sample needs to be protected from light at all times. This is essential for the protocol to work. The procedure works best when using automated liquid handling, which necessitates building light-protecting containers around the machines. If manual handling is performed, keep the light as dim as possible, and preferentially at wavelengths above ~600 nm.
When performing 4i on tissue culture cells, perform A and then repeat C and D to achieve the desired level of multiplexing. In the last 4i cycle, perform C, E, and D. 4i works well with poorly adherent cell lines, which require a coated surface (e.g., poly-lysine). However, liquid handling should be performed very cautiously.
When performing 4i on tissue sections, perform B and then repeat C and D to achieve the desired level of multiplexing.
Sample preparation, tissue culture cells
Prepare the fixation solution (FS, see Recipes) and the permeabilization solution (PS, see Recipes).
Perform experiment as desired.
Add 100 μL of FS. 1:2
Incubate at RT for 20 min.
Wash three times with 100 μL of PBS.
Aspirate to a volume of 50 μL.
Add 50 μL of PS. 1:2
Incubate at RT for 10 min.
Wash three times with 100 μL of PBS.
Aspirate to a volume of 100 μL.
Store the plate at 4 °C until proceeding to Section C.
Sample preparation, FFPE (tissue sections)
Place the microtiter plate superstructure over the glass bottom. Mark the position of the microtiter plate well on the glass bottom by running the water-resistant, ethanol-soluble pen along the inside of each well while the pen is also touching the glass bottom. Each well will contain one tissue section.
Remove the plate superstructure and turn the glass bottom over.
Using a PAP pen, encircle all well markings with one single line. This line will serve as a tension barrier in the next step.
Add 7.5 mL of Poly-L-Lysine inside the PAP pen line and ensure that the whole surface within the surface tension barrier is covered by the solution.
Incubate in a chemical hood at RT for 1 h.
Remove the Poly-L-Lysine solution from the glass bottom by aspiration or by gently tipping the glass bottom on its side.
Use a water microtome cutter to generate sections 2–6 μm thick of the precooled paraffin block and let them float in the water bath of the microtome. A detailed protocol for cutting the paraffin sections can be found in Wang and Hasnain (2017).
Insert the glass bottom at a 45° angle into the water bath, with the coated side facing the samples.
Using the well markings, place the edge of the paraffin on the slanted plate and pull the plate out at a 45° angle.
Dry the glass bottom slide in a plate at 37 °C for 2 min.
Repeat steps 7–10 until you have transferred all sections to your slides, or all positions on the slide are occupied by sections. Do not resubmerge areas that have sections recently placed on them!
Store the glass bottom slide at 37 °C overnight.
To perform deparaffinization, place the glass bottom slide in a glass container, on top of two microscopy glass slides with the sections facing upwards.
For the following steps (15–22), use volumes that ensure that the glass bottom is just about covered. Do not pour the solutions directly onto the glass bottoms.
Histoclear three times for 4 min.
Histoclear one time for 3 min.
100% EtOH two times for 2 min.
96% EtOH two times for 2 min.
80% EtOH one time for 2 min.
70% EtOH one time for 2 min.
50% EtOH one time for 2 min.
PBS one time for 2 min.
Place 40 mL of 10× Antigen Retrieval solution together with 360 mL of ddH2O in a steamer.
In the steamer, place a sieve close to the steam source.
Place the glass bottom with deparaffinized sections on or into the sieve, with the sections facing upwards.
Turn the steamer on and set it to run for 40 min. Every 10 min, rotate the sieve by 90°.
Switch off the steamer, carefully tilt the plate at an angle, and let it air dry for 1 h.
Place the glass bottom with the sections facing up on a kitchen towel, positioned on a firm surface.
To combine the glass bottom with the plastic superstructure, first expose the adhesives of the plastic superstructure and then align the wells to the sections as best as possible.
Lightly press the glass to the superstructure through the edges, to ensure that all points of interest have no bubbles in them.
Add 50 μL of ddH2O per well.
Add 50 μL of ES per well.
Incubate at RT for 90 min.
Add 33.33 μL of FS.
Wash five times with 100 μL of H2O.
Store the plate at 4 °C until proceeding to Section C.
Indirect iterative (cyclic) immunofluorescence imaging on tissue culture cells and tissue sections
Reminder: Keep the sample protected from light at all times.
Prepare the elution buffer (EB, see Recipes), the 4i blocking buffer (sBB, see Recipes), and the conventional blocking buffer (cBB, see Recipes).
Wash five times with 100 μL of ddH2O.
Aspirate to a volume of 30 μL.
Add 100 μL of EB. 1:1.3
Incubate at RT for 10 min.
Wash three times with 100 μL of ddH2O.
Aspirate to a volume of 30 μL.
Add 100 μL of EB. 1:1.3
Incubate at RT for 10 min.
Wash three times with 100 μL of ddH2O.
Aspirate to a volume of 30 μL.
Add 100 μL of EB. 1:1.3
Incubate at RT for 10 min.
Wash five times with 100 μL of PBS.
Aspirate to a volume of 30 μL.
Add 30 μL of sBB. 1:2
Incubate at RT for 1 h.
While incubating, prepare the primary antibody staining solution (1° AS, see Recipes).
Wash five times with 100 μL of PBS.
Aspirate to a volume of 30 μL.
Add 30 μL of 1° AS. 1:2
Incubate at RT for 2 h.
While incubating, prepare the secondary antibody staining solution (2° AS, see Recipes).
Wash five times with 100 μL of PBS.
Aspirate to a volume of 30 μL.
Add 30 μL of 2° AS. 1:2
Incubate at RT for 1 h.
While incubating, prepare the DNA stain solution (DSS, see Recipes) and the imaging buffer (IB, see Recipes).
Wash five times with 100 μL of PBS.
Aspirate to a volume of 30 μL.
Add 100 μL of DSS. 1:1.3
Incubate at RT for 10 min.
Wash five times with 100 μL of ddH2O.
Aspirate to a volume of 30 μL.
Add 100 μL of IB. 1:1.3
Let the samples rest at RT for ~30–45 min.
Image sample at appropriate conditions. See section D for more details.
Wash five times with 100 μL of PBS.
Repeat (in a cyclic fashion) from step 1 until every desired epitope is imaged (see Figure 1 for the workflow and Figure 2 for an example panel of epitopes) and move to Section E. Recommendation: do not exceed 25 iterations.
Figure 1. Flowchart depicting the standard workflow of 4i
Figure 2. Multiplexed images obtained by performing 4i. 184A1 cells were deprived of serum and growth factor for 16 h, and then stimulated with 100 ng/mL EGF for 5 min before fixation. Then, 4i was performed as described for the tissue culture cells. Representative composites of a 30-plex experiment are displayed with the stained epitopes indicated. The same field of view is shown in all composites. Scale bars: 20 μm.
Imaging
Align the imaging roster to the imaging roster of the previous 4i cycle.
Use the lowest possible amount of laser and light exposures (sufficient signal-to-noise ratio).
Whole protein staining
Prepare the whole protein staining solution (WPSS, see Recipes)
Wash five times with 100 μL of PBS.
Aspirate to a volume of 50 μL.
Add 100 μL of WPSS. 1:1.5
Wash five times with 100 μL of PBS.
Aspirate a volume of 100 μL.
Image the sample at appropriate conditions.
Notes
Reproducibility: when adhered to carefully (preferentially using automated liquid handling), the method achieves almost perfect technical reproducibility (Figure 3). For reference, see Gut et al. (2018) (Figure 1C–1E) and Kramer et al. (2022) (Supplementary Figure 1D and 1E).
Figure 3. 4i is reproducible across acquisition cycles. The top panel shows representative images of repeated staining against phosphorylated ERK in different cycles (01, 07, and 13) in 184A1 cells exposed to 100 ng/mL of EGF. The bottom panel shows the bivariate plot of quantified pERK intensities for each combination of indicated 4i cycles. Scale bars: 20 μm.
Cautionary points
The method described relies on employing mild conditions to elute bound antibodies from epitopes for cyclic acquisition. This is enabled by using a radical scavenging imaging buffer during fluorescence imaging. As the sample cannot be kept in this buffer at all times during processing since primary and secondary antibodies at times are bound, it is crucial to protect the sample from light exposure at these times. This is crucially the case after incubation with the primary and secondary antibodies, but before adding the radical scavenging imaging buffer.
Antibody selection
Rarely, antibodies may be incompatible with the 4i protocol. As a result, the antibody staining appears altered or no signal is detected at all. Thus, it is advisable to perform a validation experiment prior to including new antibodies in a 4i panel. The aim of the validation experiment is to compare the staining of an antibody in conventional immunofluorescence and 4i. In a first step, perform (indirect) immunofluorescence using the unvalidated antibody and image the sample in imaging buffer. Next, elute the antibody and perform one round of 4i. Next, compare the antibody staining achieved using conventional immunofluorescence and 4i.
Antibody panel design and epitope prioritization
When designing a 4i antibody panel, it is important to identify the minimal concentration with which a reliable staining can be achieved. We generally start with the manufacturer’s recommended concentration and perform serial dilutions (1:2 steps) to identify the minimal concentration using conventional IF. Then, we assess whether that concentration yields discernable and reliable staining using the 4i protocol. If needed, increase the concentration only in small steps, as keeping the antibody concentration low will help in preventing photo-induced antibody crosslinking during imaging. Another important factor to consider when designing a 4i antibody panel is the potential change in detectability of epitopes as a consequence of multiple cycles of 4i. This can be assessed using repeated mock 4i cycles, by performing all the liquid handling steps but omitting the antibodies, and imaging interchanged with true 4i cycles (see workflow in Figure 4).
Figure 4. Standard workflow of an antibody panel design experiment
The following example describes a detectability assay for a 19 cycle 4i experiment. First, perform a 4i cycle including imaging. These images serve as staining references. Then, perform a round of elution, stain only with the secondary antibody, and image the sample. These images serve as reference whether the primary antibody can be eluted from their epitopes. Then, perform four cycles of elution followed by a complete cycle of 4i. This is treated as an antibody used during the seventh cycle. Repeat this, including the elution control, in a fashion yielding images that represent the staining obtained during cycles 1, 7, 13, and 19 (for an example, see Figure 5). Compare the images and identify whether the staining becomes less reliable in later cycles of 4i. In your antibody panel, prioritize antibodies for which altered staining patterns are observed in later cycles. In general, we find that phosphorylated specific antibodies are most prone to signal decay (with exceptions) after many rounds of 4i. Thus, we would advise to prioritize them, using them in earlier cycles.
Figure 5. Images obtained during an antibody panel design experiment. Lamin B is an example of an antibody that needs to be prioritized, due to altered signal in later cycles. The top panel depicts staining at the respective cycles with mock cycles (repeated elution but no antibody incubations) with both primary and secondary antibodies. Bottom panel depicts the residual signal after elution and staining with only secondary antibody.
Residual antibody staining from previous cycles
You may discover that signal from a previous 4i cycle is still detected in your current staining. This is due to a (partially) failed elution, which in turn is normally due to photo-induced antibody crosslinking during the imaging step of the previous cycle; thus, the residual signal from the previous cycle cannot be removed anymore. Using minimal antibody concentration and photon load during imaging prevents elution failures.
Recipes
Recipes are calculated based on requirements for a standard 96-well plate and a standardized antibody dilution. Adjust accordingly for different volumes. The number in bold indicates the final concentration.
40 mL elution buffer (EB)
L-Glycine (molar mass: 75.067 g/mol), 1.5 g, 0.5 M
Guanidinium hydrochloride (molar mass: 95.53 g/mol), 11.46 g, 3 M
Urea (molar mass: 60.06 g/mol), 7.2 g, 3 M
Tris(2-carboxyethyl)phosphine hydrochloride (molar mass: 286.65 g/mol), 0.69 g, 70 mM
ddH2O, ~23 mL
12 M hydrochloric acid, ~800 μL
Total: 40 mL
Adjust pH to 2.5 using hydrochloric acid and sodium hydroxide
6 mL 4i blocking buffer (sBB)
Bovine serum albumin, 0.24 g, 4% (w/v)
Maleimide (molar mass: 97.07), 0.17 g, 300 mM
PBS, ~5.8 mL
Total: 6 mL
10 mL conventional blocking buffer (cBB)
Bovine serum albumin, 0.4 g, 4% (w/v)
PBS, ~9.9 mL
Total: 10 mL
20 mL 4i imaging buffer (IB)
N-acetyl-cysteine (molar mass: 163.19 g/mL), 2.28 g, 700 mM
1M HEPES, 2 mL, 100 mM
ddH2O, 16 mL
10 M sodium hydroxide, 2 mL
Total: 20 mL
Adjust pH to 7.4 using hydrochloric acid and sodium hydroxide
20 mL fixation solution (FS)
16% paraformaldehyde, 10 mL, 8% (v/v)
ddH2O, 10 mL
Total: 20 mL
10 mL permeabilization solution (PS)
Triton-X, 50 μL, 0.5% (v/v)
PBS, 9.5 mL
Total: 10 mL
5 mL primary antibody staining solution (1° AS)
Primary antibody solution, 25 μL, 0.5% (v/v)
cBB, 4.975 mL
Total: 5 mL
5 mL secondary antibody staining solution (2° AS)
Secondary antibody solution, 25 μL, 0.5% (v/v)
cBB, 4.975 mL
Total: 5 mL
10 mL DNA stain solution (DSS)
4’,6-Diamidino-2-phenylindole, 750 μg, 75 μg/mL
PBS, 10 mL
Total: 10 mL
10 mL whole protein staining solution (WPSS)
Succinimidyl Ester Alexa Fluor 647, 1.66 μg, 0.166 μg/mL
PBS, 10 mL
Total: 10 mL
Embedding solution (ES)
ddH2O, 6.67 mL
Acrylamide/bis-acrylamide (30%/0.8% w/v), 0.83 mL
1.5 M Tris-HCl (pH 8.8), 2.5 mL
*10% (w/v) ammonium persulfate (APS), 100 μL
*TEMED, 10 μL
Total: 10 mL
*To be added right before addition on the well
Acknowledgments
G.G. received funding from the Swiss National Science Foundation and InnoSuisse as part of the BRIDGE program, as well as from the University of Zurich through the BioEntrepreneur Fellowship. We also would like to thank the authors of Gut et al. (2018), for which the presented protocols were originally designed for. L.P. received funding from the Swiss National Science Foundation (grant 310030_192622), the European Research Council (advanced grant CROSSINGSCALES-885579), the Chan Zuckerberg Initiative (grant CZF2019-002440), and the University of Zurich. This protocol is derived from the original research paper (Kramer et al., 2022).
Competing interests
G.G. and L.P. are authors on multiple patent applications concerning the 4i technology.
References
Cole, J. D., Sarabia Del Castillo, J., Gut, G., Gonzalez-Bohorquez, D., Pelkmans, L. and Jessberger, S. (2022). Characterization of the neurogenic niche in the aging dentate gyrus using iterative immunofluorescence imaging. Elife 11: e68000.
Wahle P., Harmel C., He Z., Gut G., Santos A., Yu Q., Noser P., Fleck J. S., Gjeta B., Pavlinić, D., et al. (2022). Multimodal spatiotemporal phenotyping of human organoid development.bioRxiv. doi: https://doi.org/10.1101/2022.03.16.484396.
Wang, R. and Hasnain, S. Z. (2017). Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology. Bio-protocol 7(14): e2394.
Hickey, J. W., Neumann, E. K., Radtke, A. J., Camarillo, J. M., Beuschel, R. T., Albanese, A., McDonough, E., Hatler, J., Wiblin, A. E., Fisher, J., et al. (2022). Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nat Methods 19(3): 284-295.
Gut, G., Herrmann, M. D. and Pelkmans, L. (2018). Multiplexed protein maps link subcellular organization to cellular states. Science 361(6401): eaar7042.
Kramer, B. A., Sarabia Del Castillo, J. and Pelkmans, L. (2022). Multimodal perception links cellular state to decision-making in single cells. Science 377(6606): 642-648.
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© 2023 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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A Simple and Reproducible Stereomicroscopic Method to Assess Fungal Biofilms: Application to Antifungal Susceptibility Testing
ZS Zinnat Shahina
TD Tanya E. S. Dahms
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4713 Views: 462
Reviewed by: Wenrong HeYe Xu Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS ONE Nov 2022
Abstract
Candida albicans, a well-known opportunistic pathogen, is a major cause of human fungal infections. Biofilm formation is considered an important pathogenesis factor. Biofilms are less sensitive to antibiotics and immune responses, allowing them to colonize and persist in host niches. Biofilm screening is important in the identification of anti-biofilm drugs. However, developing nations, with limited financial resources, often do not have access to advanced scientific equipment. Here, we describe an in vitro, protocol using common materials and simple equipment to evaluate static microbial biofilms.
Keywords: Antimicrobial resistance Biofilm Candida albicans Stereomicroscope Virulence
Background
Candida albicans, a commensal fungus, is a leading cause of human fungal infections. It becomes a very resilient pathogen under low host immunity, for example in neonates or patients with AIDS or transplants (Ostrosky-Zeichner et al., 2003). Biofilm formed by this pathogen plays important roles in both its virulence and antifungal resistance (Gulati and Nobile, 2016). Various microscopic methods are routinely used to evaluate Candida, including microscopic (atomic force, epifluorescence, laser scanning confocal, and scanning electron) assays. The simple technique described herein is more cost effective, in particular for biofilm screening projects.
The most common method to evaluate biofilm formation is the microplate assay (Azeredo et al., 2017). Here, we present a low-cost visual biofilm detection protocol that uses tissue culture plates and detection in brightfield that offers comparable results to standard assays (Shahina et al., 2022a and 2022b). This assay (Figure 1) is suitable for researchers in developing countries where there is a lack of access to high throughput microscopy and microplate readers.
Figure 1. Schematic step-by-step protocol for visualizing C. albicans biofilm formation and inhibition. 1. C. albicans stock culture in 50% glycerol. 2. Colony formation on YPD media. 3. Overnight culture on YPD broth. 4. Sample in prewarmed 10% FBS with YPD diluted according to desired cell density. 5. Candida in 24-well plate (plate 1) in preparation for incubation at 37 °C for 24 h. 6. Sample plate after 24 h incubation. 7. Biofilm growth at the bottom of the 24-well plate following removal of the medium and planktonic cells. 8. Preparation of drugs/essential oils in a new plate (plate 2) and its transfer into the pre-formed biofilm (plate 1). 9. Biofilm with antifungal drugs (plate 1) is ready for incubation at 37 °C for 24 h. 10. After 24 h incubation (plate 1), the media is aspirated, and the plate is ready for stereomicroscopic imaging.
Materials and reagents
15 mL screw cap conical bottom tube (Sarstedt, catalog number: 50-809-221)
24-well tissue culture plates (Sarstedt, catalog number: 83.3922)
50 mL screw cap conical bottom tube (Sarstedt, catalog number: 50-809-218)
96-well, flat base, PS, transparent plate (Sarstedt, catalog number: 82.1581001)
Syringe filter, Filtropur S, 0.2 μm (Sarstedt, catalog number: 83.1826.001)
Test tube racks (Fisher Scientific, catalog number: 14-809-62) or any standard test tube racks
Agar (VWR, catalog number: 97064-336)
Antifungal agent; amphotericin B (Sigma-Aldrich, catalog number: 1397-89-3)
BactoTM peptone (Becton Dickinson, catalog number: 211677)
D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270)
Essential oils:
RM (Aroma force, catalog number: 6658117001)
1,8-cineole, 99% (Acros organics, catalog number: 470-82-6)
(+)–α-pinene ≥ 99% (Sigma-Aldrich, catalog number: 268070)
Fetal bovine serum (FBS) (Gibco, catalog number: 12483-020)
Parafilm (Amcor, catalog number: PM996)
Yeast extract (Sigma-Aldrich, catalog number: 70161)
TWEEN® 80, polysorbate 80 (polyoxyethylene sorbitan monooleate) (Sigma-Aldrich, catalog number: 9005-65-6)
Yeast extract peptone (YPD) plate containing freshly revive C. albicans colonies
YPD agar medium (per liter) (see Recipes)
YPD broth (per liter) (see Recipes)
YPD broth containing 10% FBS (see Recipes)
Equipment
-20 °C freezer (General Electrics)
-80 °C freezer (Revco, catalog number: ULT2586-4-A)
Autoclave (Steris Scientific, AMSCO® C series small steam sterilizer) or any standard autoclave
Balance (Mettler Toledo NewClassic MF Precision Balance, catalog number: ML303E/03)
Benchtop shaking incubator (CorningTM LSETM, catalog number: CLS6791)
Inoculating loop (FisherbrandTM, catalog number: 131045)
Laminar flow workstation (MicroZone Corporation, model: V4-MN-99-030) or any standard equipment with similar features
Microbiological benchtop laboratory incubator (Thelco, Model 4)
Micropipette 100–1,000 μL (Eppendorf, catalog number: 3123000063)
Micropipette 20–200 μL (Eppendorf, catalog number: 3123000055)
Micropipette 100–1,000 μL tips (VWR International, catalog number: 83007-376)
Micropipette 20–200 μL tips (Sarstedt, catalog number: 70.3030)
Micropipette 500–5,000 μL tips (Sarstedt, catalog number: 70.1183.002)
Petri dish, 92 mm × 16 mm (Sarstedt, catalog number: 82.1473.001)
Pipette, 500–5,000 μL (Eppendorf, catalog number: 3123000071)
Plate reader for OD determination (BioTek Instruments, Epoch- Microplate Spectrophotometer)
Note: The plate reader was used for verification of this method; a standard spectrophotometer (supplier model T, A, C. Detector: Silicon Photodiode or Thermo ScientificTM GENESYSTM Visible and UV-Visible Spectrophotometers) can be used to measure OD.
PYREX glass erlenmeyer flask, 125 mL (Corning, catalog number: 4980-125)
Stereomicroscope (Nikon’s SMZ 1500) equipped with a 2× and 4× objective and a digital camera or eyepieces for use of a mobile phone camera
Procedure
Candida growth
Use a sterile inoculating loop to remove culture from the glycerol stock culture (-80 °C) and inoculate a YPD plate (Table 1, Day 1).
Table 1. Protocol steps
Total time Workflow Condition
Day 1 Streak C. albicans on YPD plate Incubate for 24 h at 30 °C in the standby incubator
Day 2 Inoculate a small, round colony into 10 mL of YPD broth Incubate for 14–16 h at 30 °C until mid-logarithmic phase in a shaking incubator at 200 rpm
Day 3 Prepare 24-well plate with culture for biofilm growth Incubate for 24 h at 37 °C in a shaker at 75 rpm
Day 4 After removing the non-adherent cells, add antifungal agents for biofilm inhibition experiments
Again, incubate for 24 h at 37 °C in a shaker at 75 rpm
Note: Low shaking speed reduces the chance of biofilm detachment from the bottom surface of the plate.
Day 5 Assess biofilm by imaging in brightfield
Incubate the plate at 30 °C for 24–36 h.
Inoculate a single colony from the cultured plate into a sterile 100 mL conical flask containing 10 mL of YPD broth (Table 1, Day 2).
Incubate the flask at 30 °C for 16 h with constant shaking at 200 rpm (Figure 1).
Biofilm formation
Measure the OD600 of the overnight Candida culture and dilute in YPD medium supplemented with 10% FBS to a starting concentration of 107 CFU/mL (Table 1, Day 3).
Note: Use a standard CFU method (Miller, 1972) or direct microscopic count using Petroff-Hausser counting chambers.
Distribute 500 μL of the Candida suspension to each well of a 24-well tissue culture plate (plate 1) and incubate at 37 °C for 24 h without shaking. For the blank, add 500 μL of YPD medium supplemented with 10% FBS.
Note: For static biofilms, the plate must remain stationary.
The next day, gently remove the media and non-adherent cells from each well (Figure 1).
Note: Tilting the 24-well plates and gently placing the pipette tip in the corner of the well helps to prevent disruption of the fragile biofilm.
Biofilm inhibition assay
Prepare working solutions of antimicrobials (either antifungal drugs or essential oils) at minimum inhibitory concentration (MIC) with proper solubilizing agents (0.2% Tween-80 is suitable for essential oils).
In a new 24-well plate (plate 2), add 500 μL of YPD medium supplemented with 10% FBS to every well except the first. To the first well, add 1,000 μL of antimicrobial working solution (from C, step 1), followed by a serial dilution (in YPD medium supplemented with 10% FBS) to lower concentrations (e.g., 1/16 MIC) along the rows of the plate (Table 1, Day 4).
Note: Washable test tubes (less expensive) can be used to serially dilute the antifungal drugs.
Transfer drugs/plant-based essential oils (EOs) to the plate containing the pre-formed biofilm (plate 2 from step B), including a growth control and blank.
If working with volatile antifungals (e.g., EOs), cover the plate with parafilm to avoid evaporation and incubate the plate at 37 °C for 24 h in the incubator without shaking (Figure 1).
Biofilm visualization
After 24 h, gently aspirate and discard the media from each well (Table 1, Day 5).
Image samples at 4× with a stereomicroscope and capture images either with a digital camera or through the eyepiece using a cellphone camera. Representative images of C. albicans RSY150 biofilms exposed to various essential oil components are shown in Figure 2.
Figure 2. Stereoscopic brightfield images show visual differences between C. albicans RSY150 biofilm mass following exposure to rosemary essential oil, its components 1,8-cineole and α-pinene, Amp B (1/16 MIC to MIC), and the two components at 1/2 and 1 fractional inhibitory concentration index (FICI), compared to control cells. Plates were imaged at 4× with the digital camera mounted on the stereomicroscope.
Note: Assessment of biofilm formation and inhibition require experiments in triplicate, including controls, as there can be high variability in biofilm growth. Quantitative assays would require a microplate assay (Crystal violate/MTT/XTT). Many studies suggest 48 h incubation as optimal for biofilm studies (Chandra et al., 2001; Cruz et al., 2018; Ripolles-Avila et al., 2018; Cendra et al., 2019; Galdiero et al., 2020), but 24 h incubation does not require replacement of media as a result of nutrient depletion (Khelissa et al., 2017). FIC estimates the interaction between two or more drugs intended to be used in combination, and the FICI is calculated as the sum of FICs for compounds 1 and 2.
Data analysis
Following this experimental protocol, images from three independent biological replicates (see Figure 2) were used to determine the presence or absence of biofilm, compared with a blank well consisting of equal volumes of experimental media lacking Candida (Shahina et al., 2022a and 2022b).
Recipes
YPD agar medium (per liter)
Glucose 20 g in 1 L flask
Agar 18 g
Yeast extract 10 g
Peptone 20 g
dH2O 1 L
Add all of the above to a 1 L flask, except glucose. Add 800 mL of dH2O and allow the powder to dissolve with gentle stirring.
Autoclave for 40 min at 15 psi at 121 °C.
In the meantime, add 20 g of glucose to 200 mL of dH2O, use a magnetic stirrer to mix well, and sterilize by filtration.
After autoclaving the flasks, allow them to cool to 45 °C, add the sterile-filtered dextrose, and mix well.
Dispense 15 mL of the solution from step d into Petri plates inside a sterile hood and let cool to room temperature.
Note: If either glucose or dextrose are added to the YPD broth powder prior to autoclaving, the sugar will be caramelized, observed as a darker media color. It is good practice to add dextrose separately after the autoclave step once it has been dissolved in water and sterile filtered.
YPD broth (per liter)
Glucose 20 g in 1 L flask
Yeast extract 10 g
Peptone 20 g
dH2O 1 L
Follow the same procedure as described in Recipe 1, but without pouring into the Petri plates.
YPD broth containing 10% FBS
Thaw and warm up 100% FBS, stored as appropriately sized aliquots in the freezer.
Add 50 mL of 100% FBS to 450 mL of YPD broth to obtain 10% FBS in YPD.
Note: Serum is very expensive; therefore, always aliquot and freeze the serum (-80 °C) and add it to the medium just prior to its use. Store unused portions of thawed aliquots in the refrigerator (-20 °C), where they can be stored for several weeks.
Acknowledgments
This work was supported by the National Science and Engineering Research Council (NSERC) grant to TESD (228206-07). Z.S. was partially supported by the Faculty of Graduate Studies and Research at the University of Regina. The University of Regina main campus is situated on the traditional territories of the nêhiyawak, Anihšināpēk, Nakoda, Dakota, and Lakota peoples, and the homeland of the Métis/Michif Nation. This protocol is based on previous work (Shahina et al., 2022a and 2022b).
Competing interests
The authors declare that they have no competing interests.
References
Azeredo, J., Azevedo, N. F., Briandet, R., Cerca, N., Coenye, T., Costa, A. R., Desvaux, M., Di Bonaventura, G., Hébraud, M., Jaglic, Z., et al. (2017). Critical review on biofilm methods. Crit Rev Microbiol 43(3): 313-351.
Cendra, M. D. M., Blanco-Cabra, N., Pedraz, L. and Torrents, E. (2019). Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms. Sci Rep 9(1): 16284.
Chandra, J., Kuhn, D. M., Mukherjee, P. K., Hoyer, L. L., McCormick, T. and Ghannoum, M. A. (2001). Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183(18): 5385-5394.
Cruz, C. D., Shah, S. and Tammela, P. (2018). Defining conditions for biofilm inhibition and eradication assays for Gram-positive clinical reference strains. BMC Microbiol 18(1): 173.
Galdiero, E., de Alteriis, E., De Natale, A., D’Alterio, A., Siciliano, A., Guida, M., Lombardi, L., Falanga, A. and Galdiero, S. (2020). Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625. Sci Rep 10(1): 5780.
Gulati, M. and Nobile, C. J. (2016). Candida albicans biofilms: development, regulation, and molecular mechanisms. Microbes Infect 18(5): 310-321.
Khelissa, S. O., Jama, C., Abdallah, M., Boukherroub, R., Faille, C. and Chihib, N. E. (2017). Effect of incubation duration, growth temperature, and abiotic surface type on cell surface properties, adhesion and pathogenicity of biofilm-detached Staphylococcus aureus cells. AMB Express 7(1): 191.
Miller, J. H. (1972). Experiments in molecular genetics. Cold Spring Harbor Laboratory. N.Y.
Ostrosky-Zeichner, L., Rex, J. H., Pappas, P. G., Hamill, R. J., Larsen, R. A., Horowitz, H. W., Powderly, W. G., Hyslop, N., Kauffman, C. A., Cleary, J., et al. (2003). Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob Agents Chemother 47(10): 3149-3154.
Ripolles-Avila, C., Hascoët, A. S., Guerrero-Navarro, A. E. and Rodríguez-Jerez, J. J. (2018). Establishment of incubation conditions to optimize the in vitro formation of mature Listeria monocytogenes biofilms on food-contact surfaces. Food Control 92: 240-248.
Shahina, Z., Al Homsi, R., Price, J. D. W., Whiteway, M., Sultana, T. and Dahms, T. E. S. (2022a). Rosemary essential oil and its components 1,8-cineole and alpha-pinene induce ROS-dependent lethality and ROS-independent virulence inhibition in Candida albicans. PloS One 17(11): e0277097.
Shahina, Z., Ndlovu, E., Persaud, O., Sultana, T. and Dahms, T. E. S. (2022b). Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral. Microbiol Spectr 10(6): e0318322.
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Peer-reviewed
Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes
EM Eriko Matsuura-Suzuki *
HT Hirotaka Toh *
SI Shintaro Iwasaki
(*contributed equally to this work)
Published: Vol 13, Iss 13, Jul 5, 2023
DOI: 10.21769/BioProtoc.4714 Views: 618
Reviewed by: Gal HaimovichFrédéric CATEZVinay Panwar
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Original Research Article:
The authors used this protocol in eLIFE Jun 2022
Abstract
In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.
Keywords: In vitro translation Ribosome Translation elongation Post-translational modification Rabbit reticulocyte lysate (RRL) Hybrid translation system Dual luciferase assay Modified ribosome
Background
In addition to transcription, much of gene expression is regulated at the protein synthesis step. Translational regulation is central to diverse cellular and organismal processes, such as development, differentiation, memory, viral infection, tumorigenesis, stress response, and the cell cycle. A wide variety of tools have been developed to monitor the translational output in cells [reviewed in Iwasaki and Ingolia (2017)]. These include fluorescence-based techniques [e.g., fluorescent noncanonical amino acid tagging (Dieterich et al., 2010)], mass spectrometry–based approaches [e.g., pulsed stable isotope labeling by amino acids in cell culture (Schwanhäusser et al., 2009)], ribosome profiling (Ingolia et al., 2009), and single-molecule imaging by Sun tags and spaghetti monster tags (Morisaki et al., 2016; Wang et al., 2016; Wu et al., 2016; Yan et al., 2016). However, for the detailed exploration of molecular mechanisms, in vitro translation remains a powerful and valuable methodology.
Although purified factors could serve for the biochemical reconstitution of protein synthesis processes (Pestova et al., 1998; Shimizu et al., 2001; Pestova and Hellen, 2003; Alkalaeva et al., 2006; Pisarev et al., 2007; Machida et al., 2018; Yokoyama et al., 2019; Abe et al., 2020), the preparation of individual factors is not an easy task. Thus, in vitro translation with cell extracts has become an attractive and useful strategy (Gregorio et al., 2019). For mammals, lysate-based in vitro translation systems were developed with a variety of cell types, such as CHO (Brödel et al., 2014), HEK293 (Fritz et al., 2018), HeLa ( Molla et al., 1991; Bergamini et al., 2000; Witherell, 2001; Thoma et al., 2004; Mikami et al., 2006; Rakotondrafara and Hentze, 2011), and Krebs-2 (Kerr et al., 1966; Mathews and Korner, 1970; Svitkin and Agol, 1978; Svitkin and Sonenberg, 2004 and 2007). Moreover, rabbit reticulocyte lysate (RRL) (Hunt and Jackson, 1974; Pelham and Jackson, 1976; Jackson and Hunt, 1983) has been widely used due to its commercial availability and high translational capacity. However, RRL struggles in depleting proteins by gene knockout or knockdown. Although immunodepletion by antibodies (Rakotondrafara and Hentze, 2011) has been used for this purpose, this technique depends on the efficacy, specificity, and availability of the antibodies for proteins of interest.
This barrier similarly applies to the investigation of ribosomes. Given the growing evidence of modifications of ribosomal RNA (Penzo et al., 2015; Erales et al., 2017; Taoka et al., 2018), ribosomal proteins (Simsek and Barna, 2017), and the heterologous composition of the constituents (Emmott et al., 2019), specialized ribosomes with diverse functions have been proposed (Genuth and Barna, 2018a and 2018b; Guo, 2018). Although ribosome profiling and proteome analysis in vivo provide global views of the function of specialized ribosomes (Ferretti and Karbstein, 2019), biochemical assay systems complement those approaches.
A technique termed the hybrid translation system, which is based on the exchange of ribosomes, is an alternative option in such studies (Panthu et al., 2015; Penzo et al., 2016; Erales et al., 2017; Trainor et al., 2021). Given that ultracentrifugation sediments the ribosome into the pellet, RRL can be prepared as ribosome-free but translation-competent only when the ribosomes are replenished. By supplementation with purified ribosomes of interest, for example from factor-mutated human cell lines, this system makes it possible to test the function of ribosomes in the context of active protein synthesis.
Moreover, the designed reporter enables researchers to investigate the effect of specific codons or RNA sequences along mRNA on translation elongation (Kisly et al., 2018 and 2021). The reporter consists of the fusion of Renilla and firefly luciferases (Rluc and Fluc) and measures the difference in the speeds of Rluc and Fluc synthesis to determine the ribosome elongation rate. Whereas downstream firefly luciferase detection requires complete reporter protein translation (as a whole process of initiation, elongation, and termination), upstream Renilla luciferase can be monitored even in the middle of translation elongation. Thus, the synthesis rate difference between the two luciferases provides a near proxy for elongation efficacy. The insertion of a sequence of interest between the luciferases allows a motif-specific effect on ribosome processivity.
Here, we describe the step-by-step method for the concomitant use of two experimental setups. This protocol includes four major steps: 1) the preparation of ribosome-depleted RRL; 2) the purification of ribosomes from HEK293 cells; 3) the preparation of reporter mRNAs; and 4) the translation reaction. The first two steps were based on an earlier study (Panthu et al., 2015). The design of the reporter mRNA followed the work reported by Tamm and colleagues (Kisly et al., 2021). The combination of the two approaches allowed us to study the effect of the histidine methylation of ribosomal protein uL3 (or RPL3) (Matsuura-Suzuki et al., 2022). Concretely, we delineated the results of ribosomes purified from naïve HEK293T cells and ribosomes purified from cells deficient for METTL18, the enzyme responsible for the methylation of uL3 on His245 (Małecki et al., 2021; Matsuura-Suzuki et al., 2022). In the reporter mRNA, we inserted Tyr repeats between the two luciferases to monitor the ribosome processivity on those codons. Similar applications of this methodology will expand our understanding of the regulatory mechanisms of decorated or specialized ribosomes along diverse codon/RNA element contexts.
Materials and reagents
Pipette, 10 mL, graduated 1/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 607180)
Pipette, 25 mL, graduated 2/10 mL, sterile, paper-plastic packaging, single packed (Greiner Bio-One, catalog number: 760180)
10 μL, long, graduated, filter tip with system rack (pp), sterilized (WATSON, catalog number: 1252P-207CS)
20 μL hyper filter tip with system rack (pc), sterilized (WATSON, catalog number: 125-20S)
200 μL hyper filter tip with system rack (pc), sterilized (WATSON, catalog number: 125-200S)
1,000 μL, long, graduated, filter tip with system rack (pc), sterilized (WATSON, catalog number: 124-1000S)
TipXL box (IKA Works, catalog number: 0020017832)
Tube, 15 mL, pp, 17/120 mm, natural, sterile, 20 pcs/bag (Greiner Bio-One, catalog number: 188271-013)
Labcon SuperClear 1.5 mL screw cap microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 3611-870-000)
11 mm diameter Delrin tube adapter (Beckman Coulter, catalog number: 393238)
DNA LoBind tubes, 1.5 mL (Eppendorf, catalog number: 0030108418)
Nunc EasYDishes, 100 mm (Thermo Fisher Scientific, catalog number: 150466)
3.2 mL, open-top thick wall polycarbonate tube, 13 mm × 56 mm (Beckman Coulter, catalog number: 362305)
0.2 mL 8-strip PCR tube and cap (NIPPON Genetics, catalog number: FG-028FC)
Screw cap tube, 5 mL (L × Ø): 57 mm × 15.3 mm, PP (SARSTEDT, catalog number: 60.558.001)
Corning 96-well white flat bottom polystyrene not treated microplate, 25 per bag, without lid, nonsterile (Corning, catalog number: 3912)
Rabbit reticulocyte lysate (RRL), nuclease-treated (Promega, catalog number: L4960, stored at -80°C)
DMEM, high glucose, GlutaMAX supplement (Thermo Fisher Scientific, catalog number: 10566016, stored at 4°C)
Fetal bovine serum (FBS) (MERCK, catalog number: F7524, stored at -20 °C)
HEK293T (RIKEN BRC, catalog number: RCB2202)
METTL18 KO HEK293T (Matsuura-Suzuki et al., 2022)
D-PBS(-) without Ca and Mg, liquid (Nacalai Tesque, catalog number: 14249-24, stored at room temperature)
1 M HEPES-KOH buffer solution (pH 7.5) (Nacalai Tesque, catalog number: 15639-84, stored at room temperature)
Potassium acetate (KOAc), nuclease and protease tested (Nacalai Tesque, catalog number: 28434-25, stored at room temperature)
Magnesium acetate tetrahydrate (MgOAc2·4H2O), nuclease and protease tested (Nacalai Tesque, catalog number: 20849-32, stored at room temperature)
Dithiothreitol (DTT), nuclease tested (Nacalai Tesque, catalog number: 14128-62, stored at 4 °C)
UltraPure DNase/RNase-free distilled water (Thermo Fisher Scientific, catalog number: 10977-015, stored at room temperature)
Sucrose, ultra pure (FUJIFILM, catalog number: 198-13525, stored at room temperature)
5 M sodium chloride (NaCl) solution (Nacalai Tesque, catalog number: 06900-14, stored at room temperature)
KCl (2 M), RNase-free (Thermo Fisher Scientific, catalog number: AM9640G, stored at room temperature)
1 M magnesium chloride (MgCl2) solution, sterile filtered (Nacalai Tesque, catalog number: 20942-34, stored at room temperature)
2-Mercaptoethanol, nuclease tested (Nacalai Tesque, catalog number: 21438-82, stored at 4 °C)
Qubit RNA BR Assay kit (Thermo Fisher Scientific, catalog number: Q10210) (accompanied with Qubit RNA BR buffer, Qubit RNA BR reagent, and 0.5 mL PCR tubes)
1 M Tris-HCl solution (pH 6.8) (BioVision, catalog number: 2106-100, stored at room temperature)
UltraPure SDS solution, 10% (Thermo Fisher Scientific, catalog number: 15553-035, stored at room temperature)
Glycerol (Nacalai Tesque, catalog number: 17018-25, stored at room temperature)
Bromophenol blue (Nacalai Tesque, catalog number: 05808-61, stored at room temperature)
BLUE Star prestained protein ladder (NIPPON Genetics, catalog number: MWP03-8, stored at -20 °C)
SuperSep Ace, 5%–20%, 17 well (FUJIFILM, catalog number: 194-15021, stored at 4 °C)
Tris(hydroxymethyl)aminomethane (Nacalai Tesque, catalog number: 35406-91, stored at room temperature)
Glycine (Nacalai Tesque, catalog number: 17109-35, stored at room temperature)
Sodium lauryl sulfate (Nacalai Tesque, catalog number: 31606-75, stored at room temperature)
GelCode blue stain (Thermo Fisher Scientific, catalog number: 24590, stored at room temperature)
Methanol (FUJIFILM, catalog number: 131-01826, stored at room temperature)
Acetic acid (Nacalai Tesque, catalog number: 00212-85, stored at room temperature)
psiCHECK2-Y0× (Matsuura-Suzuki et al., 2022)
psiCHECK2-Y39× (Matsuura-Suzuki et al., 2022)
Primer 1, 5′-TGACTAATACGACTCACTATAGG-3′ dissolved in TE (eurofins, stored at -20 °C) (Matsuura-Suzuki et al., 2022)
Primer 2, 5′-TGTATCTTATCATGTCTGCTCGAA-3′ dissolved in TE (eurofins, stored at -20 °C) (Matsuura-Suzuki et al., 2022)
TE buffer solution (pH 8.0), nuclease and protease tested (Nacalai Tesque, catalog number: 32739-31, stored at room temperature)
PrimeSTAR Max DNA polymerase (TaKaRa, catalog number: R045A, stored at -20 °C)
0.5 M EDTA (pH 8.0) (NIPPON GENE, catalog number: 311-90075, stored at room temperature)
Agarose for ≥ 1 kbp fragment (Nacalai Tesque, catalog number: 01163-05, stored at room temperature)
10× loading buffer (TaKaRa, catalog number: 9157, stored at room temperature)
1 kb DNA Ladder (New England BioLabs, catalog number: N3232S, stored at -20 °C)
GreenView nucleic acid gel stain, 10,000× in water (RELYON, catalog number: N100, stored at 4 °C)
NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL, catalog number: 740609.50, stored at room temperature)
T7-Scribe Standard RNA IVT kit (CELLSCRIPT, catalog number: C-AS3107, stored at -20 °C) (accompanied with 10× T7-Scribe transcription buffer, 100 mM ATP, 100 mM CTP, 100 mM UTP, 100 mM DTT, 40 U/μL ScriptGuard RNase inhibitor, T7-Scribe enzyme solution, RNase-free water, and DNase I)
Agencourt RNAClean XP (Beckman Coulter, catalog number: A63987, stored at 4 °C)
Ethanol (99.5) for molecular biology (FUJIFILM, catalog number: 054-07225, stored at room temperature)
ScriptCap m7G capping system (CELLSCRIPT, catalog number: C-SCCE0625, stored at -20 °C)
ScriptCap 2′-O-methyltransferase kit (CELLSCRIPT, catalog number: C-SCMT0625, stored at -20 °C)
A-Plus Poly(A) Polymerase Tailing kit (CELLSCRIPT, catalog number: C-PAP5104H, stored at -20 °C)
RNA 1000 kit (SHIMADZU, catalog number: 292-27913-91, stored at -20 °C and 4 °C), supplied with separation buffer and marker solution
SYBR Green II RNA gel stain, 10,000× concentrate in DMSO (Thermo Fisher Scientific, catalog number: S7564, stored at -80 °C)
RNA 6000 ladder (Thermo Fisher Scientific, catalog number: AM7152, stored at -80 °C)
Formamide (Nacalai Tesque, catalog number: 16229-95, stored at room temperature)
Amino acid mixtures (Promega, catalog number: L4461, stored at -80 °C)
Recombinant RNase inhibitor (TaKaRa, catalog number: 2313A, stored at -20 °C)
Dual-Luciferase Reporter Assay System (Promega, catalog number: E1910, stored at -20 °C), supplied with passive lysis buffer, 5× (Promega, catalog number: E1941, stored at -20 °C)
Liquid nitrogen
DMEM supplemented with FBS (500 mL) (see Recipes)
1 M KOAc (5 mL) (see Recipes)
1 M MgOAc2 (1 mL) (see Recipes)
1 M DTT (5 mL) (see Recipes)
Buffer R (10 mL) (see Recipes)
Sucrose cushion solution (10 mL for eight samples) (see Recipes)
Buffer R2 (5 mL) (see Recipes)
2× Laemmli sample buffer (see Recipes)
10× SDS-PAGE running buffer (see Recipes)
1× SDS-PAGE running buffer (see Recipes)
Gel fixation buffer (see Recipes)
50× TAE (1 L) (see Recipes)
1% agarose gel (100 mL) (see Recipes)
70% ethanol (50 mL) (see Recipes)
Buffer KM (500 μL) (see Recipes)
200 μM amino acid mixture (500 μL) (see Recipes)
1× passive lysis buffer (5 mL) (see Recipes)
Equipment
PIPETMAN P-10 (Gilson, catalog number: F144802)
PIPETMAN P-20 (Gilson, catalog number: F123600)
PIPETMAN P-200 (Gilson, catalog number: F123601)
PIPETMAN P-1000 (Gilson, catalog number: F123602)
PiptPAL single-channel pipette 1,000–10,000 μL (BMBio, catalog number: PAL-10 ml)
Pipet-Aid XP2 110 V, w/Charger (Drummond, catalog number: 4-040-501)
Optima MAX-TL ultracentrifuge (Beckman Coulter, catalog number: A95761)
TLA110 rotor (Beckman Coulter, catalog number: 366735)
Tube rack (13.0 mm, tubes) (Beckman Coulter, catalog number: 348122)
CO2 incubator (PHCbi, model: MCO-170AIC-PJ)
High-speed microcentrifuge (Hitachi, model: himac CF16RN)
Swing rotor (Hitachi, model: T4SS31) and 15TCX6S adaptor (Hitachi, catalog number: S307335A)
High-speed refrigerated microcentrifuge (TOMY, model: MX-307)
High-speed refrigerated microcentrifuge rotar rack (TOMY, model: AR015-24)
Analytical balance (SHIMADZU, model AP124W)
Slim stirrer same rotation control (AS ONE Corporation, model: 1-5940-02-22)
Semimicro stir bar (value) Ø3 mm × 6 mm football (AS ONE Corporation, model: 3-6659-03)
Qubit 2.0 fluorometer (Thermo Fisher Scientific)
Mini cooling dry bath incubator (Major Science, model: MC-0203) with mini dry bath blocks (Major Science, model: MD-MINI-B02)
Power Supply Power Station III (ATTO, model: WSE-3200)
Mini-Gel Slab Electrophoresis Tank (BIO CRAFT, model: BE-211G)
LABO SHAKER (BIO CRAFT, model: BC-740)
Odyssey CLx Imager (LI-COR, model: 9140)
ProFlex 3 × 32-well PCR system (Thermo Fisher Scientific, catalog number: 4484073)
Spectrophotometer (DeNovix, model: DS-11)
Milli-Q reference A+ system (MERCK, model: Z00QSVC01)
Mupid-exU submarine electrophoresis system (ADVANCE, model: EXU-1)
LED transilluminator (Gellex International ltd., model: LB-16)
Ultraviolet transilluminator (UVP, model: M-20)
MIXER uzusio (LMS, model: VTX-3000L)
MINI centrifuge (ALLSHENG, model: Mini-6KS)
NGS MagnaStand (YS-Model) 8 Ch × 0.2 mL PCR tube (FastGene, model: FG-SSMAG2)
Microchip Electrophoresis System for DNA/RNA Analysis MultiNA (SHIMADZU, model: MCE-202), equipped with MICROCHIP, TYPE WE-C (SHIMADZU, model: 292-36010-41)
GloMax Navigator System with Dual Injectors (Promega, model: GM2010)
Software
Image Studio (LI-COR, ver. 5.2)
MultiNA Control Software (SHIMADZU, ver. 1.14.0)
MultiNA Viewer (SHIMADZU, ver. 1.14.0)
GloMax Navigator Software (Promega, ver. 3.1.0)
Excel (Microsoft, ver. 16.66.1)
Procedure
Preparation of ribosome-depleted RRL
Load 1 mL of RRL into a 1.5 mL microcentrifuge tube, place into an 11 mm Delrin tube adapter, and ultracentrifuge at 240,000× g for 2 h 15 min at 4 °C using an Optima MAX-TL ultracentrifuge with a TLA110 rotor.
Note: Keep the sample on ice as much as possible in steps 1–2. Handling the sample in a cold room should be an option.
Collect 900 μL of the supernatant, transfer to a 1.5 mL DNA LoBind tube, flash freeze with liquid nitrogen, and store at -80 °C. See Video 1 for details on the RRL supernatant collection.
Notes:
Avoid touching the precipitate that contains the ribosomes.
Consider dividing the ribosome-depleted RRL into aliquots in several tubes before the flash freezing to avoid repeated freezethaw cycles.
Video 1. Collection of the RRL supernatant
Purification of ribosomes from HEK293 cells
Seed 5 × 106 HEK293T (naïve or METTL18 KO) cells in 10 mL of DMEM with 10% FBS (see Recipes) into a 10 cm dish and incubate overnight in a humidified incubator with 5% CO2 at 37 °C. Prepare 10 dishes.
Note: The cell culture typically reaches 70%–80% confluency.
Aspirate the medium from a dish and add 5 mL of ice-cold PBS.
Aspirate the PBS immediately and add 1 mL of ice-cold PBS.
Repeat steps 2–3 for the other nine dishes.
Resuspend cells in the dishes by pipetting, transfer to a 15 mL tube, and centrifuge at 500× g for 3 min at 4 °C, using a refrigerated centrifuge with a swing rotor.
Discard the supernatant and add 1 mL of ice-cold PBS.
Resuspend cells by pipetting, transfer to a 1.5 mL DNA LoBind tube, and centrifuge at 500× g for 3 min at 4 °C, using a refrigerated centrifuge with a fixed angle rotor.
Note: Weigh the 1.5 mL tube before the cell transfer for the next step.
Discard the supernatant and weigh the tube with the cell pellet by the scale.
Notes:
From this step, handle the sample on ice or at 4 °C.
We typically have a ~300 mg cell pellet.
Resuspend the cell pellet in the same volume of buffer R (see Recipes) (e.g., 300 μL of buffer R to 300 mg of cell pellet) and incubate for 15 min on ice.
Note: Buffer R is a low-stringency buffer. Thus, the isolated ribosome may contain the associated factors.
Vortex the mixture for 30 s and centrifuge at 16,000× g for 10 min at 4 °C, using a refrigerated centrifuge with a fixed angle rotor.
Collect the supernatant in a 1.5 mL DNA LoBind tube and mix well. Keep 10 μL of the supernatant in another 1.5 mL DNA LoBind tube for Coomassie Brilliant Blue staining.
Load 300 μL of the supernatant at the bottom of the 3.2 mL polycarbonate tube and then underlay 1 mL of sucrose cushion solution (see Recipes) slowly using a PIPETMAN P-1000 with a long 1 mL tip. See Video 2 for details on the underlaying of sucrose cushion solution.
Notes:
The cell lysate should float on top of the sucrose cushion solution. The interface between the cell lysate and sucrose cushion solution should be clearly visible. If the two solutions are mixed, the ribosome pellet may contain increased contaminants.
Instead of PIPETMAN P-1000 with a long 1 mL tip, a cannula or equivalent can be used.
Video 2. Laying sucrose cushion solution under the cell lysate
Ultracentrifuge the tube at 240,000× g for 2 h 15 min at 4 °C using an Optima MAX-TL with a TLA110 rotor.
Note: Mark the outside edge of the 3.2 mL polycarbonate tube to indicate the side where the ribosome pellet should be located.
Discard the supernatant by removing thoroughly with a pipette and slowly add 100 μL of buffer R2 (see Recipes) to the bottom of the tube, avoiding disrupting the pellet.
Note: A glassy ribosome pellet should be visible on the outside edge of the tube bottom.
Discard the supernatant immediately and add 30 μL of buffer R2.
Place the tube on a tube rack, add a stir bar to the tube, and then resuspend the solution at 4 °C for 15 min on the magnetic stirrer. See Video 3 for details.
Video 3. Resuspension of the pelleted ribosomes with a stir bar and a magnetic stirrer
Transfer the solution to a 1.5 mL DNA LoBind tube. Keep 1 μL of the solution in another 1.5 mL DNA LoBind tube for Coomassie Brilliant Blue staining.
Note: If the pellet does not dissolve completely, stir additionally for 15 min.
Determine the RNA concentration by Qubit RNA BR Assay kit and then determine the ribosome concentration as described below.
Prepare 200 μL of Qubit working solution for each standard and sample by diluting Qubit RNA BR reagent 200-fold with Qubit RNA BR buffer, according to the following table below. When you have one sample to test, prepare [2 (standards) + 1 (sample) + 1] × 200 μL solution.
Reagent Final concentration Amount
Qubit RNA BR buffer n/a 800 μL
Qubit RNA BR reagent n/a 4 μL
Prepare the assay tubes (use 0.5 mL PCR tubes accompanied with Qubit RNA BR Assay kit) according to the following table.
Standards
Reagent Final concentration Amount
Qubit working solution n/a 190 μL
20× Standard 1 or 2 (accompanied with Qubit RNA BR Assay kit) 1× 10 μL
Samples
Reagent Final concentration Amount
Qubit working solution n/a 199 μL
Ribosome solution n/a 1 μL
Vortex standards and samples for 2–3 s and incubate at room temperature for 2 min.
Turn on Qubit 2.0 fluorometer and select RNA and then RNA Broad Range Assay. Calibrate with the standards and then read the sample.
Select Read Next Sample, followed by Calculate Stock Conc. and then 1 μL to calculate the concentration of RNA in the ribosome solution.
Note: We typically have 1,000–2,000 ng/μL RNA in the ribosome solution.
Convert the RNA concentration to the ribosome concentration. Given the size of rRNAs (18S rRNA, 1.9 kb; 28S rRNA, 5 kb), the formula (number of nucleotides × 320.5) provides a total rRNA molecular weight of 6.9 × 103 × 320.5 = 2.2 × 106. Thus, the molar concentration should be determined as X [ng/μL]/(2.2 × 106). For example, for a ribosome solution with 1,500 ng/μL RNA, the ribosome concentration should be 1,500 [ng/μL]/(2.2 × 106) = 0.68 μM.
Dilute the ribosome solution to 226 nM with buffer R2.
Flash freeze the purified ribosome with liquid nitrogen and store at -80 °C.
Note: Consider dividing the ribosome solution into aliquots in several tubes before the flash freeze to avoid repeated freezethaw cycles.
Determine the protein composition in the ribosome solution by Coomassie Brilliant Blue staining.
Mix the HEK293T cell lysate (from step 11) and ribosome solution (from step 17) with 2× Laemmli sample buffer (see Recipes) in each 1.5 mL DNA LoBind tube according to the table below and denature at 95 °C for 10 min in a dry bath incubator.
Reagent Cell lysate Ribosome solution
Sample 10 μL 1 μL
Buffer R2 n/a 9 μL
2× Laemmli sample buffer 10 μL 10 μL
Load 20 μL of each sample and 5 μL of BlueStar prestained protein ladder into a precast 5%–20% polyacrylamide gel in 1× SDS-PAGE running buffer (see Recipes) set up in the mini-gel electrophoresis tank. Perform electrophoresis at a constant 20 mA for 70 min at room temperature with a power supply.
Fix the gel with 15 mL of gel fixation buffer (see Recipes) for 30 min on a rotary shaker at room temperature and briefly wash the gel twice with Milli-Q water.
Stain the gel with 15 mL of GelCode Blue Safe protein stain for 60 min on a rotary shaker and wash the gel with Milli-Q water.
Destain the gel with Milli-Q water and paper towels until the background signals are reduced.
Note: This typically takes 3 h to overnight.
Visualize the gel on an Odyssey CLx imager and Image Studio with 700 nm channels with the focus at 0.5 mm, which corresponds to the center of the 1 mm thick gel. See Figure 1 for representative protein staining results.
Figure 1. Coomassie Brilliant Blue staining for proteins in ribosome solution. Representative gel image of Coomassie Brilliant Blue staining for proteins in HEK293T cell lysate (i.e., input) and in prepared ribosome solution. The staining pattern was similar to that in an earlier report (Penzo et al., 2016). We note that due to the low stringency of buffer R, the ribosome solution contained high-molecular-weight ribosome-interacting proteins in addition to ribosomal proteins.
Preparation of reporter mRNAs
Prepare the PCR mixture in a 0.2 mL PCR tube as described below to amplify the DNA fragments for the template of in vitro transcription of Rluc-Y39×-Fluc and Rluc-Y0×-Fluc RNAs (100 μL reaction).
Reagent Final concentration Amount
1 ng/μL plasmid (psiCHECK2-Y0× or psiCHECK2-Y39×) 0.01 ng/μL 1 μL
5 μM Primer 1 0.2 μM 4 μL
5 μM Primer 2 0.2 μM 4 μL
PrimeSTAR Max Premix (2×) 1× 50 μL
RNase-free water n/a 41 μL
Perform the PCR amplification in a thermal cycler with the program described below.
Temperature Time Cycles
98 °C 10 s 1×
98 °C 10 s 30×
55 °C 5 s
72 °C 35 s
72 °C 1 min 1×
4 °C ∞ 1×
Mix the PCR with 10× loading buffer, load it onto 1% agarose gel (see Recipes) (with 1 kb DNA ladder on a lane), and separate the DNA fragment by electrophoresis with TAE (see Recipes).
Stain the gel with GreenView nucleic acid gel stain in TAE and visualize the nucleic acid using a blue light LED transilluminator. See Figure 2 for the representative results for the Rluc-Y39×-Fluc reporter template.
Gel-purify the appropriate size of DNA fragments (~2.8 kbp) with NucleoSpin Gel and PCR Clean-up according to the manufacturer’s instructions.
Measure the concentration of template DNA using a DS-11 spectrophotometer.
Prepare the in vitro transcription reaction using the T7-Scribe Standard RNA IVT kit in 0.2 mL PCR tubes as described below (20 μL reaction).
Reagent Final concentration Amount
Purified DNA template (Rluc-Y39×-Fluc or Rluc-Y0×-Fluc) 1 μg X μL
10× T7-Scribe transcription buffer 1× 2 μL
100 mM ATP 7.5 mM 1.5 μL
100 mM CTP 7.5 mM 1.5 μL
100 mM UTP 7.5 mM 1.5 μL
100 mM GTP 7.5 mM 1.5 μL
100 mM DTT 10 mM 2 μL
40 U/μL ScriptGuard RNase inhibitor 1 U/μL 0.5 μL
T7-Scribe enzyme solution n/a 2 μL
RNase-free water n/a 7.5 - X μL
Incubate at 37 °C for 2 h in a thermal cycler.
Note: Extension of the incubation time to 4–6 h may increase the yield of RNA.
Add 1 μL of DNase I (a component of the T7-Scribe Standard RNA IVT kit) and incubate at 37 °C for 2 h in a thermal cycler.
Note: RNA may be stored at -20 °C before purification.
Purify the RNA as described below.
Mix the bottle of the Agencourt RNAClean XP thoroughly to achieve homogeneous resuspension.
Add 1.8× volume of bead solution to the reaction, mix well by pipetting 10 times or vortexing for 30 s, and centrifuge the tube briefly.
Incubate at room temperature for 15 min and place the tube on the magnetic stand for 5 min to separate the beads from the solution.
Discard the cleared supernatant.
Add 200 μL of 70% ethanol (see Recipes) into the tube and incubate for 30 s at room temperature.
Note: Keep the tube standing on the magnetic stand during steps e–f.
Discard the cleared supernatant.
Repeat steps e–f twice (for a total of three washes).
Centrifuge the tube briefly to collect the remaining 70% ethanol at the bottom of the tube, keep the tube on the magnetic stand for 1 min, and discard the cleared supernatant.
Open the lid of the tube and allow the beads to dry for 5 min at room temperature.
Note: Beads may be cracked after completely drying.
Caution: Overdrying the beads may result in inefficient recovery of RNA.
Add 20 μL of RNase-free water, resuspend the beads by pipetting, and incubate for 2 min at room temperature.
Place the tube on the magnetic stand for 2 min at room temperature and then transfer the cleared supernatant to a new 1.5 mL DNA LoBind tube.
Note: The RNA may be stored at -20 °C or -80 °C.
Measure the concentration of the RNA using a DS-11 spectrophotometer.
Note: We typically have ~1 μg/μL RNA solution.
Prepare the following solution in 0.2 mL PCR tubes (33.5 μL reaction volume).
Reagent Final concentration Amount
Purified RNA 20–30 μg X μL
RNase-free water n/a 33.5 - X μL
Note: Keep the remaining RNA for application to the fragment analyzer.
Incubate at 65 °C for 5 min in a thermal cycler and then immediately place on ice.
Prepare the capping reaction using the ScriptCap m7G Capping System and ScriptCap 2′-O-Methyltransferase kit in the 0.2 mL PCR tubes as described below (50 μL reaction).
Reagent Final concentration Amount
Denatured RNA 20–30 μg 33.5 μL
10× ScriptCap capping buffer 1× 5 μL
10 mM GTP 1 mM 5 μL
20 mM SAM 0.5 mM 1.25 μL
40 U/μL ScriptGuard RNase inhibitor 1 mM 1.25 μL
100 U/μL ScriptCap 2′-O-methyltransferase 4 U/μL 2 μL
10 U/μL ScriptCap capping enzyme 0.4 U/μL 2 μL
Incubate at 37 °C for 30 min in a thermal cycler.
Prepare the poly(A) tailing reaction using the A-Plus Poly(A) Polymerase Tailing Kit in 0.2 mL PCR tubes as described below (66 μL reaction volume).
Reagent Final concentration Amount
Capping reaction n/a 50 μL
40 U/μL ScriptGuard RNase inhibitor 0.18 U/μL 0.3 μL
10× A-Plus poly(A) tailing buffer 1× 6.6 μL
20 mM ATP 2 mM 6.6 μL
4 U/μL A-Plus poly(A) polymerase 0.15 U/μL 2.5 μL
Incubate at 37 °C for 30 min in a thermal cycler.
Add 2.5 μL of 0.5 M EDTA to stop the poly(A) tailing reaction (to make ~18 mM EDTA at final concentration).
Note: The poly(A) tailing reaction may be stored at -20 °C before purification.
Purify the RNA as described in step 10 above.
Note: Purified RNA may be stored at -20 °C or -80 °C before use.
Assess the purity of the RNA with the fragment analyzer MultiNA.
Note: Instead of MultiNA, Bioanalyzer, TapeStation, or electrophoresis with denaturing TBE-agarose gel can be used.
Bring separation buffer (a reagent of the RNA 1000 kit), marker solution (a reagent of the RNA 1000 kit), SYBR Green stock solution, and RNA 6000 ladder to room temperature.
Dilute SYBR Green stock solution 100-fold with TE.
Dilute RNA 6000 Ladder 6-fold with TE.
Dilute poly(A)-tailed reporter RNA (from step 18) and non-poly(A)-tailed RNA (from steps 10–11) with RNase-free to 25–250 ng/μL.
Prepare the required volume of buffer solution for MultiNA into a screw cap tube, 5 mL, as described below.
Note: The required volume depends on the sample number. The following table shows an example.
Reagent Final concentration Amount
1/100 diluted SYBR Green n/a 1 μL
Formamide n/a 80 μL
Separation buffer (a reagent of the RNA 1000 kit) n/a 319 μL
Prepare the sample and RNA 6000 ladder in 0.2 mL PCR tubes for the MultiNA run as described below.
Reagent Final concentration Amount
1/6 RNA 6000 ladder, 1/10 poly(A)-tailed reporter RNA, or 1/10 non-poly(A)-tailed reporter RNA n/a 3 μL
RNA marker solution (a reagent of the RNA 1000 kit) n/a 3 μL
Incubate at 65 °C for 5 min in a thermal cycler and immediately place on ice.
Set the 0.2-mL PCR tubes from step f and the 5 mL tube from step e (leaving the lid off) on MultiNA and start the run with MultiNA Control Software. See Figure 3 for the representative results for the Rluc-Y39×-Fluc reporter transcript.
Calculate the molar concentration of reporter mRNA as described below.
i. Take the ng/μL concentration by the result of the MultiNA. For example, since the concentration of Rluc-Y39×-Fluc with poly(A) tail in Figure 3 was 6.0 ng/μL, the stock solution should be 6.0 ng/μL × 10 (dilution factor from the stock) = 60 ng/μL.
ii. Estimate the poly(A) length by the result of MultiNA. For example, the poly(A) length of Rluc-Y39×-Fluc with a poly(A) tail in Figure 3 was ~100 nt.
iii. Calculate the molecular weight of reporter mRNA. Given that the non-poly(A)-tailed Rluc-Y39×-Fluc should be 2,787 nt, the poly(A)-tailed Rluc-Y39×-Fluc should be ~2,887 nt. Thus, the estimated molecular weight should be 2,887 × 320.5 = 0.93 × 106. For example, the molar concentration of poly(A)-tailed Rluc-Y39×-Fluc reporter in Figure 3 should be 60 [ng/μL]/(0.93 × 106) = 65 nM.
Dilute the reporter mRNA to 11 nM with RNase-free water.
Figure 2. Agarose gel electrophoresis for the PCR-amplified DNA fragments for in vitro transcription templates. Representative gel image of the 1 kb DNA ladder and the PCR product for the in vitro transcription template of the Rluc-Y39×-Fluc reporter. The area indicated by the dashed line was gel excised. Note that this image was visualized by a UV transilluminator, which should be avoided for the actual gel-excision step.
Figure 3. Quality check of the reporter transcript by fragment analyzer. Representative electropherograms of RNA 6000 ladder and Rluc-Y39×-Fluc reporter RNA with and without poly(A) tail. MultiNA Viewer software automatically assesses the size and concentration of RNA. The poly(A) tail length was estimated to be ~100 nt based on the length difference. Additionally, the RNA concentrations were determined as follows: Rluc-Y39×-Fluc with poly(A) tail, 6.0 ng/μL; Rluc-Y39×-Fluc without poly(A) tail, 23.3 ng/μL. We note that the concentration of individual mRNA measured by MultiNA may be lower than the total RNA concentration predicted by the DS-11 spectrophotometer.
Translation reaction
Prepare the in vitro translation reaction in a 1.5 mL DNA LoBind tube as described below (120 μL reaction).
Reagent Final concentration Amount
Ribosome-depleted RRL n/a 60 μL
226 nM purified ribosome (from naïve or METTL18 KO cells) 22.6 nM 12 μL
11 nM reporter mRNA (Rluc-Y39×-Fluc or Rluc-Y0×-Fluc) 1.1 nM 12 μL
Buffer KM (see Recipes)
KCl: 75 mM
MgCl2: 0.75 mM
12 μL
200 μM amino acid mixture (see Recipes) 20 μM 12 μL
40 U/μL RNase inhibitor (TaKaRa) 0.8 U/μL 2.4 μL
RNase-free water n/a 9.6 μL
Note: RRL includes an energy regeneration system (phosphocreatine and phosphocreatine kinase), hemin, tRNA mixture, etc., which are generally required for in vitro translation. Thus, further supplementation of those materials is not necessary.
Start the reaction at 25 °C, take a 10 μL aliquot from the reaction every 5 min (for 50 min total, i.e., total 11 timepoints, including time 0 min) into 1.5 mL DNA LoBind tubes, immediately mix with 20 μL of 1× passive lysis buffer (see Recipes) to quench the reaction, and keep the mixture on ice until the luminescence measurement.
Measure the Rluc and Fluc luminescence by the Dual-Luciferase Reporter Assay System in Glomax.
Prepare Luciferase assay reagent II (LAR II) working solution as described below (for 44 + 1 samples).
Reagent Final concentration Amount
Luciferase assay substrate (lyophilized product) n/a 1 vial
Luciferase assay buffer II n/a 10 mL
Notes:
i. Typically, the assay requires (number of samples +1) × 50 + 500 μL.
ii. The remaining LAR II can be stored at −80 °C for a month.
Prepare Stop & Glo working solution as described below.
Reagent Final concentration Amount
50× Stop & Glo substrate 1× 55 μL
Stop & Glo buffer n/a 2,695 μL
Note: Typically, the assay requires (number of samples +1) × 50 + 500 μL.
Wash both injectors of the GloMax Navigator System with Milli-Q water, 70% ethanol, Milli-Q water, and air, according to the manufacturer’s instructions.
Prime injectors with LAR II and Stop & Glo working solutions according to the manufacturer’s instructions.
Program the GloMax Navigator Software to perform a two-second premeasurement delay, followed by a 10-second measurement period for each assay, and to dispense 50 μL of LAR II and Stop & Glo working solutions per sample.
Transfer 20 μL of the reaction mixture (prepared in step 2 above) to each well of a white 96-well plate.
Place the white 96-well plate in the GloMax Navigator System and initiate the assay.
Export the luminescence data from the GloMax Navigator Software into a csv file. See the representative data of “GloMax_rawdata_1.csv” in Supplemental material.
Data analysis
Load the csv file into Excel software and analyze the data for the following steps. For the representative data analysis, see “Slope.xlsx” in Supplemental material. See Figure 4 for representative data analysis results.
Draw the scatter plots of Rluc and Fluc luminescence along the time.
Determine the linear part of the signal raise and calculate the slope (SlopeRluc and SlopeFluc).
Note: In this analysis, we used luminescence values at 25–45 min. The timepoint that provides a linear increase in the signal may depend on the experimental conditions.
Calculate SlopeFluc/SlopeRluc and then relative SlopeFluc/SlopeRluc (i.e., normalize the SlopeFluc/SlopeRluc of METTL18 KO ribosomes by that of naïve ribosomes).
Figure 4. Representative data of the hybrid translation. Rluc and Fluc luminescence from the Rluc-Y39×-Fluc reporter over incubation time. Data for naïve ribosomes (A and B) and for METTL18 KO ribosomes (C and D) are shown. Based on the results, the SlopeFluc/SlopeRluc values for naïve ribosomes and METTL18 KO ribosomes were determined to be 0.0259 and 0.0341, respectively. Thus, the relative SlopeFluc/SlopeRluc for METTL18 KO ribosomes was 1.32, suggesting increased processivity of elongation along Y39×.
Notes
To test whether individual ribosome-depleted RRL and purified ribosomes have no translation activity on their own, small-scale reactions (10 μL) omitting each factor should be conducted prior to the experiments. See Figure 5 for the representative result, “GloMax_rawdata_2.csv” in Supplemental material for the raw data, and “Material_check.xlsx” in Supplemental material for the analysis.
Figure 5. Control experiments for checking the quality of materials used in the hybrid translation. Rluc (A) and Fluc (B) luminescence from the Rluc-Y0×-Fluc reporter under the indicated conditions after 60 min of incubation. Data for naïve ribosomes are shown.
Recipes
DMEM supplemented with FBS (500 mL)
Reagent Final concentration Amount
DMEM n/a 500 mL
FBS 10% 50 mL
Note: Keep at 4 °C.
1 M KOAc (5 mL)
Reagent Final concentration Amount
KOAc 1 M 0.49 g
RNase-free water n/a up to 5 mL
Note: Keep at room temperature.
1 M MgOAc2 (1 mL)
Reagent Final concentration Amount
MgOAc2·4H2O 1 M 0.214 g
RNase-free water n/a up to 1 mL
Note: Keep at room temperature.
1 M DTT (5 mL)
Reagent Final concentration Amount
DTT 1 M 0.771 g
RNase-free water n/a up to 5 mL
Note: Store at -20 °C.
Buffer R (10 mL)
Reagent Final concentration Amount
1 M HEPES-KOH pH 7.5 10 mM 100 μL
1 M KOAc 10 mM 100 μL
1 M MgOAc2 1 mM 10 μL
1 M DTT 1 mM 10 μL
RNase-free water n/a 9,780 μL
Note: Prepare before use and keep on ice.
Sucrose cushion solution (10 mL for eight samples)
Reagent Final concentration Amount
Sucrose 1 M 3.4 g (corresponding to 2.2 mL)
1 M HEPES-KOH pH 7.5 10 mM 100 μL
1 M KOAc 10 mM 100 μL
1 M MgOAc2 1 mM 10 μL
1 M DTT 1 mM 10 μL
RNase-free water n/a 7,580 μL
Note: Prepare before use and keep on ice.
Buffer R2 (5 mL)
Reagent Final concentration Amount
1 M HEPES-KOH pH 7.5 20 mM 100 μL
5 M NaCl 10 mM 10 μL
2 M KCl 25 mM 62.5 μL
1 M MgCl2 1.1 mM 5.5 μL
14.3 M 2-mercaptoethanol 7.7 mM 2.7 μL
RNase-free water n/a 4819.3 μL
Note: Prepare before use and keep on ice.
2× Laemmli sample buffer (1 mL)
Reagent Final concentration Amount
1 M Tris-HCl pH 6.8 125 mM 125 μL
100% Glycerol 20% 200 μL
10% SDS solution 4% 400 μL
Bromophenol Blue 0.004% 0.04 mg
RNase-free water n/a 175 μL
14.3 M 2-mercaptoethanol 10% 100 μL
Notes:
Keep at room temperature (without 2-mercaptoethanol).
Add 2-mercaptoethanol before use.
10× SDS-PAGE running buffer (1 L)
Reagent Final concentration Amount
Tris(hydroxymethyl)aminomethane 0.25 M 30.2 g
Glycine 1.92 M 144 g
Sodium lauryl sulfate 1% 10 g
Milli-Q water n/a up to 1 L
Note: Keep at room temperature.
1× SDS-PAGE running buffer (1 L)
Reagent Final concentration Amount
10× Running buffer 1× 100 mL
Milli-Q water n/a 900 mL
Note: Keep at room temperature.
Gel fixation buffer (100 mL)
Reagent Final concentration Amount
Methanol 50% 50 mL
Acetic acid 7% 7 mL
Milli-Q water n/a 43 mL
Note: Keep at room temperature.
50× TAE (1 L)
Reagent Final concentration Amount
Tris(hydroxymethyl)aminomethane 2 M 242 g
Acetic acid 1 M 57.1 mL
0.5 M EDTA (pH 8.0) 50 mM 100 mL
Milli-Q water n/a up to 1 L
1% agarose gel (100 mL)
Reagent Final concentration Amount
Agarose 1% 1 g
50× TAE 1× 2 mL
Milli-Q water n/a 97 mL
Note: Dissolve agarose by heating with a microwave oven. Pour the reagent into the gasket and cool it until use.
70% ethanol (50 mL)
Reagent Final concentration Amount
Ethanol 70% 35 mL
RNase-free water n/a 15 mL
Buffer KM (500 μL)
Reagent Final concentration Amount
2 M KCl 750 mM 187 μL
1 M MgCl2 7.5 mM 3.7 μL
RNase-free water n/a 309.3 μL
Note: Prepare before use and keep on ice.
200 μM amino acid mixture (500 μL)
Reagent Final concentration Amount
1 mM amino acid mixtures 200 μM 100 μL
RNase-free water n/a 400 μL
Note: Store at -80 °C.
1× passive lysis buffer (5 mL)
Reagent Final concentration Amount
5× passive lysis buffer 1× 1 mL
RNase-free water n/a 4 mL
Note: Prepare before use.
Acknowledgments
We are grateful to all the members of the Iwasaki laboratory for technical help. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (a Grant-in-Aid for Transformative Research Areas [B] “Parametric Translation”, JP20H05784 to S.I.); the Japan Society for the Promotion of Science (JSPS) (a Grants-in-Aid for Young Scientists [A], JP17H04998 to S.I.; a Grant-in-Aid for Scientific Research [C], JP21K06026 to E.M.S.; a Grant-in-Aid for Research Activity start-up, JP22K20765 to H.T.); the Japan Agency for Medical Research and Development (AMED) (AMED-CREST, JP22gm1410001 to S.I.); RIKEN (Pioneering Projects “Biology of Intracellular Environments” to S.I.; Collaboration Seed Fund to E.M.S. ; Incentive Research Project to E.M.S. and H.T.). This protocol was derived from an original paper (Matsuura-Suzuki et al., 2022) and the manufacturer’s protocols for the Qubit RNA BR Assay kit (Thermo Fisher Scientific), NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL), T7-Scribe Standard RNA IVT kit (CELLSCRIPT), ScriptCap m7G Capping system (CELLSCRIPT), ScriptCap 2′-O-Methyltransferase Kit (CELLSCRIPT), A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT), Dual-Luciferase Reporter Assay System (Promega), and GloMax Navigator System with Dual Injectors (Promega).
Competing interests
The authors declare no competing interests.
Ethical considerations
No human or animal subjects were included in this study.
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Supplementary information
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4,715 | https://bio-protocol.org/en/bpdetail?id=4715&type=0 | # Bio-Protocol Content
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Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques
TK Takanobu A. Katoh
TO Toshihiro Omori
TI Takuji Ishikawa
YO Yasushi Okada
HH Hiroshi Hamada
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4715 Views: 831
Reviewed by: Pilar Villacampa AlcubierreHiroshi YokeChristina Yan Ru Tan
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Original Research Article:
The authors used this protocol in Science Jan 2023
Abstract
Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.
Keywords: Left-right symmetry breaking mRNA imaging Super-resolution imaging Optical tweezers Mathematical modeling Mechanical Simulation
Background
Primary cilia—hair-like protrusions on the cell surface—function as antennas. Cilia sense extracellular stimuli, such as flow stimulation, and regulate/organize many signals; one of which governs left-right (L-R) determination (Shinohara and Hamada, 2017). Research concerning flow sensing in cilia began with two techniques: a micropipette (Praetorius and Spring, 2001) and a flow chamber (Nauli et al., 2003) approach. Using these techniques in combination with genetic engineering, we now understand the correlation between flow and chemical reactions, such as calcium responses, within the cilia (Su et al., 2013). However, the sensory capability of cilia with respect to flow, particularly whether cilia sense flow-mediated chemical or mechanical stimuli, is difficult to determine (Ferreira et al., 2019). In this protocol, we provide a method for dissecting the forces acting on cilia.
Recently, Katoh et al. reported the application of optical tweezers (Ashkin, 1970) to a single cilium, which enabled the application of only mechanical force to cilia (Katoh et al., 2018). We further updated this technique to apply to early mouse embryos and combined them with mRNA imaging (Minegishi et al., 2021). We now provide a method for observing the responses to mechanical stimuli by cilia, such as mRNA degradation in ciliated cells (Katoh et al., 2023).
Ascertaining how forces acting on cilia are affected by fluid flow and, in particular, how these forces affect the membrane tension of cilia, is a challenging issue. Owing to the size of cilia, which have a length of ~5 μm and a diameter of only ~200 nm, direct measurement of the tension on the ciliary membrane is difficult. Previously, membrane tension of the cilium was estimated only by flow simulation (Rydholm et al., 2010; Omori et al., 2018). By controlling the extracellular flow and concurrently measuring changes in the shape of cilia using high-resolution microscopy, we first reported the membrane strain of cilia driven by the actual extracellular flow (Katoh et al., 2023).
Finally, we introduced a method for analyzing the precise location of membrane proteins in cilia using 3D stimulated emission depletion (STED) (Klar et al., 2000; Vicidomini et al., 2011), a super-resolution microscope whose lateral resolution is close to 30 nm, using a biophysical approach. In this study, we provide useful protocols for applying state-of-the-art microscopy to cilia in mouse embryos. These methods allow us to study mechanical signals in other cilia as well as in other small organelles.
Materials and reagents
Coverslip (18 mm × 18 mm or 24 mm × 24 mm; No. 1S HT, Matsunami)
400 μm silicone rubber spacer (discontinued product; alternatively, use AS ONE, catalog number: 6-9085-14)
100 μm silicone rubber spacer (AS ONE, catalog number: 6-9085-12)
Rat serum (purchased from Charles River Laboratories Japan)
Fluorescent beads for Point Spread Function (PSF) measurement (Thermo Fisher, catalog number: F8811)
Polystyrene beads for optical tweezers (diameter 3.5 μm) (Thermo Fisher, catalog number: S37224)
Wide bore tip (Funakoshi, catalog number: T-205-WB-C)
FluoroBrite Dulbecco modified Eagle medium (DMEM) (Thermo Fisher, catalog number: A1896701)
Anti-acetylated tubulin antibody (Sigma, catalog number: T6793)
Anti-green fluorescent protein antibody (Abcam, catalog number: ab13970)
Secondary antibody: STAR RED, anti-mouse (Abberior, catalog number: STRED-1001-500UG)
Secondary antibody: STAR ORANGE, anti-chick (Abberior, catalog number: STORANGE-1005-500UG)
2,2’-thiodiethanol (TDE) (Tokyo Chemical Industry, catalog number: T0202)
1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma-Aldrich, catalog number: 290734-100 ML)
Triton X-100 (Nacalai Tesque, catalog number: 35501-15)
Medium for observation: FluoroBrite DMEM supplemented with 75% rat serum (see Recipes)
PBST (see Recipes)
TDE-DABCO (see Recipes)
Equipment
Microscopy for measurement of flow-dependent changes in ciliary shape (Figure 1A and 1B)
IX83 microscope (OLYMPUS; equipped with shutter (Sh1))
Spinning disk confocal unit (CSU) (Yokogawa, CSU-W1)
Objective lens (Obj.1): UPLAPO100XOHR 1.5 N.A. (Olympus)
UV laser, 375 nm, 70 mW (Kyocera SOC, JUNO 375)
z-Piezo (Physik Instrumente, model: P-721)
FL1: red-light band-pass filter (Asahi, model: LV0630)
Dichroic mirror (DM1) (Chroma, zt405/488/561rpc)
Beam expander (Ex1) (Thorlabs, GBE15-A)
Iris (Linos, G061653000)
Shutter (Sh2) (SURUGA SEIKI, model: F116)
Lens (L1) (Thorlabs, AC254-300-A))
EM-CCD camera (Andor, iXon Ultra 888), water cooled (ASONE, LTB-125A)
Stage top incubator (Tokai Hit, STXF-IX83WX and GM3000)
Optical table (Nippon Boushin Industry, AS-1809)
Figure 1. Microscope and optical pathway. (A) Microscope for measurement of flow-dependent changes in ciliary shape. (B) Image of the microscope shown in A and C. Optical pathways are constructed on the optical table. (C) Microscope for optical tweezers and whole-cell fluorescence recovery after photobleaching (FRAP). For abbreviations, see Equipment section in the main text.
Microscope for optical tweezers and whole-cell fluorescence recovery after photobleaching (FRAP) (Figure 1B and 1C)
IX83 microscope (OLYMPUS; equipped with shutter (Sh1))
Spinning disk confocal unit (CSU) (Yokogawa, CSU-W1)
Objective lens (Obj2): UPLSAPO 60XW 1.2 N.A. (Olympus)
z-Piezo (Physik Instrumente, P-721)
Digital analog converter for regulating z-Piezo (USB-DAQ) (National Instruments, USB-6363)
Infrared (IR) laser for optical tweezers, 1,064 nm; 5 W (IPG Photonics, YLR-5-1064-LP-SF)
Blue laser for irradiation, 488 nm; 55 mW (Coherent, Sapphire)
FL1: red-right band-pass filter (Asahi, LV0630)
FL2 (Asahi, SIX870)
DM1 (Chroma, zt405/488/561rpc)
DM2 (Chroma Technology, ZT1064rdc-sp-UF3). Note that we used ZT1064rdc-sp in Katoh et al. (2023), but that DM is better than this.
Beam expander for irradiation (Ex2 and 3) (Sigmakoki, LBED-5 and LBED-3)
Beam expander for optical tweezers (Ex4) (Sigmakoki, LBED-4Y)
Iris (Linos G061653000) (Opened)
Shutter (Sh2) (SURUGA SEIKI, model: F116)
Lens for irradiation (L1) (Thorlabs, AC254-300-A)
Lens for optical tweezers (L2) (Thorlabs, AC254-200-C)
Lens for optical tweezers (L2) (Thorlabs, AC254-150-C)
Neutral density (ND) filter for calibration of optical tweezers (only use for calibration measurement): NENIR13B (Thorlabs; transmission at 1064 nm is 3.81%)
Motorized XY stage (OptoSigma, BIOS-225T and FC-101G)
Stage top incubator (Tokai Hit, STXF-IX83WX and GM3000)
Optical table (Nippon Boushin Industry, AS-1809)
Microscope for analyzing Pkd2 distribution
TCS SP8 STED 3× (Leica)
HC PL APO 100×/1.40 Oil STED white (Leica)
Software
ImageJ, Fiji (version 1.52a, NIH)
Excel (Microsoft)
Software for deconvolution of STED microscopy image (Huygens; version 21.10; Scientific Volume Imaging)
Software for control of IX83 and CSU (iQ; Version 3.6.3; Andor)
Software for control of z-Piezo (LabView 2018; Version 18.0.1f4; National instruments)
Software for analyzing Pkd2 distribution (Igor; version 8.0.4.2; WaveMetrics)
Procedure
Measurement of flow-dependent changes in ciliary shape
Mouse E7.5 embryos harboring an NDE4-hsp-5HT6-mNeonGreen-2A-tdKatushka2 (Katoh et al., 2023) transgene were isolated and roller-cultured as previously described (Behringer et al., 2014).
We prepared the medium for observation (see Recipes) by supplementing FluoroBrite DMEM with 75% rat serum. The medium was incubated in a CO2 incubator.
The distal portion of each embryo, including the node, was excised and placed into a chamber, consisting of a glass slide fitted with a thick silicone rubber spacer (thickness of 400 μm) to prevent disturbance of nodal flow, and covered with a coverslip (Figure 2). Setting the node region close to the glass surface is very important; however, do not deform the shape of the node to prevent disturbance of nodal flow.
Figure 2. Schematic of the mounting of the mouse embryonic node. (A) For imaging, the distal portion of the embryo, including the node, was carefully cut. It is preferable to cut the tissue such that the node is located at the center top of the excised tissue. For immunostaining, the ectoplacental cone was removed (see section D). (B) A silicon rubber spacer was fitted on the glass slide. Excised tissue including the node was then placed in the center of the hole with the medium. The wide bore tip is preferable for usage (see Materials and Reagents). Lastly, it was covered with a coverslip.
We perform 3D live imaging using the microscope described above (see Table 1).
Setting of the objective: The correction ring was set at 0.17 μm for 37 °C. A lens heater (37 °C) was used. Note that the deconvolution calculation (see Data analysis) is sensitive to aberrations.
Setting z-Piezo: the z-stack distance was set to 200 nm. Typically, 301 z-stacks (60 μm) were used.
Setting of the EM-CCD camera: EMGain and A/D were 999 and 10 MHz, respectively.
Note: When using a high level of EM gain, do not irradiate strong light to the EM-CCD camera to prevent damage to the image sensor.
Table 1. Equipment settings for 3D live imaging
Device Setting Parameter
Objective lens Correction ring 0.17 μm (37 °C)
z-Piezo z-stack distance 200 nm
Range 301 z-stacks (60 μm)
EM-CCD camera EM gain 999
A/D 10 MHz
The center of the node was irradiated with UV to stop the nodal flow (see Figure 1 in Katoh et al., 2023): a 375 nm laser (set at 70 mW; laser power before the objective was 23 mW) was radiated for 45 s. The irradiation duration was controlled by a shutter equipped with an IX83 microscope using iQ software. Laser protection goggles must be worn during this operation.
The procedure for 3D live imaging was repeated. The settings were identical to those of the previous imaging. In most embryos, the fluorescence intensity is weak; thus, the laser power for CSU is typically higher than before UV irradiation.
The obtained 3D images were used for analysis for flow dependent change in angle of cilia and computational mesh generation for calculating membrane tension of cilia, as described below.
Measurement of PSF for deconvolution calculation (see Table 2).
Sample: fluorescent beads were diluted 1:1,250,000 in phosphate-buffered saline [PBS(-)] solution. The beads were attached to the glass surface within several minutes.
3D imaging of a fluorescent bead was performed using the same settings as described in step 4, excluding the use of the EM-CCD camera. To increase S/N, the EM gain and A/D of the EM-CCD camera were set to 3 and 1 MHz, respectively.
Table 2. Equipment settings for PSF measurement
Device Setting Parameter
Objective lens Correction ring 0.17 μm (37 °C)
z-Piezo z-stack distance 200 nm
Range 301 z-stacks (60 μm)
EM-CCD camera EM gain 3
A/D 1 MHz
Manipulation of single cilium by optical tweezers
Mouse E7.5 embryos harboring NDE4-hsp-dsVenus-Dand5-3′-UTR (Minegishi et al., 2021) and NDE4-hsp-5HT6-GCaMP6-2A-5HT6-mCherry (Mizuno et al., 2020) were isolated and roller-cultured as previously described (Behringer et al., 2014).
The medium was prepared as described above.
The beads were prepared for trapping. Twenty microliters of polystyrene beads (diameter, 3.5 μm) were added to 980 μL of FluoroBrite DMEM (1:50 dilution). After centrifugation at 20,300× g for 15 min, isolated beads (pellets) were resuspended in 200 μL of FluoroBrite DMEM.
A distal portion of each embryo, including the node, was excised and placed into a chamber consisting of a glass slide fitted with a thick silicone rubber spacer (thickness of 400 μm) to prevent any disturbance of nodal flow (see Figure 2). The embryo was carefully trimmed to obtain a node located parallel to the glass surface to prevent deformation of the embryo during observation.
A portion of the diluted beads was carefully transferred to the medium above the node with a P2 pipette tip to expose the node to ~1–10 beads, and then carefully covered with a coverslip.
Live imaging was performed, and the target cilium was set using the ciliary GCaMP6/mCherry signal. A brightfield image was observed through a red channel of the CSU to check the shape of the embryo.
In the case of analysis by whole-cell FRAP later, 3D images of ciliary mCherry and cytoplasmic dsVenus were recorded (typically a z-axis depth of 1 μm with 30 sections).
Mechanical stimuli were applied to cilia using the microscope described above. A polystyrene bead was trapped, placed in contact with a cilium, and forced to oscillate along the dorsoventral (D-V) axis for 1.5 h with the use of optical tweezers. Laser protection goggles must be worn during this operation.
Setting of the objective: the correction ring was set at 0.17 μm for 37 °C. A lens heater (37 °C) was used. Please note that optical tweezers are sensitive to aberrations.
The setting of the IR laser was 400 mW for ZT1064rdc-sp-UF3 and 800 mW for ZT1064rdc-sp (condition in Katoh et al., 2023).
Setting z-Piezo and USB-DAQ: a 2 Hz triangle wave with 1.025 ± 0.175 V amplitude was generated using USB-DAQ controlled by the simulate signal express function in LabView and applied to the z-Piezo (Figure 3).
Note that we manually tracked the cilium using a motorized XY stage because most embryos were displaced or deformed during 1.5 h of stimulation.
To measure mRNA degradation triggered by mechanical stimuli, we performed whole-cell fluorescence recovery after photobleaching (FRAP) analysis, as described below.
Measurement of trapping stiffness:
Sample: polystyrene beads (diameter 3.5 μm) were diluted with distilled deionized water (MQ, typically 1:1,000) and placed into a flow chamber (Katoh et al., 2021) made up of a slide glass (24 mm × 60 mm, No. 1S), a coverslip (24 mm × 24 mm, No. 1S HT), and two pieces of double-sided tape for spacers (cut approximately 5 mm wide and 40 mm in length).
Measurement: a single bead was weakly trapped by optical tweezers (typically 3.81 mW: laser power set at 100 mW and a 3.81% ND filter inserted into the optical pathway), and the restricted Brownian motion was measured. Motion of the bead was observed using a brightfield image with a CSU (using the bypath mode). Here, the frame rate needs to be faster than the cut-off frequency of the motion (we typically set this to ~200 fps).
Figure 3. Setting of simulate signal express function in LabView. z-Piezo applied a 2 Hz triangle wave with an amplitude of 1.025 ± 0.175 V using USB-DAQ.
Measurement of Dand5 mRNA degradation by whole-cell FRAP
Whole-cell FRAP is a method for measuring mRNA levels using the level of fluorescence recovery after whole-cell bleaching (Katoh et al., 2023). Before bleaching, 3D images of ciliary mCherry and cytoplasmic dsVenus were recorded (typically, a z-axis depth of 1 μm and 30 sections; Table 3).
Table 3. Setting of 3D live imaging for whole-cell FRAP
Device Setting Parameter
z-Piezo z-stack distance 1 μm
Range 30 z-stacks (29 μm; typically)
EM-CCD camera EM gain 999 (typically)
A/D 30 MHz (typically)
Exposure time 200 ms (typically)
Binning 1 × 1
All cells in the node region were uniformly bleached by 3 min irradiation with a 488 nm laser (output power set at 55 mW). The irradiation duration was controlled by a shutter equipped with an IX83 microscope using iQ software (Figure 4A). Laser protection goggles must be worn during this operation.
Fluorescence recovery was measured by 3D images using the microscope described above (Figure 4B).
Setting z-Piezo: the distance of the z-stack was set to 1 μm. Typically, 30 z-stacks (29 μm) were observed.
Setting of the camera: the fluorescence intensity was quite low compared with that before bleaching. Therefore, we usually employed a 2 × 2 binning mode with a 1 s exposure.
3D images were captured 15 times at 2 min time intervals (total observation duration is 30 min).
Bleaching was repeated and fluorescence recovery was observed during the repeated process for 30 min (Figure 4A).
The intensity in the second fluorescence recovery is low; thus, finally, we observed the 3D image with strong excitation (typically 30–55 mW) and long exposure times (1–4 s in each z-stack).
Analysis of fluorescence recovery is described below (see “Analysis of Dand5 mRNA levels for whole-cell FRAP experiments”).
Figure 4. Setting of iQ. (A) Screenshot of Protocol in iQ. Steps 2–4 are automatically performed using this protocol. (B) Screenshot of Channel in iQ, representing the acquisition setting in Step 3.
Measurement of Pkd2 distribution by 3D-STED imaging
Immunostaining was performed as follows:
Dissection: mouse E7.5 embryos harboring NDE2-hsp-Pkd2-Venus (Yoshiba et al., 2012) were recovered in cold PBS(-). The ectoplacental cone was then removed from the embryo (see Figure 2A). For this procedure, we recommend using a 1.5 mL tube (although other sizes work equally well).
Fixation: embryos were immediately transferred to ice-cold PBS(-). When all embryos had been isolated, they were immediately fixed for 1 h at 4 °C in PBS containing 4% paraformaldehyde.
Rinse: embryos were washed three times with PBS containing 0.01% Triton X-100 (PBST; see Recipes).
Permeabilization: 30 min at room temperature in PBS containing 0.2% Triton X-100.
Rinse: embryos were washed three times with PBST.
Primary antibody: incubated overnight at 4 °C with acetylated tubulin (1:200 dilution) and green fluorescent protein (1:200 dilution) antibodies diluted in PBST.
Rinse and wash: antibodies were washed three times with PBST. During this, it is preferable to change the tube. Then, wash > 6 times with PBST at 1 h intervals.
Secondary antibody was incubated overnight at 4 °C with STAR RED (1:200 dilution, anti-mouse) and STAR ORANGE (1:200 dilution, anti-chick) antibodies diluted in PBST.
Mounting was performed as follows:
Mounting solution: TDE-DABCO (see Recipes)
A distal portion of each embryo, including the node, was excised and the node region was placed in PBST containing 10% TDE-DABCO. In the final step, the node should be located close to the glass surface, as described below, to prevent resolution reduction—so, carefully trim the embryo.
The node region was transferred to PBST containing 20% TDE-DABCO.
The node region was transferred to PBST containing 50% TDE-DABCO.
The node region was transferred to TDE-DABCO.
The node region was placed into a chamber consisting of a glass slide fitted with a thin silicone rubber spacer (thickness 100 μm) and then carefully covered with a coverslip (see Figure 2). In this step, it is very important for the node to be close to the coverslip without deforming the shape of the node. Please note that we index-matched samples to minimize spherical aberration.
Finally, it was sealed with nail polish. Samples were stored at 4 °C for approximately one week.
3D-STED observation: 3D-STED imaging was performed as follows:
Bit depth: 16 bit
Channel 1: excitation wavelength 561 nm (white light laser pulse), detection wavelength for HyD 571–623 nm, gating 0.3–6 ns, depletion wavelength 775 nm (pulse)
Channel 2: excitation wavelength 633 nm (white light laser pulse), detection wavelength for HyD 650–700 nm, gating 0.3–6 ns, depletion wavelength 775 nm (pulse)
z-donut: 60%–80% (typically 80%)
Pinhole setting: 0.5 AU at 640 nm
Before observation, it is better to perform Beam alignment.
Note that in STED microscopy, the excitation light is overlapped with the STED beam, quenching excited molecules in the excitation spot periphery. In this setting, we used the same wavelength of the STED beam for Channel 1 and Channel 2 (see depletion wavelength), so chromatic aberration does not occur.
Analysis of 3D images is described below in Analysis of Pkd2 distribution for STED images.
Data analysis
Analysis for flow dependent change in angle of cilia
Deconvolution calculation of 3D images: 3D deconvolution calculations were performed using DeconvolutionLab2 (Sage et al., 2017) (Figure 5A).
Settings of DeconvolutionLab2:
Algorithm: Richardson-Lucy Total Variation
Iterations (N): 100
Regularization parameter λ: 10-12
Figure 5. Setting for deconvolution and correction of the three-dimensional orientation of the embryo. (A) Screenshot of DeconvolutionLab2. (B) Define two vectors a and b from the base positions of the three cilia and calculate their outer product n = a × b. The same process is applied to two different images (before and after UV irradiation) and the images are corrected so that the respective n-vectors match.
An 8-bit transformation of the image was performed and location information on the ciliated surface was extracted from ImageJ’s Gaussian Filter and Find Edges convolution functions.
To quantify the three-dimensional orientation of the embryo, the basal positions of the three cilia were detected, which were defined as r1, r2, and r3.
Two vectors, a (= r2 - r1) and b (= r3 - r1), were defined from the three basal positions, as shown in Figure 5B, and one normal vector, n = a × b, was determined by the outer product of these vectors.
The n-vector in both the pre- and post-UV irradiation images was found, and these images were then rotated such that the two n-vectors coincide.
Ellipsoid fitting was performed on the ciliary geometry obtained from the microscopic images. The ciliary posture was defined by determining the declination of the ellipsoid major axis with respect to the dorsoventral axis.
Ciliary deformation was defined based on the change in the angle before and after UV irradiation.
Computational mesh generation for calculating membrane tension of cilia
The cross-sectional centers of the cilia in the z-stack of each 3D image were determined.
Spline interpolation was applied to represent a smooth curve connecting the centers of each z-section.
We defined circles of radius 100 nm each in the direction normal to the centerline, as shown in Figure 6.
Figure 6. Computational mesh generation to represent the ciliated membrane surface. The ciliated surface is represented as a cylindrical surface with a series of circles of radius 100 nm normal to the centerline (yellow line). Grid size, 1.6 µm.
The surface of the ciliary membrane was defined by connecting each circumference in the direction of the longitudinal axis.
A computational mesh was generated by discretizing the defined membrane surface by triangles.
The cilia shape after UV irradiation was defined as the reference shape and the shape before UV irradiation was used to calculate the elastic deformation of the cilia.
Analysis of Dand5 mRNA levels for whole-cell FRAP experiments
As described in Figure S6 and the Methods section in Katoh et al. (2023), our whole-cell FRAP system with NDE4-hsp-dsVenus-Dand5-3′-UTR transgene (Minegishi et al., 2021) can directly measure Dand5 mRNA levels as the final (plateau) intensity. In this analysis, we used three 3D images: the image captured before stimuli, the last image of first fluorescence recovery, and the final image of second fluorescence recovery.
Measurement of intensity before stimulation:
We first find a cell with a stimulated cilium and neighboring unstimulated ciliated cells (typically two unstimulated cells).
The suitable size of the region of interest (ROI) for these cells was set, and the average intensity in each of them was measured using ImageJ/Fiji.
The background intensity was measured using the region in the center of the node.
Finally, we calculated the following ratio: Ratio0 = 2 (IS - IB)/(IN1 + IN2 - 2IB), where IS, IB, IN1, and IN2 are the intensity in stimulated cells, background intensity, intensity in neighboring unstimulated cell number #1, and intensity in neighboring unstimulated cell number #2, respectively.
Measurement of the intensity in the last image of first fluorescence recovery.
The stimulated cilium and the unstimulated cells were carefully identified. This was difficult because embryos are usually deformed during long-term observations. To identify target cells, we used movies taken during optical tweezer experiments, and 3D timelapse images during the FRAP experiment.
The intensity of each cell and the background intensity were measured as described above. We then calculated the Ratio1 as described above.
Measurement of the intensity in the final image of second fluorescence recovery:
The cells were carefully identified, the intensity of each cell was measured, and the background intensity was measured as described above. We then calculated Ratio2, as described above.
Finally, the normalized values were calculated: mRNA levels 30 min after stimuli (%) = Ratio1/Ratio0, mRNA levels 1 h after stimuli (%) = Ratio2/Ratio0
Analysis of Pkd2 distribution for STED imaging
Deconvolution calculation by Huygens software
Firstly, we directly read a .lif file by Huygens and then started the deconvolution wizard.
The settings of the deconvolution wizard were as follows:
Stabilization: On
Deconvolution algorithm: classical maximum likelihood estimation (CMLE)
PSF estimation: as close guess, Max detail
The calculated image was saved.
A higher bit depth is better for subsequent analysis; therefore, we save the image as a .ome file in Huygens. The file was then opened by Fiji. When the image is opened by Fiji, it is flipped vertically, so we should reflip vertically. This image was saved as a .tiff file.
Analysis of angular distribution
We used a custom-written macro that runs in Igor software. The details of this macro are as follows:
First, using ImageJ, the location of a single cilium in a 3D image was memorized as the width, depth, height, x, y, and z values of the ROI. The threshold values in the red and green channels were measured to extract the signal. Then, the 3D tiff image was loaded with Igor.
When the cilium elongates along the z-axis, we calculated the center of gravity of the red channel in each xy plane (each z-stack). We typically use the threshold value for the red channel as the background. However, the background affects this calculation; therefore, it is preferable to carefully set the background. The resulting (x, y) coordinates represent the cilium center in each z-stack.
When the cilium elongates along the x- or y-axis, we calculated the center in yz or xz planes using the same method.
The angle of the Pkd2 channel was calculated. If the z-plane had a Pkd2 signal above the threshold, we calculated the following and saved it as an array.
Angle from the center of the cilia to the Pkd2 signal using arctangent function θ
Distance from the center of cilia to the Pkd2 signal: r
Intensity: I
The intensity was accumulated at each angle in each cilium. We extracted pixels whose r was close to the cilium (we usually set 500–1,000 nm) to exclude the non-ciliary signal. Then, all I values are accumulated at each θ angle (typically a 45° sector) in every cilium.
All data from each cilium were plotted in one figure such as Figure 4B in Katoh et al. (2023).
Recipes
Medium for observation: FluoroBrite DMEM supplemented with 75% rat serum
We usually store 1 mL of 100% rat serum in sterile tubes at -80 °C and 10 mL of FluoroBrite DMEM in Corning tubes at 4 °C (long-term storage at -80 °C). Just before use, we mix 3 mL of rat serum with 1 mL of FluoroBrite DMEM on a clean bench.
PBST
100 μL of Triton X-100
1 L of PBS(-)
TDE-DABCO
11.5 g of TDE
250 μL of DABCO
150 μL of 1 M Tris-HCl (pH 8.0)
Acknowledgments
We thank K. Mizuno, K. Minegish, X. Sai, and all members of the Laboratory for Organismal Patterning for improving the protocol; K. Kawaguchi and members of his laboratory for support in developing the analysis method for Pkd2 distribution for STED images; and D. Takao for support with STED imaging.
This study was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (No. 17H01435) and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST) (No. JPMJCR13W5) to H.H.; by a Grant-in-Aid (No. 21K15096) from the Japan Society for the Promotion of Science (JSPS); by the RIKEN Special Postdoctoral Researcher Program to T.A.K.; by a grant from Precursory Research for Embryonic Science and Technology (PRESTO) of JST (No. JPMJPR2142) to T.O. 3D-STED microscopy was supported by grants from JST (Nos. JPMJMS2025-14, JPMJCR20E2, JPMJCR15G2, and JPMJCR1852), and JSPS (Nos. 19H05794 and 16H06280) to Y.O.
This protocol is derived from the original research paper (Katoh et al., 2023).
Competing interests
The authors declare no competing interests.
Ethical considerations
All animal experiments were approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch.
References
Ashkin, A. (1970). Acceleration and Trapping of Particles by Radiation Pressure. Phys Rev Lett 24(4): 156-159.
Behringer, R., Gertsenstein, M., Nagy, K. V. and Nagy, A. (2014). Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press. ISBN: 9781936113002, 9781936113019.
Ferreira, R. R., Fukui, H., Chow, R., Vilfan, A. and Vermot, J. (2019). The cilium as a force sensor-myth versus reality. J Cell Sci 132(14): jcs213496.
Katoh, T. A., Daiho, T., Yamasaki, K., Danko, S., Fujimura, S. and Suzuki, H. (2021). Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging. Sci Rep 11(1): 13672.
Katoh, T. A., Ikegami, K., Uchida, N., Iwase, T., Nakane, D., Masaike, T., Setou, M. and Nishizaka, T. (2018). Three-dimensional tracking of microbeads attached to the tip of single isolated tracheal cilia beating under external load. Sci Rep 8(1): 15562.
Katoh, T. A., Omori, T., Mizuno, K., Sai, X., Minegishi, K., Ikawa, Y., Nishimura, H., Itabashi, T., Kajikawa, E., Hiver, S., et al. (2023). Immotile cilia mechanically sense the direction of fluid flow for left-right determination. Science 379(6627): 66-71.
Klar, T. A., Jakobs, S., Dyba, M., Egner, A. and Hell, S. W. (2000). Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A 97(15): 8206-8210.
Minegishi, K., Rothe, B., Komatsu, K. R., Ono, H., Ikawa, Y., Nishimura, H., Katoh, T. A., Kajikawa, E., Sai, X., Miyashita, E., et al. (2021). Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex. Nat Commun 12(1): 4071.
Mizuno, K., Shiozawa, K., Katoh, T. A., Minegishi, K., Ide, T., Ikawa, Y., Nishimura, H., Takaoka, K., Itabashi, T., Iwane, A. H., et al. (2020). Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry. Sci Adv 6(30): eaba1195.
Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., et al. (2003). Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33(2): 129-137.
Omori, T., Winter, K., Shinohara, K., Hamada, H. and Ishikawa, T. (2018). Simulation of the nodal flow of mutant embryos with a small number of cilia: comparison of mechanosensing and vesicle transport hypotheses. R Soc Open Sci 5(8): 180601.
Praetorius, H. A. and Spring, K. R. (2001). Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium. J Membr Biol 184(1): 71-79.
Rydholm, S., Zwartz, G., Kowalewski, J. M., Kamali-Zare, P., Frisk, T. and Brismar, H. (2010). Mechanical properties of primary cilia regulate the response to fluid flow. Am J Physiol Renal Physiol 298(5): F1096-1102.
Sage, D., Donati, L., Soulez, F., Fortun, D., Schmit, G., Seitz, A., Guiet, R., Vonesch, C. and Unser, M. (2017). DeconvolutionLab2: An open-source software for deconvolution microscopy. Methods 115: 28-41.
Shinohara, K. and Hamada, H. (2017). Cilia in Left-Right Symmetry Breaking. Cold Spring Harb Perspect Biol 9(10): a028282.
Su, S., Phua, S. C., DeRose, R., Chiba, S., Narita, K., Kalugin, P. N., Katada, T., Kontani, K., Takeda, S. and Inoue, T. (2013). Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia. Nat Methods 10(11): 1105-1107.
Vicidomini, G., Moneron, G., Han, K. Y., Westphal, V., Ta, H., Reuss, M., Engelhardt, J., Eggeling, C. and Hell, S. W. (2011). Sharper low-power STED nanoscopy by time gating. Nat Methods 8(7): 571-573.
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© 2023 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 movement > Cell motility
Developmental Biology > Morphogenesis > Motility
Biophysics > Single-molecule technique
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4,716 | https://bio-protocol.org/en/bpdetail?id=4716&type=0 | # Bio-Protocol Content
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Peer-reviewed
In situ Quantification of Cytosine Modification Levels in Heterochromatic Domains of Cultured Mammalian Cells
MA María Arroyo *
MC M. Cristina Cardoso
FH Florian D. Hastert *
(*contributed equally to this work)
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4716 Views: 568
Reviewed by: Gal HaimovichVeena Padmanaban Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Nature Communications Sep 2022
Abstract
Epigenetic modifications of DNA, and especially cytosine, play a crucial role in regulating basic cellular processes and thereby the overall cellular metabolism. Their levels change during organismic and cellular development, but especially also in pathogenic aberrations such as cancer. Levels of respective modifications are often addressed in bulk by specialized mass spectrometry techniques or by employing dedicated ChIP-seq protocols, with the latter giving information about the sequence context of the modification. However, to address modification levels on a single cell basis, high- or low-content microscopy techniques remain the preferred methodology. The protocol presented here describes a straightforward method to detect and quantify different DNA modifications in human cell lines, which can also be adapted to other cultured mammalian cell types. To this end, cells are immunostained against two different cytosine modifications in combination with DNA counterstaining. Image acquisition takes place on a confocal microscopy system. A semi-automated analysis pipeline helps to gather data in a fast and reliable fashion. The protocol is comparatively simple, fast, and cost effective. By employing methodologies that are often well established in most molecular biology laboratories, many researchers are able to apply the described protocol straight away in-house.
Keywords: DNA methylation In situ quantification Immunocytochemistry Image analysis Epigenetics Heterochromatin
Background
Methylation of the fifth carbon atom of cytosine (5′-methylcytosine; 5mC) is found in the DNA of most eukaryotes and in all human cell types. It is one of the most abundant and conserved epigenetic modifications and regulates gene expression by silencing promoter regions and facilitating the compaction of heterochromatin (Lande-Diner et al., 2007; Smith and Meissner, 2013). Mammalian DNA methylation occurs predominantly in the context of symmetric CpG dinucleotides. Consequently, of the 28 million CpG sites in the human genome, between 60% and 80% are methylated, which corresponds to approximately 5% of all cytosines (Smith and Meissner, 2013). However, up to 200 base-pair long stretches of these dinucleotides, so-called CpG-islands, are often found in the context of promoters and transcription start sites, where they are sparsely methylated (Bird, 1986; Smith and Meissner, 2013).
Moreover, DNA methylation contributes to the integrity of heterochromatin by serving as a binding platform for various proteins that can recognize and bind 5mC, thereby shaping the heterochromatin landscape (Ludwig et al., 2016). On the other hand, the members of the Tet (Ten-eleven-translocation)-protein family, Tet1, Tet2, and Tet3, were found to oxidize 5mC to 5′-hydroxymethylcytosine (5hmC), 5′-formylcytosine (5fC), and 5′-carboxylcytosine (5caC). All oxidative derivatives of 5mC were found to act as intermediates in the active removal of DNA methylation and are themselves removed via different glycosylases (Ludwig et al., 2016). Tet proteins share the same catalytic activity (Bauer et al., 2015) but different physiological roles (Santiago et al., 2014). An example is Tet1 and its short isoform, which exhibit distinct nuclear localization during DNA replication. This results in aberrant cytosine modification levels in heterochromatic regions of mouse and human cells and LINE 1 activation (Arroyo et al., 2022). While in murine cells heterochromatin rich domains are found on most chromosomes, in human cells long stretches of pericentric heterochromatin are mostly found on chromosomes 1, 9, and 16 (Meneveri et al., 1993; Bizhanova and Kaufman, 2021).
Changes in cytosine modifications and, in particular, hypomethylation, are a hallmark of many cancers (Vidal et al., 2017). A reproducible method to measure changes in cytosine modification levels in euchromatic vs. heterochromatic regions is highly desirable in this context. For this purpose, microscopy techniques are more revealing than mass spectrometry or ChIP-seq methods, since direct images from single cells provide information about the abundance, subnuclear distribution, and cell cycle variation of different epigenetic marks. Therefore, we developed a pipeline to address cytosine modification levels and applied it to MCF7 human breast adenocarcinoma cells, expressing higher levels of TET1 proteins, and MCF10a non-transformed human breast epithelial cells (Figure 1). Our method, performed in Arroyo et al. (2022), includes immunodetection of cytosine modifications with antibodies against 5mC and either 5hmC, 5fC, or 5caC, followed by confocal microscopy imaging and image analysis. The implemented image analysis includes nucleus and heterochromatin and (euchromatic) nucleoplasm segmentation, achieved by creating different masks based on the DNA counterstaining (DAPI) signal. Heterochromatin segmentation using DAPI as a proxy for differences in chromatin compaction has been extensively used in quantitative analyses of the nuclear landscape (Schmid et al., 2017; Pradhan and Cardoso, 2023). The following protocol can be applied to a variety of experimental models and cell lines in order to study and quantify differences in cytosine modification patterns in different cellular backgrounds.
Figure 1. Model of study. TET protein catalytic activity in heterochromatin regions results in aberrant oxidation of 5-methylcytosine in human cells, depending on the level of TET proteins.
Materials and reagents
P100 dishes (Merck KGaA, catalog number: CLS430293)
6-well dishes (Merck KGaA, catalog number: CC302)
Glass coverslips (Carl Roth, catalog number: P232.1)
Microscopy slides (Thermo Fisher Scientific, catalog number: ABAA000080)
Petri dish (Carl Roth, catalog number: EL50.1)
Gel-blotting paper (Carl Roth, catalog number: A126.1)
Aluminum foil (Carl Roth, catalog number: 1770.1)
Parafilm (Merck KGaA, catalog number: P7793)
MCF7 cells (ATCC, catalog number: HTB-22) (Soule et al., 1973)
MCF10a cells (ATCC, catalog number: CRL-10317) (Soule et al., 1990)
DMEM (Merck KGaA, catalog number: D6429), store at 4 °C
DMEM/F12 (Merck KGaA, catalog number: D6421), store at 4 °C
Fetal bovine serum (FBS) (Gibco, catalog number: A5256701), aliquot and store at -20 °C
Horse serum (Merck KGaA, catalog number: H1138), aliquot and store at -20 °C
Pen/Strep (Merck KGaA, catalog number: P4333), store at 4 °C
L-Glutamine (Merck KGaA, catalog number: G7513), store at 4 °C
hEGF (Merck KGaA, catalog number: E9644)
Hydrocortisone (Merck KGaA, catalog number: H0888)
Cholera toxin (Merck KGaA, catalog number: C8052)
Insulin human (Merck KGaA, catalog number: I2643)
PBS (VWR International, catalog number: 0780)
Trypsin (Merck KGaA, catalog number: T4049), store at 4 °C
Gelatine (Sigma-Aldrich, catalog number: G2500)
Formaldehyde (Merck KGaA, catalog number: F8775)
EDTA (Merck KGaA, catalog number: E5134)
MgCl2·6H2O (Merck KGaA, catalog number: 1374248)
CaCl2 (Merck KGaA, catalog number: C1016)
Trizma® hydrochloride (Merck KGaA, catalog number: T2319)
Tween 20 (Merck KGaA, catalog number: P9416)
Triton X-100 (Merck KGaA, catalog number: X100)
Methanol (Merck KGaA, catalog number: 32213)
RNase A (Merck KGaA, catalog number: 10109169001)
BSA (Merck KGaA, catalog number: 10735094001)
DNase I (Merck KGaA, catalog number: D5025)
Monoclonal mouse α-5mC (CF-MAB - Helmholtz Munich, 32E2) (Weichmann et al., 2020)
Polyclonal rabbit α-5-hmC (Active Motif, catalog number: 39769)
Polyclonal rabbit α-5-fC (Active Motif, catalog number: 61223)
Polyclonal rabbit α-5-caC (Active Motif, catalog number: 61225)
Goat α-mouse IgG (H+L) Alexa Fluor 488-conjugated (Thermo Fisher Scientific, catalog number: A32723) (suggested antibody, which worked well in this protocol)
Goat α-rabbit IgG (H+L) Alexa Fluor 594-conjugated (Thermo Fisher Scientific, catalog number: A32740) (suggested antibody, which worked well in this protocol)
DAPI, 4’,6-diamidino-2-phenylindole (Merck KGaA, catalog number: D9542)
Mowiol® 4-88 (Merck KGaA, catalog number: 81381)
Wet chamber for staining (Cardoso and Leonhardt, 1996)
Solutions
0.1% gelatine (see Recipes)
3.7% formaldehyde (see Recipes)
88% methanol (see Recipes)
0.5% Triton X-100 in PBS (see Recipes)
0.01% PBS-T (see Recipes)
0.01% PBS-TE (see Recipes)
4%, 2%, 1% BSA in PBS (see Recipes)
100 µg/mL RNase in 1× PBS (see Recipes)
2× DNase I Buffer (see Recipes)
Recipes
0.1% gelatine
Reagent Final concentration Amount
Gelatine 0.1% 0.5 g
1× PBS 1× 500 mL
Total n/a 500 mL
Sterilize by autoclaving at 121 °C for 30 min.
3.7% formaldehyde
Reagent Final concentration Amount
36.5%–38% formaldehyde solution 3.7 % 2.5 mL
1× PBS 1× 22.5 mL
Total n/a 25 mL
Prepare fresh and use within one week.
88% methanol
Reagent Final concentration Amount
Methanol 88% 88 mL
ddH2O 12% 12 mL
Total n/a 100 mL
Store at -20 °C
0.5% Triton X-100 in PBS
Reagent Final concentration Amount
1× PBS 1× 99.5 mL
Triton X-100 0.5% 0.5 mL
Total n/a 100 mL
Prepare at least two days prior to use and store at room temperature.
0.01% PBS-T
Reagent Final concentration Amount
1× PBS 1× 249.975 mL
Tween 20 0.01% 0.025 mL
Total n/a 250 mL
Store at room temperature.
0.01% PBS-TE
Reagent Final concentration Amount
1× PBS 1× 249.75 mL
Tween 20 0.01% 0.025 mL
EDTA 100 mM
Total n/a 250 mL
Store at room temperature
4% BSA in PBS
Reagent Final concentration Amount
BSA 4% 0.6 g
1× PBS 1× 15 mL
Total n/a 15 mL
Dissolve BSA completely, prepare 1.5 mL aliquots, and store at -20 °C.
If 1% or 2% BSA are needed, prepare appropriate dilutions with 1× PBS.
100 µg/mL RNase in 1× PBS
Reagent Final concentration Amount
RNase A - 0.025 g
ddH2O - 250 µL
Total 100 mg/mL 250 µL
Dissolve RNase A powder completely in ddH2O and store in 25 µL aliquots at -20 °C.
Before use, prepare a 1:1,000 dilution with 1× PBS to yield a concentration of 100 µg/mL.
2× DNase I Buffer
Reagent Final concentration Amount
Trizma® hydrochloride pH 7.5 20 mM 2 mL
MgCl2 5 mM 0.102 g
CaCl2 1 mM 0.011 g
ddH2O - 98 mL
Total n/a 100 mL
Prepare 2 mL aliquots and store at -20 °C.
Equipment
Laminar flow cabinet (neoLab Migge GmbH, BDK 1200-SK, catalog number: 83 99 51200)
Cell culture CO2 incubator (Binder, catalog number: C150-230V)
Water bath (GFL Technology, catalog number: 10865892E)
Hybridization oven (Appligene, catalog number: 22012105); alternatively, use 37 °C incubator
Confocal microscope (Perkin Elmer Spinning Disk/Nikon Ti-E, PerkinElmer Life Sciences) equipped with high magnification objective (oil immersion 100× Plan-Apochromat, NA 1.49)
Software
Image acquisition software [in our case, Volocity 6.3 (Perkin Elmer, USA) was used] “Fiji Is Just ImageJ” Fiji (NIH/http://Fiji.sc/Fiji) or ImageJ (NIH/https://imagej.nih.gov/ij/download.html) for image pre-processing and analysis
RStudio (https://rstudio.com/) for plotting and statistical analysis
Procedure
Cell culture (all steps in laminar flow cabinet)
Culture cells by following established protocols for adherent cell lines (for more details see Note 1).
Split cells 2–3 times per week or as needed (for more details see Note 2).
Seeding cells for immunostaining (all steps in laminar flow cabinet)
Fill appropriate cell culture dish (6-well) with ddH2O (2 mL).
Sterilize glass coverslips by dipping multiple times into 70% ethanol using sterilized tweezers.
Transfer glass coverslips to a ddH2O-filled dish. Make sure to submerge coverslips.
Aspirate ddH2O.
Cover glass coverslips with 100 μL of 0.1% gelatine each and incubate for at least 15 min (preferably overnight) at 37 °C.
Aspirate remaining gelatine solution.
Split cells as usual and seed 0.25 × 106 cells in a glass coverslip–containing dish.
Incubate for at least 18 h at 37 °C, 5% CO2.
Immunostaining for DNA base modifications
Remove medium from cells that were seeded the day before at a density of 0.25 × 106 per p35 dish.
Carefully wash cells with 1 mL of 1× PBS and discard PBS.
Fix cells with 1 mL of 3.7% formaldehyde for 10 min at room temperature.
Remove formaldehyde solution.
Wash three times with 2 mL of PBS-T (for more details see Note 3).
Permeabilize cells with 1 mL of 0.5% Triton X-100 in PBS for 20 min.
Remove Triton X-100.
Add ice-cold 88% methanol and incubate for 5 min at room temperature.
Remove methanol.
Wash as before three times with PBS-T.
Prepare a wet chamber with moist gel-blotting paper and Parafilm on top (for more details see Note 4 and Figure 2).
Figure 2. Diagram of a wet chamber for immunostaining
Prepare a 100 μg/mL RNase A in 1× PBS solution.
Place coverslips cell side down in 20 μL droplets of RNase A solution on Parafilm and incubate for 1 h at 37 °C.
Wash three times with PBS-T, by carefully adding 100 μL of PBS-T droplets under the coverslips (for more details see Note 5).
Block for 30 min at 37 °C by placing coverslips in 20 μL of 1% BSA in 1× PBS cell side down.
Prepare primary antibody solution (detection solution) per coverslip. For one coverslip use:
10 μL of 2% BSA in PBS.
10 μL of DNase buffer (2×).
0.25 μL of DNase I (2,000 U/mL).
Antibody against 5mC (1:250).
Antibody against 5-hmC, 5-fC, or 5-caC (1:250, 1:100, or 1:100, respectively).
Place coverslips cell side down in detection solution.
Incubate at 37 °C for 70 min.
Wash three times with 100 μL droplets of PBS-TE (0, 5, and 10 min).
Dilute secondary antibodies.
Prepare 20 μL of secondary antibody solution (detection solution) per coverslip. For one coverslip use:
20 μL of 2% BSA in PBS.
Secondary antibody goat α-mouse IgG (H+L) Alexa Fluor 488-conjugated (1:500).
Secondary antibody goat α-rabbit IgG (H+L) Alexa Fluor 594-conjugated (1:500).
Incubate with secondary antibodies at room temperature for 45 min.
Wash three times with 100 μL droplets of PBS-T (0, 5, and 10 min).
Counterstain with 20 μL of 1 μg/mL DAPI for 10 min.
Meanwhile, take out a glass slide and put a 20 μL drop of Mowiol mounting medium on top.
After DAPI staining, briefly wash coverslip by dipping in ddH2O.
Dry excess water by carefully tipping the edge of the coverslip on a paper tissue.
Place on top of the mounting medium with the cells side facing down.
Microscopy
Set up the image acquisition settings for each channel and use the same settings for all images, avoiding oversaturation.
Acquire images from higher to lower wavelength, starting with red, green, and blue at the end. See Note 6 for setting details.
Acquire Z-stack images with a fixed Z-step length, preferably 0.5 μm. Acquire enough Z-stack to image the whole nuclei from “top” to “bottom.” For the majority of the cells, 10–12 Z-stacks are enough.
For further image processing in Fiji, data should be exported as .tiff files as Multicolor Z-stack.
Image analysis (manual procedure using Fiji)
The purpose of this image analysis pipeline is to use DAPI intensities to segment the cell nuclei, creating a nuclear mask, and then segment heterochromatic regions (HCmask). Euchromatic regions are defined by heterochromatin subtraction from the nucleus mask. Once these subnuclear compartments are defined, the final aim of the analysis is to measure cytosine modification levels within these different regions. Minor optimizations to the following procedure can be applied for different cell types or imaging systems.
Pre-processing
Analyze → Set Measurements → select the desired properties to be measured (see Note 7).
Edit → Options → Colors… → Foreground: black; Background: black; Selection: white.
Open a Multicolor Z-stack image.
Image → Stacks → Z Project… → Choose first slice as Start slice and last slice as Stop slice and Max Intensity from Dropdown Menu.
Right-mouse click → Rename to “Master.”
Right-mouse click → Duplicate → Title: “nucleus_mask;” Tick: Duplicate hyperstack; Channels (c): leave as is.
Nucleus segmentation
Select window “nucleus_mask.”
Choose DAPI channel using channel slider.
Image → Type → 8-bit.
Process → Filters → Gaussian Blur… → Sigma (Radius): 1.0 → “OK” → Click “Yes” in dialogue “Process Stack?”.
Process → Enhance Contrast… → Saturated pixels: 0.3% → “OK.”
Right-mouse click → Duplicate → Title: “forHCmask;” Tick: Duplicate hyperstack; Channels (c): leave as is.
Select window “nucleus_mask.”
Process → Binary → Make Binary → Method: Triangle; Background: Dark; Tick: Black background (of binary masks).
Process → Binary → Watershed → “OK” → Click “Yes” in dialogue “Process Stack?”.
Select “Wand (tracing) tool.”
Select window “nucleus_mask.”
Select nucleus of interest.
Edit → Clear Outside → Click “Yes” in dialogue “Process Stack?”.
Analyze → Tools → ROI manager… → Add [t] → Rename… to “nucleus_mask.”
Heterochromatin segmentation
Select window “forHCmask.”
Select window “ROI manager.”
Select ROI “nucleus_mask.”
Edit → Clear Outside → Click “Yes” in dialogue “Process Stack?”.
Image → Adjust → Threshold… → Dropdown 1: Triangle; Dropdown 2: B&W; Tick: Dark background → adjust to DAPI dense areas → Apply.
Click “OK” in dialogue “Convert Stack to Binary.”
Edit → Selection → Create selection.
Select window “ROI manager” → Add [t] → Rename… to “HCmask.”
Close window “Threshold.”
Heterochromatin subtraction
Select window “nucleus_mask.”
Select window “ROI manager” and select ROI “HCmask.”
Edit → Clear.
Edit → Selection → Create selection.
Select window “ROI manager” → Add [t] → Rename… to “Nucleoplasm_mask.”
Close windows “nucleus_mask” and “forHCmask.”
Measure intensities in the respective regions of interest (ROIs)
Select window “Master.”
Select window “ROI manager.”
Select desired ROI.
Press “Ctrl + M” to measure the first channel.
Use the channel slider to move to the next channel and press “Ctrl + M” to measure again.
Repeat until all ROIs are measured in all channels and remember the series.
Save the measurements as .csv files (Ctrl + S).
The pipeline for image analysis is illustrated in Figure 3.
Figure 3. Image processing and in situ quantitative analysis pipeline for cytosine modifications levels. After immunofluorescence detection of DNA base modifications and image acquisition by confocal microscopy, the preprocessing of the multicolor Z-stack is performed in Fiji. Afterwards, the nucleus, heterochromatin, and the euchromatic nucleoplasm are segmented into regions of interest (ROIs) based on their respective DAPI signals, and binary masks are generated. Signals of all channels in the respective ROIs are then measured based on the binary masks and results exported as .csv files.
Image analysis (semi-automated procedure)
In addition to the step-by-step analysis procedure described in section E, in this section we describe a semi-automated analysis pipeline using a macro in Fiji. The semi-automated procedure speeds up the analysis process along with a higher standardization for different samples.
Start the semi-automated image analysis pipeline macro by opening it in Fiji (see File S1).
Open a Multicolor Z-stack image of interest.
Click “Run” in the Macro UI.
You will be prompted to select the DAPI channel. Use the channel slider to move to the DAPI channel and click “OK” in the dialogue box.
You will be prompted to choose a nucleus of interest. After clicking on the nucleus of interest, confirm by clicking “OK” in the dialogue box.
You will be prompted to set a threshold for the heterochromatin regions. Use the sliders in the threshold menu to adjust and confirm by clicking “OK” in the dialogue box.
Based on the DAPI signal, the macro generates ROIs for (I) the nucleus, (II) the heterochromatin, (III) the euchromatic nucleoplasm. Based on these ROIs, the macro quantifies the fluorescence intensities in channel 1 for ROI I, II, and III, in channel 2 for ROI I, II, and III, and in channel 3 for ROI I, II, and III.
You are prompted to save the measurements as a .csv file. Save with “Ctrl + S” and confirm by clicking “OK” in the dialogue box.
All windows will be closed, and you can continue with the next image, starting from step 2.
Data analysis
After the image analysis, we obtain .csv files as output, with a total of nine rows corresponding to measurements in three channels (DAPI, 5mC, and 5hmC) and for the three different masks (nucleus, heterochromatin, nucleoplasm). The output includes values for “Area,” “Mean,” “StdDev,” “IntDen,” etc., which depend on the selected settings of the measurement in Fiji. For our analysis, described in Arroyo et al. (2022), the respective mean cytosine modification levels were normalized against the respective normalized 5mC levels to compensate for the epigenetic heterogeneity in cancer cells (Guo et al., 2019). Normalized mean intensities for at least 20 cells were plotted with RStudio (Version 1.1.447). For the statistics, an independent two-group comparison was made for all groups, using a Wilcoxon–Mann–Whitney test. Boxplots showing normalized mean intensity of cytosine modifications in heterochromatin regions are shown in Figure 4A and representative images in Figure 4B.
Figure 4. Cytosine modification levels in heterochromatic regions in MCF cell lines. (A) Boxplots showing normalized mean intensities of cytosine modifications in heterochromatin regions. n (5mC) = 28 (MCF10a) - 27 (MCF7), n (5hmC) = 25 (MCF10a) - 24 (MCF7), n (5fC) = 27 (MCF10a) - 19 (MCF7), n (5caC) = 18 (MCF10a) - 19 (MCF7) cells. For all boxplots, the box represents 50% of the data, starting in the first quartile (25%) and ending in the third (75%). The line inside represents the median. The whiskers represent the upper and lower quartile. Statistical significance was tested with a paired two-samples Wilcoxon test [n.s. (not significant) is given for p-values ≥ 0.05; one star (*) for p-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005; three stars (***) is given for values < 0.0005]. (B) Representative confocal mid-Z sections of DAPI-stained nuclei of MCF10a and MCF7 cells showing levels of 5mC and 5hmC, or 5fC or 5caC, respectively. Confocal Z-stacks were analyzed with Fiji. Scale bar = 2 μm.
General notes and troubleshooting
Cells are cultured in a humidified atmosphere under 5% CO2 at 37 °C. Media are supplemented with pen/strep (100 IU/mL penicillin and 100 μg/mL streptomycin) and 2.5 mM L-glutamine. MCF10a non-tumorigenic mammary gland cells are cultured in DMEM/F12 supplemented with final concentrations of 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 mg/mL insulin. MCF7 breast cancer cells are cultured in DMEM containing 10% FBS.
Trypsinization of cells: Aspirate the medium and discard; wash cells with PBS/EDTA 0.02% (w/v), aspirate, and discard; add enough warmed 1× trypsin–EDTA solution to cover the cell monolayer and place the dish in a CO2 incubator at 37 °C for 2–5 min; once detached, resuspend the cells in growth medium containing serum; gently pipette the cells up and down to disrupt cell clumps and transfer an appropriate amount of cell suspension to a new dish and top up with normal medium.
No incubation time is necessary. PBS-T is added and removed from the dish.
As a wet chamber, a plastic box of different dimensions can be used. For example, a box of 1.5 cm × 12 cm × 12 cm (depth × width × height) covered with aluminum foil to protect the sample from light exposure. Soak one extra thick gel-blotting paper (10 cm × 10 cm) with ddH2O inside the box and remove excess water. Then, cut a 10 cm piece of parafilm and place it on top of the soaked gel-blotting paper.
For washing steps in droplets, lift the coverslips with tweezers, carefully remove the droplet of PBS-T, and add a new one, using a pipette.
Image acquisition settings for confocal microscope (Perkin Elmer Spinning Disk/Nikon Ti-E):
Channel 1: TexasRed (Set “Binning” to “x1.” Set “Exposure” to “350 ms.” Set “UltraVIEW 561 nm Laser Power” to “15.0%.”
Channel 2: GFP (Set “Binning” to “x1.” Set “Exposure” to “350 ms.” Set “UltraVIEW 488 nm Laser Power” to “15.0%.”
Channel 3: DAPI (Set “Binning” to “x1.” Set “Exposure” to “350 ms.” Set “UltraVIEW 405 nm Laser Power” to “16.5%.”
We recommend measuring the following properties: Area, Standard deviation, Min and max gray values, integrated density, and Mean gray value. However, depending on the image analysis and the user, additional properties can be required, for example Shape descriptors.
Acknowledgments
This protocol was used in Arroyo et al. (2022). The research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grants CA 198/10-1 Project number 326470517 and CA 198/16-1 Project number 425470807 to M.C.C.
Competing interests
The authors declare no competing interests.
References
Arroyo, M., Hastert, F. D., Zhadan, A., Schelter, F., Zimbelmann, S., Rausch, C., Ludwig, A. K., Carell, T. and Cardoso, M. C. (2022). Isoform-specific and ubiquitination dependent recruitment of Tet1 to replicating heterochromatin modulates methylcytosine oxidation. Nat Commun 13(1): 5173.
Bauer, C., Göbel, K., Nagaraj, N., Colantuoni, C., Wang, M., Müller, U., Kremmer, E., Rottach, A. and Leonhardt, H. (2015). Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT). J Biol Chem 290(8): 4801-4812.
Bird, A. P. (1986). CpG-rich islands and the function of DNA methylation. Nature 321(6067): 209-213.
Bizhanova, A. and Kaufman, P. D. (2021). Close to the edge: Heterochromatin at the nucleolar and nuclear peripheries. Biochim Biophys Acta Gene Regul Mech 1864(1): 194666.
Cardoso, M. C. and Leonhardt, H. (1996). Immunofluorescence Techniques in Cell Cycle Studies. In Cell Cycle — Materials and Methods (pp. 15-28). Springer Berlin Heidelberg.
Guo, M., Peng, Y., Gao, A., Du, C. and Herman, J. G. (2019). Epigenetic heterogeneity in cancer. Biomark Res 7: 23.
Lande-Diner, L., Zhang, J., Ben-Porath, I., Amariglio, N., Keshet, I., Hecht, M., Azuara, V., Fisher, A. G., Rechavi, G. and Cedar, H. (2007). Role of DNA methylation in stable gene repression. J Biol Chem 282(16): 12194-12200.
Ludwig, A. K., Zhang, P. and Cardoso, M. C. (2016). Modifiers and readers of DNA modifications and their impact on genome structure, expression, and stability in disease. Front Genet 7: 115.
Meneveri, R., Agresti, A., Marozzi, A., Saccone, S., Rocchi, M., Archidiacono, N., Corneo, G., Della Valle, G. and Ginelli, E. (1993). Molecular organization and chromosomal location of human GC-rich heterochromatic blocks. Gene 123(2): 227-234.
Pradhan, S. K. and Cardoso, M. C. (2023). Analysis of cell cycle and DNA compaction dependent subnuclear distribution of histone marks. Methods Mol Biol 2589: 225-239.
Santiago, M., Antunes, C., Guedes, M., Sousa, N. and Marques, C. J. (2014). TET enzymes and DNA hydroxymethylation in neural development and function - how critical are they? Genomics 104(5): 334-340.
Schmid, V. J., Cremer, M. and Cremer, T. (2017). Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy. Methods 123: 33-46.
Smith, Z. D. and Meissner, A. (2013). DNA methylation: roles in mammalian development. Nat Rev Genet 14(3): 204-220.
Soule, H. D., Maloney, T. M., Wolman, S. R., Peterson, W. D., Brenz, R., McGrath, C. M., Russo, J., Pauley, R. J., Jones, R. F. and Brooks, S. C. (1990). Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50(18): 6075-6086.
Soule, H. D., Vazguez, J., Long, A., Albert, S. and Brennan, M. (1973). A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51(5): 1409-1416.
Vidal, E., Sayols, S., Moran, S., Guillaumet-Adkins, A., Schroeder, M. P., Royo, R., Orozco, M., Gut, M., Gut, I., Lopez-Bigas, N., et al. (2017). A DNA methylation map of human cancer at single base-pair resolution. Oncogene 36(40): 5648-5657.
Weichmann, F., Hett, R., Schepers, A., Ito-Kureha, T., Flatley, A., Slama, K., Hastert, F. D., Angstman, N. B., Cardoso, M. C., König, J., et al. (2020). Validation strategies for antibodies targeting modified ribonucleotides. RNA 26(10): 1489-1506.
Supplementary information
The following supporting information can be downloaded at here:
File S1.
Article Information
Copyright
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
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Monitoring Group 2 Innate Lymphoid Cell Biology in Models of Lung Inflammation
JB Jana H. Badrani
AS Allyssa N. Strohm
YH Yung-An Huang
TD Taylor A. Doherty
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4717 Views: 739
Reviewed by: Meenal SinhaJulie WeidnerLuis Alberto Sánchez Vargas
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Original Research Article:
The authors used this protocol in Nature Communications Jul 2022
Abstract
Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4+ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify Lineage−Thy1.2+ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology.
Key features
• In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33.
• Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.
• During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels of Gata-3 and Ki-67 and produce greater amounts of IL-5 and IL-13.
Graphical overview
Keywords: Innate lymphoid cells Lung inflammation Alternaria alternata Flow cytometry Intracellular staining
Background
Innate lymphoid cells (ILCs) are a heterogeneous population of lymphocytes that lack common lineage markers. ILCs are differentiated by their transcription factor expression profiles and cytokine production capabilities (Wirtz et al., 2021). ILCs are categorized into three major subpopulations, ILC1s, ILC2s, and ILC3s, which are the innate counterparts to CD4+ Th1, Th2, and Th17 cells (Artis and Spits, 2015). ILCs and their T-cell counterparts share similar functions, transcription factor profiles, and cytokine production. ILC1s and Th1s express T-bet and produce IFNγ in response to intracellular pathogens. ILC2s and Th2s express Gata-3 and produce IL-4, IL-5, and IL-13 in response to extracellular pathogens and allergens. ILC3s and Th17s express RORγt and produce IL-17a in response to extracellular pathogens (Drake et al., 2014; Vivier et al., 2018).
In the lung, ILC2s are the most common ILC subpopulation (Gasteiger et al., 2015). Lung-resident ILC2s can be identified by their expression of Thy-1, CD127 (IL-7 receptor), CD25 (IL-2 receptor), and T1ST2 (IL-33 receptor) (Monticelli et al., 2011). Lung inflammation is associated with the epithelial release of alarmins, including IL-33, IL-25, and thymic stromal lymphopoietin (Rossi et al., 2022). ILC2s respond to alarmins and mediate inflammation within the lungs; in response to environmental allergens, ILC2s produce their effector cytokines and expand locally (Lai et al., 2016). ILC2-derived IL-4 is important for the differentiation of naïve T cells into Th2 cells and the transition to adaptive immunity (Pelly et al., 2016). IL-5 plays a role in maintaining and promoting eosinophils (Ikutani et al., 2017), while IL-13 promotes mucus secretion (Varela et al., 2022). With inflammation, lung ILC2s are more motile and accumulate in peribronchial and perivascular spaces (Puttur et al., 2019).
In addition to being a rare cell population, ILCs lack specific and unique surface markers, which presents a challenge in identifying ILCs in tissues. Flow cytometry is the most established technique for identifying and characterizing ILCs, including the different subpopulations. Multicolor flow cytometry allows for the identification of multiple populations based on multiple parameters. For ILC populations, a combination of surface markers, transcription factors, and cytokines serves as the basis for identifying these populations. Here, murine ILCs are identified as Lineage−Thy1.2+ lymphocytes. IL-5 and IL-13 production is used to further identify pulmonary ILC2s. In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33. Bronchoalveolar lavage and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.
Materials and reagents
21 G needle (BD, catalog number: 305167)
18 G needle (BD, catalog number: 305196)
1.5 mL Eppendorf tubes (Genesee Scientific, catalog number: 24-282S)
Flat-bottom 96-well plate (Genesee Scientific, catalog number: 25-109)
70 μm filter (Corning, catalog number: 431751)
15 mL conical tubes (FroggaBio, catalog number: TB15-25)
50 mL conical tubes (Falcon, catalog number: 352098)
Suture (Look, catalog number: SP105)
gentleMACS c-tube (Miltenyi Biotec, catalog number: 130-093-237)
Polystyrene FACS tubes (Fisher Scientific, catalog number: 14-959-5)
C57BL/6 mice (6–8 weeks old; female)
Miltenyi Lung Digest kit (Miltenyi Biotec, catalog number: 130-095-927)
Mouse recombinant IL-33 (Thermo Fisher, catalog number: 14-8332-80)
Alternaria alternata extract (Fisher Scientific, catalog number: NC1620293)
1× PBS (Gibco, catalog number: 10010-023)
RPMI (Gibco, catalog number: 21870-076)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-100G)
Fetal bovine serum (FBS) (Corning, catalog number: 35-015-CV)
Sodium azide (Fisher Scientific, catalog number: BP922I-500)
Penicillin/streptavidin (Gibco, catalog number: 15140122)
L-Glutamine (Gibco, catalog number: 25030-081)
β-Mercaptoethanol (Gibco, catalog number: 21985-023)
Cell stimulation cocktail plus protein transport inhibitor (Thermo Fisher Scientific, catalog number: 00-4975-93)
PE anti-mouse IL-13 (Thermo Fisher, catalog number: 12-7133-82; clone: eBio13A, dilution: 1:20)
PE anti-mouse/human IL-5 (BioLegend, catalog number: 504304; clone: TRFK5, dilution: 1:20)
PE Ki-67 monoclonal antibody (Thermo Fisher, catalog number: 12-5698-82; clone: SolA15, dilution: 1:50)
APC anti-mouse Ly6G-Ly6C (GR1) (BioLegend, catalog number: 108412; clone: RB6-8C5, dilution: 1:50)
PE anti-mouse SiglecF (BD Biosciences, catalog number: 552126; clone: E502440, dilution: 1:50)
PerCP anti-mouse CD45.2 (BioLegend, catalog number: 109828; clone: 104, dilution: 1:50)
FITC anti-mouse TCR γ/δ (BioLegend, catalog number: 118106; clone: GL3, dilution: 1:200)
FITC anti-mouse TCR β Chain (BioLegend, catalog number: 109206; clone: H57-597, dilution: 1:200)
FITC anti-mouse FceR1α (BioLegend, catalog number: 134306; clone: MAR-1, dilution: 1:200)
FITC anti-mouse CD5 (BioLegend, catalog number: 100606; clone: 53-7.3, dilution: 1:200)
FITC anti-mouse NK-1.1 (BioLegend, catalog number: 108706; clone: PK136, dilution: 1:200)
FITC anti-mouse CD11c (BioLegend, catalog number: 117306; clone: N418, dilution: 1:200)
FITC anti-mouse Lineage (BioLegend, catalog number: 133302; clone: 145-2C11; RB6-8C5; RA3-6B2; Ter-119; M1/70, dilution: 1:40)
Purified anti-mouse CD16/32 (Fc Block) (BioLegend, catalog number:101302; clone: 93, dilution: 1:50)
BD Cytofix/Cytoperm Fixation/Permeabilization kit (Fisher Scientific, catalog number: BDB554714)
FoxP3/Transcription Factor Staining Buffer Set (FoxP3 Perm kit and buffer) (ThermoFisher, catalog number: 00-5523-00)
32% paraformaldehyde (PFA), EM grade (Electron Microscopy Sciences, catalog number: 15714) (diluted to 4% with DI water)
FACS buffer (see Recipes)
T-cell media (see Recipes)
Equipment
Single-channel pipettes (Rainin: P10, P200, P1000)
Multi-channel pipette (Thermo Scientific)
Catheter tubing (BD, catalog number: 427411)
Fine forceps (Fine Science Tools, catalog number: 11412-11)
Curved forceps (Fine Science Tools, catalog number: 11054-10)
Surgical scissors (Fine Science Tools, catalog number: 14060-10)
Centrifuge (Beckman Coulter, model: Allegra X-15R, swinging bucket rotor)
gentleMACS dissociator, octo dissociator (Miltenyi Biotec, catalog number: 130-096-427)
gentleMACS mixer, octo dissociator (Miltenyi Biotec, catalog number: 130-096-427)
Incubator (Panasonic, catalog number: MCO-18ACL-PA)
Novocyte 3000 (Acea, catalog number: 2010011)
Software
FlowJo (Version 10.8.0)
NovoExpress (Agilent)
Prism (GraphPad, Version 9.2.0)
Procedure
Intranasal challenges
Prepare challenge solution at the correct dosage by diluting Alternaria alternata or recombinant IL-33 in sterile PBS.
Notes:
Alternaria alternata concentration may vary depending on the lot potency. With new lots of Alternaria, multiple concentrations should be tested to determine the optimal concentration. In our models of Alternaria challenge, we typically use 25–50 μg per 40 μL. For models using recombinant IL-33, concentrations of 10–50 ng per 40 μL are recommended.
The volume prepared should be reflective of challenges to be completed within a one-week period. For example, with a three-day challenge model, the volume prepared would be enough for all three days of challenge.
Mix the solution well and aliquot for each day of challenge. Additional aliquoted solutions should be stored at -20 °C until needed.
Note: Before challenging, the solution should be completely thawed and mixed well.
Anesthetize 6–8-week-old female C57BL/6 wild-type mice one at a time using inhaled isoflurane. Mice are properly anesthetized when their breathing is slow and regular.
Note: If overly anesthetized, the mouse will begin bucking its head. If this occurs, remove the mouse from the isoflurane and allow it to recover before placing it back in the isoflurane chamber.
Remove the anesthetized mouse from the isoflurane chamber and pipette 40 μL of challenge solution onto its nose.
Notes:
The mouse should be held in an upright position until all of the challenge solution has been inhaled.
To avoid bubbling of the solution and/or the mouse sneezing out the solution, the challenge should be timed to match the mouse’s breathing (i.e., pipette the solution when the mouse is inhaling).
Repeat the steps above for all challenge days of the model.
Note: The timing of the challenges should be consistent each day.
Tissue collection
Prepare RPMI and 2% BSA in PBS. Keep reagents on ice for the tissue collection.
Euthanize the mouse using carbon dioxide.
Expose the trachea and remove any connective tissue around the trachea. Using an 18 G needle, make a hole at the top of the trachea.
Note: The needle should be kept parallel to the trachea to avoid puncturing the back of the trachea.
Place a 21 G catheter through the trachea and flush 0.5 mL of 2% BSA. Allow approximately 10 s before collecting the lavage from this first draw. Collect the bronchoalveolar lavage (BAL) from the first draw in an individual FACS tube and place on ice.
Notes:
The 21 G catheter should be created before euthanizing the mouse by threading the 21 G needle through the catheter tubing.
The supernatant from the first draw of BAL can be used to measure cytokine levels within the airway using ELISA.
BAL supernatant should be clear when collected. Bloody BAL may be indicative of lung damage occurring during euthanasia.
Repeat step B4 four more times using 0.6 mL of 2% BSA. The lavage from these draws should be collected together in a separate tube and placed on ice.
Note: After collecting the supernatant from the first draw for future ELISA analysis, the cells from all five draws can be pooled together and used for flow cytometry staining. Staining these cells using the granulocyte master mix (below) is helpful for measuring the levels of airway eosinophils and monitoring the extent of lung inflammation.
Expose the chest and pierce the diaphragm. Cut away the diaphragm and cut through the middle of the rib cage.
Pull open the rib cage to expose the lungs. Collect the lungs in 1 mL of RPMI and place on ice.
BAL processing
Centrifuge all of the BAL samples (first draw and pooled consecutive draws) at 500× g for 5 min at 4 °C.
Collect the supernatant from the first draw samples and place in an Eppendorf tube.
Note: The supernatant from the first draw should be stored at -20 °C and can be used for ELISA.
Aspirate the supernatant from the pooled consecutive draws samples, taking care not to disturb the pellet.
Using 500 μL of FACS buffer (see Recipes), resuspend the pellet from the first draw and combine with the pellet from the pooled consecutive draws.
Lung processing
Place the lungs in a gentleMACS c-tube with 2.5 mL of lung digest buffer, 50 μL of enzyme D, and 7.5 μL of enzyme A.
Note: The lung digest buffer is a part of the Miltenyi Lung Digest kit and is prepared per the manufacturer’s instructions. Enzymes D and A are also a part of the Miltenyi Lung Digest kit and are prepared per the manufacturer’s instructions.
Place the c-tubes in the gentleMACS dissociator and run at the lung_01 setting.
Note: Tissue can get stuck within the blender top. After removing the tubes from the dissociator, make sure that all of the tissue is in the digestion buffer.
Incubate the tubes at 37 °C for 30 min with end-over-end mixing.
Place the tubes in the gentleMACS dissociator and run at the lung_02 setting.
Filter the solution through a 70 μm filter and into a 50 mL conical tube. Wash the tubes with 2 mL of RPMI to remove any additional cells.
Centrifuge the tubes at 500× g for 5 min at 4 °C. Remove the supernatant and resuspend the pellet in 1 mL of RPMI.
Cell counts
To count the cells in the BAL and lung samples, create a 1:100 dilution by diluting 5 μL of the sample in 495 μL of FACS buffer.
Run 100 μL of the diluted sample on the Novocyte at high speed.
Gate the live cells based on forward scatter (FSC) and side scatter (SSC) and use the gated event total to calculate the cell total in the sample, as shown below (Figure 1).
For the BAL, distribute 1 million cells to a separate FACS tube for surface staining.
Note: If the BAL cell total is less than 1 million cells, then stain the whole sample.
For the lung, split the sample into parts based on the desired stains.
For surface stains, distribute 1 million cells per stain to separate FACS tubes.
For nuclear staining, distribute 2 million cells per stain to separate FACS tubes.
For cytokine staining, set aside 10 million cells in a separate FACS tube to culture.
Notes:
It is important to include controls for the nuclear and cytokine stains for accurate gating of these populations. Fluorescence minus one (FMO) and/or isotype controls may be utilized, although isotype controls may be more accurate for intracellular stains. Controls follow the same staining protocol as the samples; however, the antibody of interest (i.e., Ki-67, Gata-3, IL-5, and IL-13) are either omitted (as in the FMO) or replaced with the antibody isotype (as per the manufacturer’s information). If using isotype controls, it is important to use the same concentration of the isotype as the original antibody for an accurate comparison.
Samples can be pooled together for the controls. For example, the controls for the nuclear stains were made by pooling together cells from all the samples. Because samples are being pooled together, only a fraction of the sample volume used is needed in order to ensure that the total cells stained do not exceed the number of cells used for the samples.
Figure 1. Sample gating and calculation for cell totals. Diluted samples are gated based on size for live cells in (A) bronchoalveolar lavage (BAL) and (B) lung in order to obtain the live event count. (C) Calculation for cell totals within the samples based on event count, dilution factor, and the total volume.
Culture for cytokine staining
Centrifuge the tubes with 10 million cells at 500× g for 5 min at 4 °C.
Remove the supernatant and resuspend in 2 mL of T-cell media.
Note: Important! The T-cell media should be at room temperature when used and should be kept sterile.
Add 4 μL of cell stimulation cocktail plus protein transport inhibitor (500×) to each tube and mix well by pipetting the solution.
Distribute 200 μL of the solution per well within a flat bottom 96-well plate.
Note: The goal is to culture 10 wells with 1 million cells per well in cell stimulation cocktail and T-cell media. The volumes noted above are calculated for a starting cell total of 10 million; however, these can be modified depending on the number of cells needed for the cytokine stains.
Incubate the plate at 37 °C for 3 h.
Note: During this incubation period, the surface stains and nuclear stains can be completed.
After 3 h, collect the cells for each sample by pooling the wells together.
Centrifuge at 500× g for 5 min at 4 °C, remove the supernatant, and resuspend in 1 mL of RPMI.
Split the sample evenly between the tubes for the cytoplasmic stains and isotype controls.
Note: These samples are now ready to be surface stained and intracellularly stained as described below.
Surface staining
Wash the tubes with 400 μL of FACS buffer and centrifuge at 500× g for 5 min at 4 °C.
Remove the supernatant and resuspend in 50 μL of FACS buffer with Fc block for approximately 10–15 min.
Note: During this incubation, the master mixes for the surface stains can be prepared as detailed below.
Add 50 μL of the master mix consisting of FACS buffer and the appropriate antibodies for each stain, as noted below.
Granulocyte: CD11c (FITC), SiglecF (PE), CD45.2 (PerCP), GR-1 (APC)
ILC: Lineage (FITC), CD45.2 (PerCP), Thy1.2 (APC)
Lineage consists of lineage cocktail, CD11c, NK1.1, CD5, FceR1a, TCRβ, and TCRγd
Cover samples in foil and keep at 4 °C for 30 min.
Note: While staining, compensation beads can be prepared for each fluorochrome used, by staining with 100 μL of FACS buffer, 25 μL of compensation beads, and 1 μL of an antibody in the selected fluorochrome for 15–30 min.
Wash samples with 400 μL of FACS buffer and centrifuge at 500× g for 5 min at 4 °C.
Remove the supernatant and resuspend the samples with the surface stain only in 150 μL of FACS buffer. Surface-stained samples can now be run on the Novocyte.
Notes:
Samples that need to be intracellularly stained (nuclear or cytoplasmic) will be permeabilized and stained as noted below.
Important! If samples will be run the next day, the cells should be fixed in 100 μL of 4% PFA for 15 min. Then, they can be washed and resuspended in 150 μL of FACS buffer.
Nuclear staining
Prepare the working solution of the FOXP3 permeabilization kit by combining one part concentrate and three parts diluent.
Add 300 μL of the working solution to each sample and place in the dark at room temperature for 30 min.
Note: Important! Mix the samples well with the working solution.
Wash the samples with 400 μL of FOXP3 permeability buffer (1×). Centrifuge at 500× g for 5 min at 4 °C and remove the supernatant.
Stain the samples with 100 μL of FOXP3 permeability buffer (1×) and the nuclear antibody (i.e., Ki-67, Gata-3, or other transcription factors of interest). Cover samples in foil and keep at 4 °C for 30 min.
Wash the samples with 400 μL of FOXP3 permeability buffer (1×). Centrifuge at 500× g for 5 min at 4 °C and remove the supernatant. Resuspend in 150 μL of FACS buffer. Nuclear-stained samples can now be run on the Novocyte.
Cytoplasmic staining
Add 300 μL of the BD Cytofix/Cytoperm working solution to each sample and place in the dark at 4 °C for 20 min.
Wash samples with 400 μL of BD permeability buffer and centrifuge at 500× g for 5 min at 4 °C.
Remove the supernatant and resuspend in 100 μL of BD permeability buffer and the cytoplasmic antibody (i.e., IL-5 and IL-13). Cover samples in foil and keep at 4 °C for 30 min.
Wash the samples with 400 μL of permeability buffer. Centrifuge at 500× g for 5 min at 4 °C and remove the supernatant. Resuspend in 150 μL of FACS buffer. Cytoplasmic-stained samples can now be run on the Novocyte.
Data analysis
Analysis of FCS files was completed with FlowJo. All populations were gated off of the live CD45+ population. Live cells were determined based on size (FSC vs. SSC). Eosinophils were identified as CD11c−SiglecF+ (Figure 2A). Neutrophils were identified as SiglecF−Gr-1+. ILCs were identified as Lineage−Thy1.2+ lymphocytes (Figure 2B). Transcription factor expression and cytokine production within the ILC population was gated relative to an isotype or FMO control (Figure 3).
Figure 2. Alternaria challenge results in increased eosinophils and innate lymphoid cells (ILCs). Alternaria-challenged mice were challenged with 25 μg of Alternaria for three days (days 0, 1, and 2) and were sacrificed on day 3. (A) Gating scheme for the identification of eosinophils within lung tissue of naïve and Alternaria-challenged mice. Eosinophils were identified as CD11c−SiglecF+. (B) ILC gating scheme within naïve and Alternaria-challenged mice. ILCs were identified as Lineage−Thy1.2+ lymphocytes. The lineage cocktail consists of markers for granulocytes, mast cells, B cells, T cells, macrophages, and NK cells, which are all excluded when gating for the lineage-negative population. Data representative of four mice per group.
Figure 3. Alternaria challenge results in an increased innate lymphoid cells (ILC) population with greater proliferation and Th2 cytokine production. Alternaria-challenged mice were challenged with 25 μg of Alternaria for three days (days 0, 1, and 2) and were sacrificed on day 3. (A) Representative FACS plots of Ki-67 expression within ILCs in naïve and Alternaria-challenged mice compared to FMO control. (B) Representative FACS plots of Gata-3 expression within ILCs in naïve and Alternaria-challenged mice compared to isotype control. (C, D) Representative FACS plots of IL-5 and IL-13 levels within ILCs in naïve and Alternaria-challenged mice compared to isotype controls. Data representative of four mice per group.
Recipes
FACS buffer
Note: Stored at 4 °C; use within six months of preparation.
1× PBS
2% FBS
0.02% sodium azide
T-cell media
Note: Stored at 4 °C; use within four weeks of preparation.
RPMI
10% FBS
1% Penicillin/streptavidin
1% glutamine
0.05 mM β-Mercaptoethanol
Acknowledgments
T.A.D. supported by NIH AI171795 and Veterans Affairs BLR&D BX005073. Graphical overview was created with BioRender.com.
Competing interests
There are no conflicts of interest or competing interests.
Ethical considerations
All studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee.
References
Artis, D. and Spits, H. (2015). The biology of innate lymphoid cells. Nature 517(7534): 293-301.
Drake, L.Y. and Kita, H. (2014). Group 2 Innate Lymphoid Cells in the Lung. Adv Immunol 124: 1-16.
Gasteiger, G., Fan, X., Dikiy, S., Lee, S. Y., Rudensky, A. Y. (2015). Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs. Science 350(6263): 981-985.
Ikutani, M., Tsuneyama, K., Kawaguchi, M., Fukuoka, J., Kudo, F., Nakae, S., Arita, M., Nagai, Y., Takaki, S. and Takatsu, K. (2017). Prolonged activation of IL-5-producing ILC2 causes pulmonary arterial hypertrophy. JCI Insight 2(7): e90721.
Lai, D. M., Shu, Q. and Fan, J. (2016). The origin and role of innate lymphoid cells in the lung. Mil Med Res 3: 25.
Monticelli, L. A., Sonnenberg, G. F., Abt, M. C., Alenghat, T., Ziegler, C. G., Doering, T. A., Angelosanto, J. M., Laidlaw, B. J., Yang, C. Y., Sathaliyawala, T., et al. (2011). Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol 12(11): 1045-1054.
Pelly, V. S., Kannan, Y., Coomes, S. M., Entwistle, L. J., Rückerl, D., Seddon, B., MacDonald, A. S., McKenzie, A. and Wilson, M. S. (2016). IL-4-producing ILC2s are required for the differentiation of TH2 cells following Heligmosomoides polygyrus infection. Mucosal Immunol 9(6): 1407-1417.
Puttur, F., Denney, L., Gregory, L. G., Vuononvirta, J., Oliver, R., Entwistle, L. J., Walker, S. A., Headley, M. B., McGhee, E. J., Pease, J. E., et al. (2019). Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans. Sci Immunol 4(36): eaav7638.
Rossi, G. A., Ballarini, S., Salvati, P., Sacco, O. and Colin, A. A. (2022). Alarmins and innate lymphoid cells 2 activation: A common pathogenetic link connecting respiratory syncytial virus bronchiolitis and later wheezing/asthma? Pediatr Allergy Immunol 33(6): e13803.
Varela, F., Symowski, C., Pollock, J., Wirtz, S. and Voehringer, D. (2022). IL-4/IL-13-producing ILC2s are required for timely control of intestinal helminth infection in mice. Eur J Immunol 52(12): 1925-1933.
Vivier, E., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J. P., Eberl, G., Koyasu, S., Locksley, R. M., McKenzie, A. N. J., Mebius, R. E., et al. (2018). Innate Lymphoid Cells: 10 Years On. Cell 174(5): 1054-1066.
Wirtz, S., Schulz-Kuhnt, A., Neurath, M. F. and Atreya, I. (2021). Functional Contribution and Targeted Migration of Group-2 Innate Lymphoid Cells in Inflammatory Lung Diseases: Being at the Right Place at the Right Time. Front Immunol 12: 688879.
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Immunology > Immune cell staining > Immunodetection
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Chromatin-RNA in situ Reverse Transcription Sequencing (CRIST-seq) Approach to Profile the Non-coding RNA Interaction Network
SZ Shilin Zhang
XW Xue Wen
LZ Lei Zhou
HL Hui Li
WL Wei Li
AH Andrew R. Hoffman
JH Ji-Fan Hu
JC Jiuwei Cui
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4718 Views: 721
Reviewed by: Gal HaimovichRohini NairMarion Hogg
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Cited by
Original Research Article:
The authors used this protocol in The Journal of Cell Biology Feb 2022
Abstract
Non-coding RNAs (ncRNAs) are defined as RNAs that do not encode proteins, but many ncRNAs do have the ability to regulate gene expression. These ncRNAs play a critical role in the epigenetic regulation of various physiological and pathological processes through diverse biochemical mechanisms. However, the existing screening methods to identify regulatory ncRNAs only focus on whole-cell expression levels and do not capture every ncRNA that targets certain genes. We describe a new method, chromatin-RNA in situ reverse transcription sequencing (CRIST-seq), that can identify all the ncRNAs that are associated with the regulation of any given gene. In this article, we targeted the ncRNAs that are associated with pluripotent gene Sox2, allowing us to catalog the ncRNA regulation network of pluripotency maintenance. This methodology is universally applicable for the study of epigenetic regulation of any genes by making simple changes on the CRISPR-dCas9 gRNAs.
Key features
• This method provides a new technique for screening ncRNAs and establishing chromatin interaction networks.
• The target gene for this method can be any gene of interest and any site in the entire genome.
• This method can be further extended to detect RNAs, DNAs, and proteins that interact with target genes.
Graphical overview
Keywords: CRIST-seq Noncoding RNA RNA-seq Regulatory element RNA–DNA interaction Epigenetics
Background
Non-coding RNAs (ncRNA), which include long non-coding RNA (lncRNA), small nuclear RNA, small nucleolar RNA, micro RNA, and other unknown RNA, play a critical role in the regulation of gene expression through multiple molecular mechanisms (Huang et al., 2016; Sridhar et al., 2017). RNA chips were initially used to identify RNAs; later, single-cell RNA sequencing expanded the ability to catalog the RNAs transcribed in a cell (Sajti et al., 2020). However, the existing RNA screening methods only focus on whole-cell RNA expression (Liang et al., 2019, Nicot, 2020) and are not sensitive enough to capture every known (or heretofore unknown) ncRNA that targets an individual gene.
To profile the ncRNA regulatory network at a specific gene locus, we developed a chromatin RNA in situ reverse transcription-associated sequencing (CRIST-seq) assay (S. Zhang et al., 2019). CRIST-seq combines the simplicity of intranuclear in situ reverse transcription–associated biotin labeling of ncRNA molecules with the specificity of catalytically inactive CRISPR dCas9 gene targeting. The assay includes 1) targeting a given gene using lentiviral dCas9-gRNAs, 2) nuclear in situ labeling of ncRNAs by reverse transcription with biotin-dCTP, 3) pulldown of chromatin-associated cDNAs by Cas9-Flag immunoprecipitation, 4) isolation of the target gene–associated cDNAs with streptavidin beads, and 5) Illumina cDNA library sequencing. We have used this approach to identify several novel ncRNAs that regulate the pluripotency of stem cells through various molecular mechanisms, including Platr10 (Du et al., 2021), Oplr16 (Oct4 promoter-interacting long noncoding RNA) (Jia et al., 2020), Peblr20 (Pou5F1 enhancer binding lncRNA 20) (C. Wang et al., 2020), Osblr8 (Oct4-Sox2 binding long noncoding RNA 8) (Zhu et al., 2020), Osilr9 (Oct4-Sox2 interacting lncRNA 9) (Zhu et al., 2023), and Peln1 (pluripotency exit lncRNA 1) (Y. Wang et al., 2022).
In this paper, we have used the Sox2 gene, a critical stem cell factor required for pluripotent reprogramming, as an example to study and verify the feasibility of this method. This CRIST-seq approach may be broadly used to map ncRNA interaction networks at target loci anywhere in the genome by creating gene-specific gRNAs. CRIST-seq can be used to identify new ncRNAs and to study how these regulate gene expression in normal, stem, and cancer cells.
Materials and reagents
Consumables and reagents
6-well cell culture plates (Corning, catalog number: 3516)
100 mm cell culture dishes (BD Falcon, catalog number: 353003)
100 mm Petri dishes (BD Falcon, catalog number: 351005)
1.5 mL microcentrifuge tubes (Pierce, catalog number: 69715)
0.2 mL 8-tube stripes (Bio-Rad, catalog number: TBC0803)
15 mL centrifuge tubes (Labselect, catalog number: CT-012-15)
DH5α competent E. coli (NEB, catalog number: ST10018)
Chloroform (Fisher Chemical, catalog number: C298-500)
Isopropanol (Thermo Scientific, catalog number: AC610080040)
Ethanol (Thermo Scientific, catalog number: 10617864)
Phenol/Chloroform/Isoamyl Alcohol (25:24:1) (Fisher Bioreagents, catalog number: BP1752I-100)
Glycogen (5 mg/mL) (Invitrogen, catalog number: AM9501)
RNase inhibitor (Invitrogen, catalog number: AM2694)
Random hexamers (Invitrogen, catalog number: N8080127)
dNTP mix (2.5 mM) (Invitrogen, catalog number: R72501)
Hybrid-Q Plasmid Rapidprep (GeneAll, catalog number: 100-102)
FastDigest Restriction endonucleases Age I (BshT I), Not I, and DpnI (Thermo Scientific, catalog numbers: FD1464, FD0596, and FD1703)
10× FastDigest buffer (Thermo Scientific, catalog number: B64)
T4 DNA ligase (5 U/μL) (Thermo Scientific, catalog number: EL0011)
PEG-it virus precipitation solution (System Biosciences, catalog number: LV825A-1)
PEI (Polysciences, catalog number: 24765-1)
Polybrene (Sigma, catalog number: TR-1003)
Formaldehyde, 37% by weight (Fisher Chemical, catalog number: F79-500)
2 M glycine (Sigma, catalog number: G8898)
NP-40 (Thermo Scientific, catalog number: 85124)
HEPES (Sigma, catalog number: H3375)
0.5 M EDTA (Thermo Scientific, catalog number: R1021)
10% SDS (Sigma, catalog number: L3771)
Halt protease inhibitor cocktail (100×) (Thermo Scientific, catalog number: 87785)
PMSF protease inhibitor (Thermo Scientific, catalog number: 36978)
Triton X-100 (Thermo Scientific, catalog number: A16046AE)
Tween 20 (Thermo Scientific, catalog number: J20605AP)
Proteinase K (Invitrogen, catalog number: 25530049)
Dynabeads M-280 streptavidin (Invitrogen, catalog number: 60210)
Formamide (Sigma, catalog number: F9037)
Biotin-14-dCTP (Invitrogen, catalog number: 19518018)
Anti-Flag antibody (Sigma, catalog number: F1804)
Anti-IgG antibody (Abcam, catalog number: ab171870)
PureProteome Protein A/G mix magnetic beads (Millipore, catalog number: LSKMAGAG10)
RNase A (20 mg/mL) (Invitrogen, catalog number: 12091021)
AMPure XP beads (Beckman Coulter, catalog number: A63881)
NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (NEB, catalog number: E6240)
NEBNext Multiplex Oligos for Illumina Index Primers Set 1 & Set 2 (NEB, catalog number: E7335 and E7500)
Maxima reverse transcriptase (200 U/μL) (Thermo Scientific, catalog number: EP0743)
NEBNext mRNA Second Strand Synthesis Module (NEB, catalog number: E6111)
1 M TRIS-HCl pH 8.1 (Biyuntian, catalog number: ST781)
1 M TRIS-HCl pH 7.5 (Biyuntian, catalog number: ST775)
1 M TRIS-HCl pH 8.5 (Biyuntian, catalog number: ST785)
KCl (Thermo Scientific, catalog number: AC418200025)
NaCl (Fisher Scientific, catalog number: 15915)
MgCl2 (Thermo Scientific, catalog number: FERR0971)
Plasmids
pMD2.G (Addgene plasmid, catalog number: 12259)
psPAX2 (Addgene plasmid, catalog number: 12260)
P-GreenPuro (SBI plasmid, catalog number: SI505A-1)
Lenti dCas9-mSox2-gRNA #1-2-puro (constructed in our lab, see Procedure)
Cell culture and cell culture reagents
HEK293T cells (ATCC, catalog number: CRL-3219), stored in liquid nitrogen
Mouse FIB cells (muscle-derived fibroblasts), cultured from a 129 mouse fetus (Zhai et al., 2015) and stored in liquid nitrogen
Mouse induced pluripotent stem cells (iPSCs) were reprogrammed by Pou5f1-Sox2-Klf4-Myc (OSKM) cocktail factors in our lab using FIB cells (M. Chen et al., 2012; H. Zhang et al., 2013; Zhai et al., 2015; X. Chen et al., 2016), and stored in liquid nitrogen
KnockOut DMEM (Gibco, catalog number: 10829018)
KnockOut SR (Gibco, catalog number: 10828010)
DMEM (Gibco, catalog number: 11995-065)
FBS (Gibco, catalog number: A31605)
MEM-NEAA (Gibco, catalog number: 11140050)
Glutamine (Sigma, catalog number: G7513)
Penicillin-streptomycin (Sigma, catalog number: P4458)
β-mercaptoethanol (Sigma, catalog number: M3148)
ESGRO Mouse LIF (Millipore, catalog number: ESG1107)
Puromycin (Invivogen, catalog number: ant-pr-1)
Mitomycin C solution (1 mg/mL) (Nacalai Tesque, catalog number: 20898-21)
2% gelatin solution (Sigma, catalog number: G1393)
PBS (Gibco, catalog number: 10010023)
Trypsin-EDTA (0.05%), phenol red (Gibco, catalog number: 25300054)
Opti-MEM (Gibco, catalog number: 51985034)
Knockout growth media (see Recipes)
Normal growth media (see Recipes)
Primers
See Table 1
Table 1. List of primers or products
Primer/Product 5′→3′ sequence
pSox2-gRNA1 GGGGTTGAGGACACGTGCTG
pSox2-gRNA2 GAGCCAATATTCCGTAGCAT
gCT1 GTTCCCTGCAAGAGTGCCCA
gCT2 GCACTACCAGAGCTAACTCA
JH3915 TAGTAATGAGTTTAAACAAGGTCGGGCAGGAAGAGGGCCT
JH3986 CCAGCACGTGTCCTCAACCCCGGTGTTTCGTCCTTTCCACAAG
JH3987 GGGGTTGAGGACACGTGCTGGTTTTAGAGCTAGAAATAGCAAGTT
JH3988 ACATGCTACGGAATATTGGCTCGGATCCAAGGTGTCTCATACAG
JH3989 GAGCCAATATTCCGTAGCATGTTTTAGAGCTAGAAATAGCAAGTT
J441 CAACTTCTCGGGGACTGTGGGCGAT
pSox2-F GAGCCAATATTCCGTAGCATG
pSox2-R CGCTGGGGAACCTTTGTATC
5′CT-F GAGCCAATATTCCGTAGCATG
5′CT-R CGCTGGGGAACCTTTGTATC
Off-target-F AGCCATCCTGTCCTCCGCCTG
Off-target-R CTGCACGGAAGGTCACGATG
Solutions
Hypotonic buffer (see Recipes)
Sonication buffer (see Recipes)
ChIP dilution buffer (see Recipes)
Protein A/G beads binding & washing buffer (see Recipes)
Protein A/G beads elution buffer (see Recipes)
2× M-280 beads binding & washing buffer (see Recipes)
M-280 beads elution buffer (see Recipes)
Recipes
Knockout growth media
KnockOut DMEM supplemented with 15% KnockOut SR
1% MEM-NEAA
2 mM glutamine
1% penicillin-streptomycin
200 μM β-mercaptoethanol
ESGRO Mouse LIF
Normal growth media
DMEM supplemented with 10% FBS and 1% penicillin-streptomycin
Hypotonic buffer
10 mM HEPES
1.5 mM MgCl2
10 mM KCl
0.4% NP-40 (add before use)
Sonication buffer
50 mM Tris-HCl (pH 7.5)
5 mM EDTA
0.5% SDS
1× Halt protease inhibitor cocktail (add before use)
0.5 mM PMSF protease inhibitor (add before use)
ChIP dilution buffer
0.01% SDS
1.1% Triton X-100
1.2 mM EDTA
16.7 mM Tris-HCl (pH 8.1)
167 mM NaCl
1× Halt protease inhibitor cocktail (add before use)
Protein A/G beads binding & washing buffer
0.05% Tween 20
PBS, pH 7.4
Protein A/G beads elution buffer
0.2 M Glycine-HCl, pH 2.5
2× M-280 beads binding & washing buffer
10 mM Tris-HCl (pH 7.5)
1 mM EDTA
2 M NaCl
M-280 beads elution buffer
10 mM EDTA (pH 8.2)
95% formamide
Equipment
Centrifuge (Eppendorf, model: 5415D)
Vibra-Cell Ultrasonic liquid processors (Sonics, model: VCX-130)
Thermomixer (Eppendorf, catalog number: vwrCA21516-176)
DNA spectrophotometer (NanoDrop, model: ND-1000)
Laboratory inverted microscope (Carl Zeiss, model: Axiovert 40 CFL)
pH meter (Mettler Toledo, model: SevenEasy S20)
PCR instrument (Eppendorf, model: Mastercycler pro PCR System)
Electrophoresis power supply (Pharmacia Biotech, model: EPS 300)
Labquake shaker (Lab Industries, catalog number: 400-110)
Gel documentation system (Axygen, model: GD-1000)
Software
FASTX (Cold Spring Harbor Laboratory, http://hannonlab.cshl.edu/fastx_toolkit/)
TopHat (Center for Computational Biology at Johns Hopkins University, http://tophat.cbcb.umd.edu)
Cufflinks (Cole Trapnell’s lab at the University of Washington, http://cole-trapnell-lab.github.io/cufflinks/)
UCSC Genome Browser (UCSC Genomics Institute, https://genome.ucsc.edu)
RIPSeeker software (University of Toronto, http://www.bioconductor.org/packages/2.12/bioc/html/RIPSeeker.html)
Procedure
Cell culture
HEK293T culture
Grow HEK293T cells (2 × 106) in a 6-well plate and culture in normal growth media (see Recipe 2) at 37 °C and 5% CO2. Use 293T cells to prepare lentivirus.
FIB culture
Grow FIB cells (1 × 107) in a 100 mm plate and culture in normal growth media (see Recipe 2) at 37 °C and 5% CO2. Use FIB cells as control cells of iPSC.
iPSC culture
Note: Feeder cells can enhance the growth and reproduction of iPSCs, maintain pluripotency, and inhibit differentiation. In this assay, the feeding layer is made with FIB cells. After being treated with mitomycin, FIBs lose their mitotic ability and will die during digestion and inoculation, which has no impact on subsequent experiments.
Coat a 100 mm plate with 2 mL of 0.1% gelatin and place in an incubator at 37 °C for 2 h.
Wash the plate twice with PBS and discard PBS before use.
Seed FIB cells (served as feeder layer) on the gelatin-coated plate and grow to ~70% confluency.
Add mitomycin C (final concentration of 10 μg/mL) and incubate for 2 h to block mitosis.
Wash the plate twice with PBS.
Grow iPSCs (1 × 106) on feeder layer and culture in knockout growth media (see Recipe 1) at 37 °C and 5% CO2.
Lenti-dCas9 construction (Figure 1A)
Design gRNAs using Broad Institute website (https://portals.broadinstitute.org/gppx/crispick/public).
Synthesize pU6-gRNA1-pH1-gRNA2 cassette using JH3915-JH3986, JH3987-JH3988, and JH3989-J441 primers (1 cycle at 95 °C for 5 min, 32 cycles at 95 °C for 20 s, 62 °C for 30 s, and 72 °C for 15 s, and 1 cycle at 72 °C for 10 min).
Use Age I and Not I restriction enzymes (37 for 30 min) and T4 DNA ligase (22 for 10 min) to insert Sox2 gRNAs or gCTs downstream from U6 promotor in the lenti-dCas9 vector.
Use DH5α competent cells for transformation.
Confirm the correct cloning by sequencing and extract the plasmid with GeneAll Hybrid-Q Plasmid Rapidprep.
Figure 1. Plasmid maps and sample preliminary detection. (A) Map of lenti-dCas9 plasmid. Sox2-gRNA1 and Sox2-gRNA2 were inserted into the vector using Age I and Not I. (B) Preliminary detection of gRNA specific binding sites in chromatin-RNA in situ reverse transcription (CRIST) samples by PCR (using pSox2-F and pSox2-R primers). The sequence of gRNA specific binding sites cannot be detected in anti-IgG sample but can be detected in anti-Flag sample and input control, which demonstrates the specificity of the method. The anti-IgG here served as background control. (C) Sample specific testing was performed by real-time PCR using specific primers derived from the pSox2 targeting site, 5′-Ct control site, and off-target site. pSox2: targeting site in the Sox2 promoter where the Cas9 gRNAs are designed; 5′-Ct: fragment that is 14.6 kb away from the pSox2 target site and is used as the control site. Cas9 vector: cells that were treated with the Cas9 control vector that lacks the gRNAs; Cas9-gRNA: cells that were targeted by both Cas9 and Sox2 gRNAs; Cas9-gCT: cells that were treated with the random control gRNA vector. Off-target: CRIST control site that is 33.8 kb upstream of the housekeeping gene GAPDH. All data shown are mean ± SEM from three independent experiments by normalization over the IgG control. (∗∗) P < 0.01 as compared with the Cas9 Vector and Cas9-gCT controls.
Lentiviral packaging and transfection
Grow 293T cells to ~70% confluency for lentiviral packaging.
Change the normal growth media to Opti-MEM 1h before packaging.
Put 5 μg of PEI and 100 μL of Opti-MEM into a tube. Add 0.8 μg of psPAX2, 0.4 μg of PMD2.G, and 0.8 μg of lenti-dCas9 (carrying pSox2-gRNAs or gCTs).
Mix and let stand for 20 min; then, add them to the culture media of 293T cells.
Change to normal growth media (see Recipe 2) 6 h later.
Collect virus supernatants at 24, 48, and 72 h, add PEG-it to the collected medium, incubate overnight at 4 °C, and spin at 1,500× g for 30 min.
Discard the supernatant and resuspend viral particles in 100 μL of PBS.
Grow iPSCs and fibroblasts (FIB) in 100 mm plates to ~50% confluency.
Mix 20 μL of lentivirus solution with polybrene (final concentration of 5–10 ng/μL) and add it to culture medium.
Change medium after 24 h and use puromycin (0.5–2 μg/mL) to select puromycin-resistant cells.
Intranuclear RNA reverse transcription
Note: In order to protect unstable RNAs, we performed intranuclear in situ reverse transcription prior to the subsequent steps and labeled RNAs with biotin-dCTP during reverse transcription.
Collect iPSCs and fibroblasts (~1 × 107), resuspend with 1 mL of PBS, and add 54 μL of 37% formaldehyde (final concentration of 2%) for 10 min at room temperature. Quench the formaldehyde with 60 μL of 2 M glycine (with a final concentration of 0.125 M) and incubate for 5 min at room temperature.
Wash both iPSCs and fibroblasts with PBS twice, resuspend with 800 μL of hypotonic buffer (see Recipe 3), and incubate for 5 min on ice to lyse the cell membranes.
Centrifuge samples for 10 min at 1,500× g and wash twice with hypotonic buffer (without NP-40).
Conduct intranuclear RNA reverse transcription at 65 °C for 30 min in a 20 μL reaction with biotin-dCTP (1 μL of 50 μM random hexamer, 1 μL of 10 mM dNTP, 1 μL of 0.4 mM biotin-dCTP, 1 μL of Maxima reverse transcriptase, 0.5 μL of RNase inhibitors, 1 μL of 0.1 M DTT, 4 μL of 5× cDNA synthesis buffer, and RNase-free water to 20 μL). Use 4 μL of 0.5 M EDTA to stop the reaction.
Centrifuge samples at 1,600× g for 10 min at 4 °C. Wash twice with ice-cold PBS.
Ultrasonic disintegration
Resuspend cell pellets with 300 μL of sonication buffer (see Recipe 4).
Note: Using too much liquid may lead to splashing, and using too little liquid may produce foam.
Clamp the sample tubes and the ultrasonic probe together and place them on ice.
Put the 2 mm ultrasonic probe 1 cm below the liquid level.
Note: In order to avoid foaming, the probe should always be kept below the liquid level
Shear DNAs into segments of 200–1,000 base pairs for 15 min (10 s on and 20 s off) at an amplitude of 40%.
Divide 300 μL of sonicated DNAs into three portions (149, 149, and 2 μL). Use the two equal portions for immunoprecipitation (using anti-Flag and anti-IgG antibody respectively) and the 2 μL portion for input control.
Immunoprecipitation of biotin-cDNA and dCas9
To reduce non-specific background, use Protein A/G mix magnetic beads (5 μL/ChIP) to pre-clear the sonicated samples.
Put 5 μL of protein A/G beads in a new tube and wash twice with beads binding & washing buffer (see Recipe 6).
Dilute sonicated samples (10-fold) with ChIP dilution buffer (see Recipe 5), mix with 5 μL of washed protein A/G beads, and incubate at 4 °C with rotation for 30 min.
Discard protein A/G magnetic bead pellet in a magnetic rack and transfer sonicated samples to new tubes.
Add anti-Flag and anti-IgG antibodies (2–5 μg/ChIP) to experimental and control groups, respectively, and incubate overnight at 4 °C with rotation.
To enrich the immunoprecipitation samples, add 10 μL of pre-cleared protein A/G magnetic beads to samples and incubate at room temperature with rotation for 30 min. Wash the beads three times with Protein A/G Beads binding & washing buffer.
Discard the supernatant and elute the immunoprecipitation samples twice with 100 μL of elution buffer (see Recipe 7).
Streptavidin-biotin capture
Put M-280 streptavidin beads in a new tube and wash twice with beads binding & washing buffer (see Recipe 8).
Transfer 200 μL of eluent samples to the washed beads, add 50 μL of 5 M NaCl (final concentration of 1 M), and incubate for 30 min with rotation at room temperature.
Discard the supernatant, elute the samples with 100 μL of elution buffer (see Recipe 9) twice, and collect 200 μL of eluate.
De-crosslink chromatin complex
Transfer 200 μL of eluate into a new tube.
Neutralize the elution buffer with 20 μL of 1 M Tris-HCl (pH 8.5).
Incubate samples and the input at 70 °C for 60 min with 20 μL of 5 M NaCl, 10 μL of 0.5 M EDTA, 20 μL of 1 M Tris-HCl (pH 7.5), 2 μL of 10 mg/mL Proteinase K, 2 μL of RNase inhibitor, and 226 μL of RNase-free water using an Eppendorf thermomixer.
Note: This step can also be used to extract proteins for identification and analysis. This is an extended use of this method.
Purify cDNAs: Mix samples with equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and stand for 30 min at 4 °C. Centrifuge at 1,600× g for 10 min at 4 °C, collect supernatant, mix with equal volume of chloroform, add 2 μL glycogen, and stand for 30 min at 4 °C. Centrifuge at 1,600× g for 10 min at 4 °C, precipitate DNAs with 0.6× volumes of isopropanol for 30 min at 4 °C, and wash with 70% (v/v) ethanol.
Preliminary detection of CRIST samples
Note: In order to identify ncRNAs binding to the Sox2 gene promoter, we selected two sites from the Sox2 gene promoter to design gRNAs. Therefore, the ds-cDNAs obtained by the above method must contain the binding site of gRNAs on the Sox2 gene promoter, so that the ncRNA combined with it can be precipitated together, and the sample quality can be preliminarily determined as qualified and used for subsequent experiments.
Resuspend purified samples in 30 μL of ultrapure water and use pSox2 primers (Table 1) to detect the promotor binding site in anti-Flag and anti-IgG samples by PCR (1 cycle at 95 °C for 5 min, 28 cycles at 95 °C for 20 s, 60 °C for 15 s, and 72 °C for 15 s, and 1 cycle at 72 °C for 10 min), and use input DNA as positive control (Figure 1B). Cas9 enrichment signals were quantitated by real-time PCR using specific primers derived from the pSox2 targeting site, 5′-Ct control site, and off-target site (Figure 1C). After confirming the specificity of the Cas9 gRNA, the Cas9 Sox2-gRNA iPSCs were then used for CRIST-seq assay.
Synthesis of double-stranded cDNA
For long-term stability, the single-strand cDNAs should be converted into double-stranded cDNAs.
Synthesize ds-cDNAs at 16 °C for 2.5 h with 20 μL of purified samples, 2 μL of synthesis enzyme mix, 4 μL of 10× reaction buffer, and 14 μL of RNase-free water. Use NEBNext mRNA Second Strand Synthesis Module.
Purify DNAs again use phenol/chloroform/isoamyl alcohol (25:24:1) as previously described.
Construction of CRIST-seq library
To facilitate sequencing, use DpnI to cut cDNAs into fragments of approximately 300 bps: add 1 μL of Fastdigest DpnI and 3 μL of 10× FastDigest Buffer to 26 μL of purified cDNAs and incubate at 37 °C for 20 min.
Purify DNAs with phenol/chloroform/isoamyl alcohol (25:24:1) and resuspend in 22 μL of ultrapure water.
Follow the protocol of NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina to prepare samples for next-generation sequencing.
Note: Follow the protocol step by step from 1.1-1.8A.
Use Index primers in NEBNext Multiplex Oligos for Illumina Index Primers Set 1 & Set 2 to amplify samples and get the cDNA libraries (1 cycle at 98 °C for 30 min, 15 cycles at 98 °C for 10 s and 65 °C for 75 s, and 1 cycle at 65 °C for 5 min, then hold at 4 °C).
Sequence the cDNA library and use index primers as sequencing tags.
CRIST-seq data analyses (Figure 2)
Filter raw data and the low-quality data using FASTX software (v0.0.13) (Du et al., 2018).
Use TopHat software (version 2.0.9) to map clean reads to the mouse mm10 genome (Trapnell et al., 2009).
Quantitate the mapped reads as “fragments per kilobase of transcript per million fragments mapped” (FPKM) using Cufflinks (version 2.1.1) (Trapnell et al., 2010).
Call and annotate the peak using RIPSeeker software (Li et al., 2013) and adjust over the peaks overlapping with the IgG control enriched regions.
Normalize the CRIST-seq signal intensities over the nontargeting Cas9 gCT control using the DiffBind package (Ross-Innes, Stark et al. 2012) [fold change difference ≥ 2 and p-value < 0.05, with false discovery rate (FDR) < 0.1].
Figure 2. Sequencing analysis. (A) Analysis of ncRNA sequencing results. Preliminary screening for non-coding RNAs (ncRNAs) that are highly expressed in iPSC and much less expressed in FIB, which are related to pluripotency maintenance. Data regarding the RNAs that were changed greater than two-fold were combined with the CRIST-seq data using a VENN program. A cut-off threshold of peak enrichment FPKM > 50 was arbitrarily set to select CRIST-seq RNAs for VENN analysis. (B) Cross matching sequencing results with RNA-seq. (C) CRIST-seq identifies the top 42 Sox2 promoter-interacting RNAs. The Sox2 interacting RNAs are listed in order of the enrichment fold of the top 42 CRIST-seq data.
Acknowledgments
Funding Statement: This work was supported by the National Key R&D Program of China (2018YFA0106902, 2020YFA0707704), the Innovative Program of National Natural Science Foundation of China (82050003), National Natural Science Foundation of China (31871297, 82273191, 81874052, 32000431, 81900327, 82001670, 82101675, 82900701), Fund of Jilin Provincial Science and Technology Department (20210101311JC, 20190303146SF, 20200602032ZP, 20200201390JC), Provincial Science Fund of Jilin Province Development and Reform Commission (2021C10 and 2020C038-4), Natural Science Fund of Jilin Provincial Finance Department (JLSWSRCZX2020-023, JLSWSRCZX2020-100), the Youth Fund of Jilin Provincial Health Commission (2016Q035), Fund of Changchun City Science and Technology Bureau (21ZGY28), China Guanghua Fund and the Youth Fund of First Hospital of Jilin University (2020-CXM-01, JDYYGH2019004, JDYY 102019002, JDYY 102019043), California Institute of Regenerative Medicine (CIRM) grant (RT2-01942), and the Department of Veterans Affairs (BX002905).
This protocol is derived from the original research paper (Y. Wang et al., 2022; DOI: 10.1083/jcb.202009134).
Competing interests
The authors declare no conflict of interest.
References
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Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments
NI Nur Kusaira Khairul Ikram
AZ Ali Muhammad Zakariya
MS Mohd Zuwairi Saiman
AK Arman Beyraghdar Kashkooli
HS Henrik Toft Simonsen
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4719 Views: 615
Reviewed by: Alba BlesaNazrin Abd AzizKrishna Saharan
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Original Research Article:
The authors used this protocol in Frontiers in Bioengineering and Biotechnology Aug 2017
Abstract
The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua–derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.
Keywords: Physcomitrium patens Protoplast Protoplast transformation Artemisinin Metabolite analysis
Background
Artemisinin is a secondary metabolite produced by the plant Artemisia annua. It is a sesquiterpene lactone with a unique endoperoxide 1,2,4-trioxane ring. This natural compound has been demonstrated to possess a range of biological activities, of which the most known is as an antimalarial drug. However, its low accumulation in the native plant resulted in a high market price, which becomes a major hindrance in its commercial production. Chemical synthesis is difficult due to its complex chemical structure, hence being not economically feasible. Efforts have been made toward the utilization of microbial production systems for increased artemisinin yield, which resulted in a partial synthesis of the compound requiring additional chemical steps (Ro et al., 2006; Paddon and Keasling, 2014). Plant heterologous hosts such as tobacco have been explored, requiring extensive bioengineering (Ting et al., 2013; Malhotra et al., 2016; Wang et al., 2016), yet producing limited artemisinin (Ikram et al., 2015).
Physcomitrium patens is a plant-based production system with important applications in biotechnology research (Simonsen et al., 2009; Ikram et al., 2015). It is a non-vascular plant that has low metabolic and chemical diversity compared to the higher plants (Bach et al., 2014), due to the low number of cytochromes P450 and UDP glycosyltransferases present in its genome (Yonekura-Sakakibara and Hanada, 2011; Hamberger and Bak, 2013). This attribute confers the advantage of reduced risks in unspecific modification by endogenous enzymes and products via pathways in higher plants for the detoxification of xenobiotics (Bach et al., 2014). P. patens is characterized by a simple terpenoid profile with a genome that possesses only a single diterpene synthase (Chen et al., 2011). Its genome is fully sequenced, has a haploid life cycle, and has an efficient homologous recombination machinery similar to yeast, making it an attractive industrial production system compared to other plant heterologous hosts (Lang et al., 2018; Decker and Reski, 2020). In addition, King et al. (2016) developed an orthodox transformation technology that involved the in vivo arrangement of DNA fragments in P. patens, further giving credence to its application as a photosynthetic chassis for heterologous natural products.
To produce artemisinin in P. patens, five genes—namely amorpha-4,11-diene synthase (ADS), cytochrome P450 (CYP71AV1), alcohol dehydrogenase 1 (ADH1), double-bond reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1)—were introduced into the moss protoplast, being responsible for the biosynthesis of dihydroartemisinic acid that is subsequently converted into artemisinin by photooxidation (Figure 1). The five genes, consisting of three transformation sets, were transformed into P. patens using in vivo homologous recombination that allows multiple DNA fragments to be transformed at once into the genome (King et al., 2016; Khairul Ikram et al., 2017; Ikram et al., 2019). Here, we describe the techniques employed toward the stable production of artemisinin using P. patens as a heterologous host. P. patens culture preparation and maintenance, transformation technique, and metabolite analysis of artemisinin and intermediate compounds are provided. The graphical representation of the overall procedure is presented in Figure 2.
Figure 1. Artemisinin biosynthesis pathway occurs in the glandular trichomes of Artemisia annua. The pathway intermediates are defined as: FPP, farnesyl diphosphate; AD, amorpha-4,11-diene; AAOH, artemisinic alcohol; AAA, artemisinic aldehyde; AA, artemisinic acid; DHAAA, dihydroartemisinic aldehyde; DHAA, dihydroartemisinic acid. Figure adapted from Ikram et al. (2019).
Figure 2. Graphical representation of the protocols
Materials and reagents
Cultivation of Physcomitrium patens
Wildtype Physcomitrium patens (Gransden ecotype #40001) can be obtained from the International Moss Stock Centre at the University of Freiburg, Germany (https://www.moss-stock-center.org/).
Growth room or growth chamber with standard growth condition of a continuous light source with light intensities of 20–50 W/m2 and temperature of 25 °C.
Petri dishes, 90 mm
Serological pipette (Eppendorf)
Sterile pipette (5–50 mL)
Forceps
Note: Wrap forceps separately in foil and autoclave at 121 °C for 20 min.
Cellophane discs
Note: Interleave cellophane discs with filter paper, wrap them in foil paper, and autoclave at 121 °C for 20 min.
Falcon tubes, 50 mL (Thermo Fisher)
Distilled water
Aluminum foil
Erlenmeyer flasks, 50–250 mL
3M surgical tape, 1.25 cm (Micropore, 3M 1533-0)
Tube rack
Parafilm
Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670-500G)
Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: C4955-500G)
MgSO4·7H2O (Sigma-Aldrich, catalog number: 63138-1KG)
FeSO4·7H2O (Sigma-Aldrich, catalog number: 215422-250G)
(NH4)2C4H4O6 (Sigma-Aldrich, catalog number: 09985-1KG)
KH2PO4 (Sigma-Aldrich, catalog number: 229806-250G)
KOH (Sigma-Aldrich, catalog number: 60377-1KG)
CuSO4·5H2O (Sigma-Aldrich, catalog number: C3036-250G)
ZnSO4·7H2O (Sigma-Aldrich, catalog number: 221376-500G)
H3BO3 (Sigma-Aldrich, catalog number: B0394-500G)
MnCl2·4H2O (Sigma-Aldrich, catalog number: M3634-500G)
CoCl2·6H2O (Sigma-Aldrich, catalog number: 31277-100G)
KI (Sigma-Aldrich, catalog number: 221945-5G)
Na2MoO4·2H2O (Sigma-Aldrich, catalog number: M1003-100G)
Agar (Duchefa, catalog number: G1101-0500G)
MES (Sigma-Aldrich, catalog number: M3671)
PhyB medium (see Recipes)
1 M CaCl2 (see Recipes)
DNA preparation for Physcomitrium patens transformation
ADS (a gift from Professor Dae Kyun Ro, University of Calgary, Canada)
CYP71AV1 (DQ268763) (GenScript)
ADH1 (JF910157.1) (GenScript)
DBR2 (EU704257.1) (GenScript)
ALDH1 (FJ809784.1) (GenScript)
LP4/2A peptide linker (GenScript)
Ubiquitin promoter from Arabidopsis thaliana (CP002686.1)
Ubiquitin terminator from Arabidopsis thaliana (CP002686.1)
Maize Ubiquitin 1 promoter from the pMP1355 vector
G418 selection cassettes from the pMP1355 vector
Rice actin promoter from the pZAG1 vector
Hygromycin selection cassette from the pZAG1 vector
Microfuge tubes (1.5 mL)
Phusion® High Fidelity DNA Polymerase (New England Biolabs, catalog number: M0530L)
DpnI (New England Biolabs, catalog number: R0176S)
Primers (IDT, USA)
QIAquick PCR Purification kit (QIAGEN GmbH, catalog number: 28104)
Physcomitrium patens transformation
Falcon tubes, 15 mL (Thermo Fisher, catalog number: 14-959-53A)
Serological pipette (Eppendorf, catalog number: 0030127714)
Sterile pipette (5–50 mL)
Cell strainer (100 μm pore size mesh) (Sigma-Aldrich, catalog number: CLS352360)
P. patens culture (five days old)
CaCl2 (Sigma-Aldrich, catalog number: C5670-500G)
MgCl2·6H2O (Sigma-Aldrich, catalog number: M2670-500G)
Driselase® enzyme (Sigma-Aldrich, catalog number: D9515-1G)
D-Mannitol (Sigma-Aldrich, catalog number: M4125-100G)
Tris-Cl (Sigma-Aldrich, catalog number: 10812846001-500G)
Polyethylene glycol (PEG) (MW 6000) solution (Sigma-Aldrich, catalog number: 14504-1G-F)
8.5% D-Mannitol (see Recipes)
Driselase® enzyme solution (see Recipes)
Protoplast wash (PW) solution (see Recipes)
MMM solution (see Recipes)
MCT solution (see Recipes)
Polyethylene glycol (PEG) solution (see Recipes)
Protoplast regeneration medium (bottom layer; PRMB) (see Recipes)
Protoplast regeneration medium (top layer; PRMT) (see Recipes)
DNA isolation and PCR
Microfuge tubes (1.5 mL)
Mortar and pestle
Liquid nitrogen
10% SDS (w/v)
3 M sodium acetate (pH 5.2)
EDTA (Sigma-Aldrich, catalog number: E9884-100G)
Tris (Sigma-Aldrich, catalog number: 10708976001-1KG)
NaCl (Sigma-Aldrich, catalog number: S9888-500G)
Ice
Sea sand (Sigma-Aldrich, catalog number: 1.07711)
Isopropanol (Sigma-Aldrich, catalog number: 563935-1L)
Ethanol (Sigma-Aldrich, catalog number: 459836-2L)
Extraction buffer (see Recipes)
Metabolite analysis
Membrane filter (0.45 μm) (Sartorius, Minisart® RC4)
HP-5MS UI column (20.0 m × 0.18 mm × 0.18 μm) for GC-MS (Agilent)
BEHC18 column (100 mm × 2.1 mm × 1.7 μm) UPLC-MS (Waters)
Artemisinin (Dafra Pharma, Belgium)
Decane (Sigma-Aldrich, catalog number: D901-500ML)
Methanol (Sigma-Aldrich, catalog number: 34860-2.5L)
Acetonitrile (Sigma-Aldrich, catalog number: 34851-2.5L)
Formic acid (Sigma-Aldrich, catalog number: F0507-1ML)
Distilled water
Trans-caryophyllene (Sigma-Aldrich, catalog number: 22075-5ML)
Ethyl acetate (Sigma-Aldrich, catalog number: 270989-2L)
Citrate phosphate buffer (Sigma-Aldrich, catalog number: P4809-50TAB)
Viscozyme® (Sigma-Aldrich, catalog number: V2010-50ML)
Filter paper (Whatman® qualitative filter paper, Grade 1) (Sigma-Aldrich, catalog number: WHA1001325)
Separation funnel
75% (v/v) methanol:water
Acetonitrile:water with formic acid [1:1,000 (v/v)]
Dihydroartemisinin (Dafra Pharma, Belgium)
Artemisinic acid (Chiralix, Nijmegen)*
Artemisinic alcohol (Chiralix, Nijmegen)*
Dihydroartemisinic alcohol (Chiralix, Nijmegen)*
*Synthesized from dihydroartemisinic acid by Chiralix (Nijmegen)
Procedure
Cultivation of Physcomitrium patens (Bach et al., 2014)
Note: Cultivation of P. patens protonema tissue on solid and liquid media should be performed under sterile conditions according to the standard method. All materials should be sterilized by autoclaving prior to use. We recommend a dedicated sterile bench (laminar air flow bench or Biosafety cabinet) for plant tissue culture to reduce the risk of microbial contamination, since no antibiotics are supplemented in the culture media.
Cultivation on solid media
Pour 25–30 mL of PhyB media into a 90 mm Petri dish or until it reaches the half-way point of the Petri dish and leave to solidify for 5–10 min.
Overlay solidified media with sterilized cellophane discs and allow it to settle for 5–10 min.
Note: If the cellophane discs wrinkle when touching the agar inside the plate, use the forceps to straighten them out so the entire disc is in contact with agar. Avoid air bubbles.
Scrap protonema tissue from the previous culture using forceps and place it into a 50 mL Falcon tube containing 10 mL of sterilized distilled water.
Note: Be sure to sterilize the forceps using the glass beads sterilizer at intervals. Allow forceps to cool before applying.
Homogenize the tissue for 30 s using a homogenizer.
Note: Be careful not to over-blend the tissue; excessive blending will lead to poor regeneration of the moss tissues.
Clean the homogenizer tips by blending them into sterile distilled water in a Falcon tube.
Transfer 2 mL of homogenized tissue using a serological pipette into each Petri dish overlaid with cellophane discs.
Note: Ensure that the tissue is well distributed on the plates by swirling adequately.
Allow excess water to evaporate by leaving the Petri dish’s lid slightly open in the laminar air flow bench for 5–15 min.
Note: Do not over dry the plates. The cellophane will curl, and the moss will not survive.
Seal the plates with 3M surgical tape and incubate in a growth chamber under standard conditions. For longer storage, use plastic film for sealing.
Note: Using 3 M surgical tape will give a high growth rate, but the media will dry quickly within 2–4 weeks. For longer storage (up to six months), seal the plates with a plastic kitchen cling wrap.
For routine subculture on solid media, repeat steps A1a–A1h.
Note: We recommend subculturing every two weeks.
Cultivation in liquid media
Add 2–5 mL of blended P. patens tissue into 20–100 mL of PhyB liquid media in a 250 mL Erlenmeyer flask.
Cover the flask with aluminum foil and seal using parafilm.
Place it on a rotary shaker at 90 rpm in a growth chamber under standard growth conditions (25 °C with continuous 20–50 W/m2 light intensity).
After every 1–2 weeks, harvest the tissues, homogenize them for 30 s using homogenizer, and inoculate in fresh PhyB liquid media.
Note: This will enhance growth rates and maintain the tissue in the haploid stage.
DNA preparation for Physcomitrium patens transformation
Note: P. patens has an efficient homologous recombinant machinery comparable to the yeast Saccharomyces cerevisiae. It can assemble multiple DNA fragments in one single transformation event. The five artemisinin genes are transformed into P. patens using the novel in vivo homologous recombination (King et al., 2016).
The synthetic genes CYP71AV1 (DQ268763), ADH1 (JF910157.1), DBR2 (EU704257.1), and ALDH1 (FJ809784.1) are codon-optimized according to the P. patens codon usage by GenScript, USA.
The genes were synthesized together with the LP4/2A peptide linker from Impatiens balsamina and foot-and-mouth disease virus (FMDV) (François et al., 2004).
Note: The synthetic artemisinin genes are linked with LP4/2A linker to facilitate the expression of several proteins under the control of a single promoter: CYP71AV1-LP4/2A-ADH1 and DBR2-LP4/2A-ALDH1. Furthermore, it minimizes the transformation event from five to three (1) ADS, (2) CYP71AV1-LP4/2A-ADH1, and (3) DBR2-LP4/2A-ALDH1).
The DNA fragment is amplified with short overlapping sequences by adding 25–50 base overhangs via PCR primers without pre-assembly of vectors in E. coli (King et al., 2016). The DNA fragments are amplified using Phusion® High Fidelity DNA Polymerase.
For PCR reactions using 1 μL of plasmid DNA as a template, the PCR product is digested with 1 μL of DpnI for 1 h at 37 °C, followed by inactivation at 65 °C for 20 min.
Note: PCR reaction using plasmid as a template is treated with DpnI to digest the methylated DNA template and select the newly synthesized DNA. The primer list is available in Ikram et al. (2017).
The PCR products are purified using QIAquick PCR Purification kit.
Physcomitrium patens transformation
The five artemisinin genes are transformed into P. patens via three independent transformation events (Figure 3). The first transformation cassette is ADS, under the control of maize Ubiquitin1 with a geneticin (G418) selection marker. The second transformation cassette is CYP71AV1-LP4/2A-ADH1, controlled by the rice actin promoter with hygromycin selection marker; the third transformation cassette is DBR2-LP4/2A-ALDH1 construct, controlled by the Arabidopsis Ubiquitin promoter and the G418 selection marker. The genome homologous overhang for the second transformation is targeted to remove the previously integrated G418 cassette, while the third transformation is targeted to remove the second selection cassette, hygromycin.
Note: The targeted integration in the P. patens nuclear genome utilizes 500 bases or longer homologous sequences between the genome and inserted DNA. We recommend using a minimum of 750 base pairs of homologous genomic sequences flanking the targeted insertion site.
Figure 3. Schematic representation of the different transformation sets into P. patens genome (1–3) using homologous recombination. Grey arrow: P. patens genomic locus (Pp108) homologous recombination flanking regions. Blue: artemisinin genes. Yellow: artemisinin genes linked with 2A/LP4 linker. Green: Promoters. Red: Terminators. Purple: G418/NPTII selection cassette. Orange: hygromycin selection cassette. The dotted lines point to the insertion between each transformation cassette and the P. patens genomic locus.
Protoplast isolation (Liu and Vidali, 2011; Bach et al., 2014)
Prepare a fresh PEG solution and let it sit for 2–3 h. Filter sterilize using a 0.22 μm syringe filter just before use.
Note: For transformation, all working solutions (PEG, Mannitol, Driselase, MMM, PW) need to be filter-sterilized using a 0.22 or 0.45 μM syringe filter just before use.
Pour PRMB into the Petri dishes and overlay them with a sterile cellophane disc.
Scrape 5-day-old protonema tissue from the agar plates overlaid with cellophane (approximately 1.5 g fresh weight) and add 1 mL of Driselase® enzyme solution for every 40 mg of P. patens tissue.
Note: We had inconsistent Driselase®enzyme quality from different batches. Hence, it is best to check the efficacy of the Driselase®upfront, or use our new method using Cellulase ONOZUKA R10 and Macerozyme R10 (Batth et al., 2021).
Incubate the tissue at room temperature in normal laboratory light conditions with occasional gentle shaking for 30–60 min.
After incubation, filter the tissue through a sterilized cell strainer (100 μm pore mesh filter) to collect the protoplast and separate it from the undigested tissues. Transfer the protoplast into a 15 mL tube.
Centrifuge the filtrate at 150–200× g for 4 min at 22 °C, with slow acceleration or breaking.
Note: Protoplast is highly sensitive to external force; hence, centrifugation needs to be slow with gentle breaking.
Slowly remove the supernatant with a serological pipette. Be careful not to disturb the protoplast pellet.
Resuspend the protoplast pellet in PW solution using the same volume of Driselase solution that was used in step C1c.
Repeat steps C1f–C1g.
Resuspend the protoplasts in 8.5% D-mannitol (i.e., half of the original volume) and estimate the protoplast density using a hemacytometer (Batth et al., 2021).
Note: The number of protoplasts is used to calculate the final protoplasts concentration in step C2b (Liu and Vidali, 2011; Batth et al., 2021).
Protoplast transformation
Centrifuge at 150–200× g for 4 min at 22 °C with slow breaking and remove the supernatant (see steps C1f and C1g).
Resuspend the protoplast in sterile MMM solution at a concentration of 1.6 × 106 protoplast/mL.
Add 10 μg of total DNA to the bottom of a 15 mL tube, followed by adding 300 μL of protoplast suspension from step C2b and 300 μL of sterilized PEG solution (see step C1a). Mix by gently flicking the tube.
Note: The multi-DNA fragment is added in an equimolar ratio. The total volume of DNA should be less than 30 μL for a successful transformation.
Incubate the mixture in the water bath for 5 min at 45 °C and another 5 min at room temperature.
Dilute protoplast suspension with 300 μL of 8.5% D-mannitol five times with 1 min waiting interval between each dilution.
Add 1 mL of 8.5% D-mannitol another five times with 1 min waiting interval between each dilution.
Pellet the transformed protoplast by centrifugation at 150–200× g for 4 min with slow breaking and remove the supernatant with a pipette.
Resuspend the protoplast in 500 μL of 8.5% D-mannitol, followed by 2.5 mL of protoplast regeneration media (PRMT), and mix by pipetting up and down.
Note: To keep the agar melted, placed the PRMT in a 45 °C water bath, cool enough to ensure protoplast survival.
Evenly dispense 1 mL of the mixture onto the Petri dishes containing protoplast regeneration media (bottom layer) overlaid with cellophane discs (see step C1b). Each transformation will result in three plates.
Seal the plates with 3M surgical tape and incubate them in the growth chamber under standard conditions for 5–7 days.
Selection and regeneration of P. patens transformants
After 5–7 days, transfer the cellophane with regenerating protoplast onto PhyB media containing appropriate selection marker and incubate for two weeks under standard conditions.
After two weeks on PhyB selective media, transfer the cellophane disc with recovered transformants onto a normal PhyB media without antibiotics for another two weeks.
Repeat steps C3a and C3b twice to obtain stable transformants. For characterization, verify transformants using PCR amplification of transgenes and metabolic profiling.
DNA isolation and PCR
Isolation of DNA from transformed P. patens is performed according to the modified method (King et al., 2016; Khairul Ikram et al., 2017).
Place 50–100 mg of fresh P. patens tissue together with a few grains of sea sand into 1.5 mL microfuge tubes and snap-freeze in liquid nitrogen.
Grind the frozen tissue using a microfuge tube pestle and add 400 μL of extraction buffer followed by 80 μL of 10% SDS.
Vortex the mixture and incubate at 65 °C for 30 min.
After incubation, add 180 μL of 3 M sodium acetate (pH 5.2) to the mixture and mix by pipetting up and down.
Centrifuge mixture at 1,500× g at room temperature for 10 min to pellet the debris.
Transfer supernatant to a new tube, add an equal volume of isopropanol, and mix by inversion.
Incubate mixture on ice for 20 min.
Pellet the DNA by centrifugation at 1,500× g for 30 min and discard the supernatant.
Wash the pellet with 80% (v/v) ethanol.
Air dry the sample and re-suspend in 50 μL of sterile water. Store at -20 °C for later use.
For PCR, use 1 μL of DNA as a template.
Metabolite analysis
Extraction of metabolites from P. patens cells
Snap-freeze one-week-old fresh P. patens tissue with liquid nitrogen and pulverize into fine powder using mortar and pestle.
Transfer 3,000 mg of the tissue powder into a 15 mL Falcon tube and add 3 mL of citrate phosphate buffer (pH 5.4).
Vortex and sonicate the mixture for 15 min.
Add 1 mL of Viscozyme® to the mixture and incubate at 37 °C overnight.
Extract the mixture by adding 3 mL of ethyl acetate into the mixture and mix by inverting the tube multiple times.
Centrifuge the mixture at 2,000× g at room temperature for 10 min.
Transfer the extract (supernatant) to a new tube.
Repeat steps E1e–E1f three times and combine all extract into one tube.
Concentrate and dry the extract using nitrogen gas flow in the fume hood. Store at -20 °C until further analysis.
Before analysis, resuspend the dried extract in 300 μL of 75% v/v methanol:water and filter through a 0.45 μm membrane filter.
Extraction of metabolites from P. patens liquid culture
Harvest 500 mL of P. patens liquid culture by passing through a filter paper (Whatman® qualitative filter paper, Grade 1).
Extract the liquid culture with 200 mL of ethyl acetate in a separation funnel.
Concentrate ethyl acetate portion using a rotary evaporator to a volume of 1 mL.
Repeat steps E1f and E1g.
Quantification of Amorpha-4,11-diene in transformed P. patens
Amorpha-4,11-diene is the first product of amorphadiene synthase (ADS), the first gene in the artemisinin biosynthetic pathway. It is a volatile compound, and its analysis can be conducted using a HS-SPME (headspace solid phase microextraction) coupled to a GCMS (Shimadzu, QP2010 Plus) (Drew et al., 2011; Andersen et al., 2015). Quantification of amorpha-4,11-diene is performed according to the published protocol Rodriguez et al. (2014).
Blend one-week-old P. patens in sterile distilled water using a homogenizer.
Normalize the tissue concentration to 0.2 mg/mL (fresh weight).
Add 2 mL of the tissue homogenate into 20 mL of liquid PhyB media in a 100 mL Erlenmeyer flask.
Incubate on a shaker at 90 rpm under standard growth conditions for four days.
Add 2 mL of decane into the culture and allow for a two-week incubation on a shaker (90 rpm) under standard growth conditions.
After two weeks, harvest 100 μL of decane and dilute it twice with ethyl acetate spiked with trans-caryophyllene as internal standard.
Inject 1 μL of the extract in split mode and separate with a HP-5MS UI column (20.0 m × 0.18 mm × 0.18 μm). Hydrogen gas is used as the carrier gas, with injection temperature of 250 °C, oven temperatures of 60 °C (3 min), and 60–320 °C at 40 °C/min.
The concentration of amorpha 4,11-diene can be calculated based on the internal standard run in parallel [see Rodriguez et al. (2014)].
Quantification of artemisinin and intermediates using UPLC-MRM-MS
Artemisinin and artemisinin biosynthesis intermediates can be measured in a targeted approach using tandem quadrupole mass spectrometer that is equipped with an electrospray ionization source and coupled to an Acuity UPLC system (Ting et al., 2013).
We use a BEH C18 column (100 mm × 2.1 mm × 1.7 μm) for chromatographic separation by applying acetonitrile (solvent A) and water with formic acid [1:1,000 (v/v)] (Solvent B) in gradient system.
The gradient starts with solvent A, 5% (v/v) for 1.25 min, followed by a gradient increase to 50% in 2.35 min, and further increase to 90% (v/v) at 3.65 min.
Maintain for 0.75 min before returning to 5% (v/v) solvent A for 0.15 min gradient.
Use the same solvent system as in step E4b to equilibrate the column for 1.85 min at a flow rate of 0.5 mL/min while maintaining the column temperature at 50 °C.
UPLC-MRM-MS condition: set injection volume at 10 μL; desolvation and cone gas flow set to 1,000 and 50 L/h; desolvation and temperatures set to 650 and 150 °C, respectively; capillary voltage set to 3.0 kV; and mass spectrometer operated in positive ionization mode.
Use collision induced dissociation by Argon gas for fragmentation.
Artemisinin and compound intermediates are identified and quantified by multiple reaction monitoring (MRM) with optimized measurement settings for MRM channels (see Table 1).
Relative level of artemisinin and compound intermediates are measured using external calibration curves of reference standards.
Perform metabolite profiling twice and two months apart using the same original cell lines sub-cultivated on PhyB media.
Table 1. Optimized multiple reaction monitoring transition settings for UPLC-MRM-MS measurement of artemisinin, artemisinic acid, dihydroartemisinic acid, artemisinic alcohol, and dihydroartemisinic alcohol (Khairul Ikram et al., 2017)
Parent (m/z) Daughter (m/z) Cone voltage Collision voltage
Artemisinin 283.19 265.22
247.19
219.22
12
12
12
14
8
8
Artemisinic acid 235.16
217.21
199.25
189.22
18
18
18
14
16
10
Dihydroartemisinic acid 237.16
163.17
107.12
81.10
16
16
16
28
26
18
Artemisinic alcohol 221.16
203.27
147.09
14
14
20
10
Dihydroartemisinic alcohol 223.22
205.27
109.13
95.07
14
14
14
24
14
12
Data analysis
Validation of transformants
The first transformation event resulted in 11 transformants; all were positive transformants containing the first gene, ADS. The ADS product amorpha-4,11-diene was detected in the 11 lines, with an average content of 200 mg/L. The second transformation resulted in 47 transformants. Eleven out of the 47 were selected for genotyping and all 11 had the ADS and CYP71AV1-ADH1 genes. The final transformation event resulted in three transformants, all having the five genes, ADS, CYP71AV1-ADH1, and DBR2-ALDH1. Figure 4 shows the stable transformants of the three transformation events after two rounds of selection. Genotyping of the three independent transformants showed that the five genes in the biosynthesis of dihydroartemisinic acid were integrated into the genome [Khairul Ikram et al. (2017), refer to Figure S1 in Supplementary Material]. The PCR analysis showed there was no untargeted integration for the selected lines, indicating a uniform integration of the five genes with one copy of each.
Figure 4. Stable P. patens transformants of the three transformation events after two rounds of selection. (A) transformants with ADS gene; (B) transformants with ADS and CYP71AV1-ADH1 genes; (C) transformants with ADS, CYP71AV1-ADH1, and DBR2-ALDH1.
Metabolite profiling
Artemisinin was detected in the transgenic P. patens extracts using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometer operated in MRM mode (UPLC-MRM-MS) (Figure 5). Only artemisinin was detected with no intermediates, and this was confirmed by comparison with an artemisinin standard as shown in Figure 5. The analysis was performed twice, two months apart, in triplicates each time. Quantification of artemisinin yield using external calibration was 0.21 mg/g dry weight (DW), which is higher than what was initially obtained via other plant heterologous expression such as Nicotiana tabacum (0.0068 mg/g DW) (Farhi et al., 2011) and Nicotiana benthamiana (0.003 mg/g DW) (Wang et al., 2016). Analysis by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) shows no glycosylated or glutathione-conjugated products were detected in the P. patens extracts and culture medium. This could be due to the lack of glycosyltransferase 1 (GT1) genes in P. patens compared to higher vascular plants. As no pathway intermediates (conjugated or not) were detected in our extracts, we conclude that the pathway operates efficiently in P. patens.
Figure 5. UPLC-MRM-MS analysis of transgenic Physcomitrium patens. Chromatogram of artemisinin standard (top), transgenic P. patens (middle), and wildtype P. patens (bottom). Figure taken from Khairul Ikram et al. (2017).
Recipes
PhyB media
Reagent Final concentration Amount (for 1 L)
Ca(NO3)2 4.59 mM 800 mg
MgSO4·7H2O 1.01 mM 250 mg
FeSO4·7H2O 44.96 μM 12.5 mg
(NH4)2C4H4O 2.72 mM 500 mg
KH2PO4 1.84 mM 250 mg
CuSO4·5H2O 0.11 μM 0.0275 mg
ZnSO4·7H2O 0.10 μM 0.0275 mg
H3BO3 4.96 μM 0.307 mg
MnCl2·4H2O 0.99 μM 0.195 mg
CoCl2·6H2O 0.12 μM 0.0275 mg
Na2MoO4·2H2O 0.06 μM 0.0125 mg
KI 0.08 μM 0.014 mg
Distilled H2O n/a Add up to 1 L
Total n/a 1 L
Adjust the pH of PhyB media to 6.5 using 4 M KOH. Solidify with 0.7% (w/v) agar and sterilize by autoclaving at 121 °C. Add 1 mL of sterile 1 M CaCl2 solution just before use.
Note: Adding CaCl2 freshly before use minimizes precipitation of the media.
The PhyB media (without the addition of CaCl2) can be stored at room temperature under dark conditions for up to one month. The excess media (with CaCl2) can be used or stored for up to two weeks.
1 M CaCl2
Reagent Final concentration Amount (for 100 mL)
CaCl2 1 M 11.1 g
Distilled H2O Add up to 100 mL
Total n/a 100 mL
8.5% D-Mannitol
Reagent Final concentration Amount (for 500 mL)
D-mannitol 8.5% 25.75 g
Distilled H2O Add up to 500 mL
Total n/a 500 mL
Driselase enzyme solution
Reagent Final concentration Amount
Driselase 0.5% 10 mg
8.5% mannitol 8.5% 20 mL
Total n/a 20 mL
Note: Filter sterilize with 0.22 μm or 0.45 μm before use.
Protoplast wash (PW) solution
Reagent Final concentration Amount (for 500 mL)
D-mannitol 8.5% 42.5 g
Calcium chloride (1 M CaCl2) 10 mM 5 mL
Distilled H2O n/a Add to 500 mL
Total n/a 500 mL
Note: Add the calcium chloride immediately before use and filter sterilize.
MMM solution
Reagent Final concentration Amount (for 20 mL)
D-mannitol 10.3% 9.1% 17.7 mL
Magnesium chloride (1M MgCl2) 15 mM 300 μL
MES 10% 2 mL
Total n/a 20 mL
Note: 2-[N-morpholino]ethanesulfonic acid (MES) (1% w/v, pH 5.6). Dissolve the D-mannitol in the H2O, sterilize by autoclaving, and store at room temperate. On the day of transformation, add the MES and MgCl2 to the D-mannitol solution and filter sterilize.
MCT solution
Reagent Final concentration Amount (for 10 mL)
Ca(NO3)2·4H2O 100 mM 236 mg
8.5% mannitol 7.65% 10 mL
Tris-Cl (1 M, pH 8.0) 10 mM 100 μL
Total n/a 10 mL
Note: Prepare fresh on the day of transformation and filter sterilize after adding the PEG.
Polyethylene glycol (PEG) solution
Reagent Final concentration Amount (for 5 mL)
PEG (MW 6000) 67 mM 2 g
MCT solution n/a 5 mL
Total n/a 5 mL
Note: Mix well as soon as the MCT is added to avoid recrystallization. Allow the mixture to stand for 2–3 h at room temperature and filter sterilize before use.
Protoplast regeneration media bottom layer solution (PRMB)
Reagent Final concentration Amount (for 1 L)
(NH4)2C4H4O6 5 mM 920 mg
D-Mannitol 6% 120 g
PhyB media solution Add up to 1 L
Agar 0.7% 7 g
Total n/a 1 L
Note: Sterilize by autoclaving at 121 °C. Add 1 mL of sterile 1 M CaCl2 solution just before use.
Protoplast regeneration media top layer solution (PRMT)
Reagent Final concentration Amount (for 250 mL)
(NH4)2C4H4O6 5 mM 230 mg
D-Mannitol 8% 20 g
PhyB media solution Add up to 250 mL
Agar 0.4% 1 g
Total n/a 250 mL
Note: Sterilize by autoclaving at 121 °C. Add 5 mL of sterile 1 M CaCl2 solution just before use.
Extraction buffer
Reagent Final concentration Amount (for 250 mL)
Tris (1 M, pH 7.5) 200 mM 50 mL
NaCl (5M) 250 mM 12.5 mL
EDTA (0.5M, pH 8.0) 25 mM 12.5 mL
SDS 0.5% 0.0625 mL
Distilled H2O n/a Add up to 250 mL
Total n/a 250 mL
Acknowledgments
The authors would like to thank Professor Mark Estelle, Professor Yuji Hiwatashi, Professor Dae Kyun Ro and Dafra Pharma (Belgium) for kindly providing the pMP1355, PZAG1 vector, the ADS template and the artemisinin and dihydroartemisinic acid, respectively. The work was supported by Ministry of Higher education Malaysia. The protocol was referred to Khairul Ikram et al. (2017), Bach et al. (2014), and King et al. (2016).
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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Intravital Imaging of Intestinal Intraepithelial Lymphocytes
SM Sara McArdle
GS Goo-Young Seo
MK Mitchell Kronenberg
ZM Zbigniew Mikulski
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4720 Views: 835
Reviewed by: Alessandro DidonnaBrahma MuluguMartin V Kolev
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Original Research Article:
The authors used this protocol in Science Immunology Jul 2022
Abstract
Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.
Keywords: Intravital microscopy Intraepithelial lymphocyte Tracking Cell labeling Suction ring Intestine Confocal reflection
Background
Intestinal intraepithelial lymphocytes (IEL), sometimes also known as intraepithelial T cells, are one of the largest populations of T lymphocytes in the body. They are located within the epithelium and interact extensively with intestinal epithelial cells by actively patrolling the basement membrane and migrating into the lateral intercellular space (Edelblum et al., 2012; Wang et al., 2014; Hoytema van Konijnenburg et al., 2017; Sumida et al., 2017). IEL are important for the maintenance of integrity of the intestinal barrier, repair of wounds, and protection from pathogenic invasion (Cheroutre et al., 2011). They include innate lymphoid cells but are mostly T lymphocytes.
In previous work, we focused on the role of herpes virus entry mediator (HVEM) as a regulator of CD8α+ intraepithelial T cells in the small intestinal (SI) epithelium. We used intravital imaging to track lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice (Edelblum et al., 2012; Wang et al., 2014). In the previous work, by using mice with deficiency for HVEM expression exclusively in the intestine epithelium we showed that in the SI, epithelial HVEM expression is required for the normal patrolling movement of CD8α+ IEL. This correlated with a decreased response to bacterial infection (Seo et al., 2018). Here, we describe a protocol to analyze CD8α+ IEL in the SI epithelium using intravital imaging.
Materials and reagents
#1.5 12 mm round coverslips (Electron Microscopy Sciences, catalog number: 72230-01)
31G 0.5 mL insulin syringe for retroorbital (RO) and anesthesia injection (BD, catalog number: 328447)
Vacuum grease, MOLYKOTE® High Vacuum Grease (McMaster-Carr, catalog number: 2966K52)
10 mL syringe (BD, catalog number: 303134)
Lab tape (Fisher Scientific, catalog number: 15-950)
Antibodies: anti-CD8α-AF488, clone 53-6.7 (eBioscience, catalog number: 53-0081-82); anti-CD8α-AF647, clone 53-6.7 (BD Biosciences, catalog number: 557682); anti-EpCAM-AF647, clone G8.8 (BioLegend, catalog number: 118211)
Sterile phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 10010023)
Isoflurane (Covetrus, National Drug Code: 11695-6777-2, 250 mL, catalog number: 029405)
Oxygen (100% compressed oxygen gas)
Ketamine, Ketaset, 100 mg/mL (Zoetis, National Drug Code: 54771-2013-1)
Xylazine, XylaMed, 100 mg/mL (VetOne, National Drug Code: 13985-612-50)
Vaseline (Fisher Scientific, catalog number: 17-986-496)
70% isopropanol (Covetrus, National Drug Code: 11695-2178-6, reorder number 002498)
Mice on a C57BL/6 background, available from Jackson Laboratories, kept in specific pathogen-free conditions. We used Hvemflox-neo/flox-neo and Hvemfl/fl mice (Seo et al., 2018)
Equipment
Animal heating controller and 15 cm × 10 cm heating plate (World Precision Instruments, catalog number: ATC2000 and 61830)
Rodent rectal temperature probe (World Precision Instruments, catalog number: RET-3)
Isoflurane vaporizer and mouse isoflurane mask
Link7 (Patterson Scientific, catalog number: 78919043)
Uniflow Single Manifold (Patterson Scientific, catalog number: 78924212)
Surgical scissors for skin, small surgical scissors, surgical forceps, Dumont #5/45 forceps
Hair clippers (Wahl, catalog number: 88420) or Nair hair remover sensitive formula
Thermal Cauterizer Unit (Geiger Instruments, model: 150-I or similar unit)
Suction ring assembly
CNC-machined suction ring (parts Prototype Master, Angle Arm, Post, and Extension from Zera Development Co., n.d., Santa Clara, CA)
Indicator swivel clamp with T-handle adjustment, 1/2” diameter × 3/8” diameter (McMaster-Carr, catalog number: 5148A28)
Polyethylene 160 tubing fitted with blunted 18G needle (Warner Instruments, catalog number: 64-0755)
Male Luer lock to 1/4” barb connector (Amazon, catalog number: B0BB649WM2)
Thick-walled vacuum tubing, internal diameter 3/8” or 9.7 mm (VWR, catalog number: MFLX06404-36)
Thick-walled vacuum tubing, internal diameter 5/16” or 7.9 mm (VWR, catalog number: MFLX06404-35)
Vacuum waste gate [can be repurposed from 2-way stopcock (Amazon, catalog number: B09P8GNP7S) or aquarium pump control valve (Amazon, catalog number: B08LGHWB77)]
5/16” T-splitter (usplastic.com, catalog number: 62095)
Small vacuum trap with fittings for 7.9 mm ID tubing (SP Bel Art, catalog number: F19919-0000)
1/4” NPT female to 3/8” barb connector (Amazon, catalog number: B09Q813WN1)
Coarse vacuum adjustment valve: 1/4” male-male NPT Valve (Amazon, catalog number: B07YFS1WFX)
1/4” Barstock tee female adapter (Amazon, catalog number: B07MTWH2W9)
2” male-male extension nipple (Amazon, catalog number: B000BOA2V2)
1/4” NPT female-female coupler (Amazon, catalog number: B09651GMQ3)
Vacuum dry pressure gauge, lower mount, 1/4” NPT (Carbo Instruments, catalog number: D25-CSL-V00)
1/4” NPT male to 5/16” barb connector (Amazon, catalog number: B07RKM6VR8)
Teflon tape (Amazon, catalog number: B095YCMHNX)
Hydrophobic filter, Millex-FG, 0.20 μm, hydrophobic PTFE, 50 mm (Millipore, catalog number: SLFG85010)
Upright Leica SP5 or SP8 confocal microscope or equivalent
Resonant scanner
25× (0.95) water-dipping, coverslip-corrected objective (Leica 506375)
Piezo Z controller (Piezosystem Jena, catalog number: 0-350-01 or similar)
488 and 638 nm laser lines
Motorized Movable Base Plate or equivalent for support of the animal stage (Scientifica, model: MMBP-7200-00)
Software
Leica SP8 software LAS X
Fiji (Schindelin et al., 2012) with the Bio-formats and MISTICA Image Alignment (Ray et al., 2016)
A version of Fiji packaged with MISTICA can be found at github.com/saramcardle/IEL. Otherwise, use the latest Fiji version.
Imaris (Bitplane, version 9.4 or higher)
MATLAB (Mathworks, version 2021a) or the compiled SpiderPlot application (github.com/saramcardle/IEL)
Procedure
Mouse preparation
Please consult your Institutional Animal Care Committee or equivalent body to ensure that all procedures were reviewed and approved according to the governing laws. All procedures described here were approved by the La Jolla Institute for Immunology Animal Care and Use Committee.
The guiding principle is to induce the least amount of stress on the animal. Handle animal cages with care so as not to startle the inhabitants. Avoid any strong scents, perfumes, and deodorants (Mouse Room Conditions, n.d.). Practice handling of the mice to be confident, accurate, and gentle—this will minimize the stress to both animals and researchers. From the perspective of the mouse, repeated attempts to grab it are likely worse than a single successful capture. It is crucial to practice restraining the animal to deliver RO injections properly. In intravital microscopy, your biggest responsibility is to care for animals and to minimize the potential harm coming from your interventions. You will have a chance to glimpse the fascinating world of cellular behavior in their natural environment, but beware of false conclusions steaming from stressed, injured, damaged, hypoxic, unperfused, and photodamaged tissues.
For antibody-based cell labeling, inject the antibodies 4 h before imaging to allow for sufficient time for antibody penetration. With shorter incubations, a significant fraction of the antibody remains in the blood vessels. Over time, surface-bound antibodies will be internalized, and distinct membrane staining will be replaced by a more uniform labeling in the cytosol (Figure 1). In our hands, longer incubation times do not significantly improve cell labeling but are a cause of concern for biological actions mediated by antigen-antibody binding and other effects.
Figure 1. Effect of varying antibody penetration times. Seven hours after RO injection of CD8α antibody (left), intestinal intraepithelial lymphocytes (IEL) show a classic membrane staining pattern. In contrast, 31 h after injection (right), IEL have internalized the CD8α antibody, leading to smaller, punctate signals. Scale bar = 100 μm. Brightness of each image was adjusted independently, and a green lookup table was used to improve visibility of the features.
If you are only using one antibody, we recommend choosing a far-red fluorophore (e.g., Alexa Fluor 647) due to that channel’s lower autofluorescent background. For the two-channel imaging used here, we also used a green fluorophore (Alexa Fluor 488) to minimize channel bleed through.
Use IgG antibodies directly conjugated to organic dyes, such as Alexa Fluor 488, 555, or 647. Avoid antibodies conjugated to bulky fluorescent proteins (such as phycoerythrin or allophycocyanin), as their large molecular weight impedes efficient diffusion into tissues and, while they are initially bright, they bleach quickly.
Dilute 15–20 μg of antibody (here, anti-CD8α and/or anti-EpCAM antibody) in 50 μL of sterile PBS.
Anesthetize the mouse with isoflurane and inject the antibody solution retroorbitally (Yardeni et al., 2011) It is important to inject the full volume of labeled antibody for consistent labeling without introducing any air bubbles in the mouse’s circulation.
Return mice to cages for recovery. Mice need to be fasted for 4 h to slow down peristalsis.
Suction ring setup
Suction rings, originally developed for lung imaging (Looney et al., 2011), offer a convenient way of stabilizing many mouse tissues. The rings we used were custom made at a CNC machine shop (see Notes for recommended supplier). An alternative design utilizes a resin 3D printer (Ahl et al., 2019) and contains an embedded electrical heater to provide more physiological thermal conditions during imaging. Ahl et al. (2019) provides an elegant description of the complete vacuum system and includes the 3D stereolithography [files for the 3D printer (in .stl format)]. An alternative device, which uses clamping instead of vacuum pressure, is described in Koike et al. (2021).
In this protocol, we based our part selection on United States American National Standard Pipe Thread (NPT). In this standard, fittings are based in inches. According to the standard, 3/8” barbed fitting will have a good fit for a 3/8” (9–10 mm) internal diameter vacuum tubing. The barb diameter is slightly larger than the tubing diameter to ensure a proper seal. You can modify the design to use materials available in your country or already existing in your laboratory. For the best performance of the vacuum system, you should try to maximize the internal diameter of the parts, minimize the total length of the circuit, and eliminate all leaks. Online suppliers such as Amazon, Fittings, Inc., or US Plastic Corporation offer a convenient source of parts. A local hardware or automotive stores can also serve as valuable resources for users who need additional help in procuring the parts or assembling the circuit.
Assemble the vacuum system (Figure 2). The suction ring is connected to a small vacuum trap to collect any biological fluids. From there, it is connected to a fine adjustment valve, a coarse adjustment valve, and a gauge to read the vacuum pressure. Finally, it is connected to the central building vacuum through a filter as a secondary method to prevent any contamination. Connect the pieces in the following order:
Connect the hydrophobic filter disk to the central vacuum shutoff valve with the 3/8” thick-walled vacuum tubing.
Attach the tubing to the barb of the 1/4” NPT female connector, and couple it to a male-male coarse adjustment valve using Teflon tape.
Couple the NPT connector with the female Tee connector.
Add the male-male extension nipple to the top of the Tee connector and fit the pressure gauge on top with the female-female 1/4” NPT coupling.
Attach the 1/4” NPT male to 5/16” barb connector to the Tee connector and fit the 5/16” vacuum tubing.
Add the plastic T-splitter and attach the 2-way stopcock fine adjustment valve on a length of tubing that will allow placement of the stopcock within reach of the surgeon. This valve can be opened to reduce the vacuum or closed to increase it. An alternative design can use an aquarium pump valve.
Attach the small vacuum trap with 5/16” vacuum tubing and secure it upright.
Add required length of the vacuum tubing to reach the microscope and connect the 1/4” barb to Luer lock connector. Add Teflon tape if needed to ensure proper seal.
Figure 2. Schematics of vacuum circuit. A. Parts with female NPT connectors are indicated in orange; parts with male NPT connectors are indicated in blue. Grey parts are plastic luer-type or barbed connectors. B. Example build with vacuum gauge and adjacent parts.
Remove the plunger from a 10 mL syringe and squeeze a large bead of the vacuum grease into the syringe. Insert the plunger and apply a thin line of grease on the edge of the ring. Use Dumont #5/45 forceps to distribute the grease evenly on the rim of the ring. Remove any grease that got into the groove of the ring.
Clean a #1.5 12 mm diameter coverslip with 70% alcohol and wipe on lens paper to remove any traces of dust and lint. Place the coverslip on ring and press down to seal (Figure 3A).
Place the suction ring assembly on a post raised above the microscope stage and secure it with a right-angle post clamp or swivel clamp. We use a custom-made aluminum block with the post attached, screwed into the microscope stage for maximum stability, which can be requested at a machine shop. Make sure the cover glass is perpendicular to the optical axis of the microscope by aligning the ring parallel to the lens of the objective (Figure 3B).
Figure 3. Assembling the suction ring. A. Attach a #1.5 coverslip to suction ring with vacuum grease and attach PE160 tubing. B. Adjust the ring to be parallel to the front lens of the objective. C. Position the suction ring and the nose cone on the heated platform.
Connect the suction ring via 18 G blunted needle end to the Luer lock of the vacuum circuit. Set the system to 5–20 mbar vacuum pressure. The exact value is dependent on the preparation and the way in which the system is integrated. Use the lowest vacuum pressure sufficient to stabilize the tissue.
Microscope setup
Insert the objective onto the piezo Z controller.
Open the acquisition software and initialize the resonant scanner.
Turn on appropriate laser lines and initially set the power to a low setting (~5%). In our case, we imaged using the 488 and 638 nm lines.
Select a dichroic filter that will give you the ability to switch between laser lines without needing to move any hardware. In our case, it was the 488/552/638 triple dichroic mirror for the SP8 system. For optimal use of confocal reflection microscopy for label-free detection of tissue structure, use the Reflection/Transmission mirror.
Set up the internal detector wavelengths, assigning the fluorescence gathered from antibody labeling on your most sensitive detectors (in our case, HyD detectors). These wavelengths were selected for AF488, AF647, and the confocal reflection channel:
AF488: 494–563 nm
Reflection: 619–651 nm
AF647: 651–722 nm
Either the tissue reflection or EpCAM staining for epithelium can be used to identify SI anatomy, although they provide slightly different information (Figure 4).
Figure 4. Comparison of visible structures from tissue reflection (left) and EpCAM staining (right) at three depths through the small intestinal (SI) villi. Brightness is adjusted in each image individually to accent details. Scale bar = 100 μm. Blue dotted lines = location of basal lamina. Blue arrows = basal lamina visible by reflection microscopy. Orange arrows = autofluorescent debris or non-specific binding of EpCAM antibody to macrophages.
Set the system to perform XYZT imaging, with the Z stepper controlled by the piezo element.
Adjust the pinhole size to two Airy units, enable bidirectional scanning, and find the appropriate phase correction. Enable line averaging during live acquisition setting and set the data acquisition mode to 8-bit. Set the zoom to the minimum allowed by the system for biggest field of view and adjust the line averaging to at least 8×. Our pixel size was set to approximately 0.6 μm with a corresponding field of view of approximately 370 μm.
Surgery and intravital microscopy
Please ensure all mouse handling and surgery steps are written in your Animal Care Committee–approved protocol. The following procedure is rather complex, with the scientist needing to simultaneously pay attention to the mouse anesthesia, the vacuum window pressure, and the image acquisition. We highly recommend practicing each part of the procedure slowly before attempting the full intravital imaging workflow. For example, a novice user can test the labeling, vacuum window, and image acquisition by practicing with a mouse that has been euthanized 4 h after antibody injection.
We found that preparations with isoflurane alone had too much movement (Video 1). Ketamine/xylazine anesthesia followed by isoflurane inhalation creates the best conditions for imaging with reduced motion artifacts (Figure 5, Video 2). Prepare the ketamine/xylazine mixture in sterile PBS (10 mg/mL ketamine and 1.5 mg/mL xylazine). Weigh the mouse and deliver 10 μL of the prepared solution per gram of body weight (100 μg of ketamine and 15 μg of xylazine per gram of mouse weight) via intraperitoneal injection.
Figure 5. Example images from a well stabilized (left) and poorly stabilized (right) intravital movie. The image on the left was taken using a mouse anesthetized with both ketamine/xylazine and isoflurane; the image on the right was from a mouse anesthetized only with isoflurane. These are Z projections of single time points. Yellow arrows point to cell shadows that are caused by significant tissue motion between sequential Z steps, compromising the accuracy of the analysis. Green = CD8α; White = EpCAM. See also supplementary movies. Brightness was adjusted in each image. Scale bar = 100 μm.
Video 1. Representative movie from an insufficiently stabilized preparation (isoflurane-only) that cannot be accurately quantified. Maximum intensity Z projection of a 10 min time series. Green = CD8α; White = EpCAM. Scale bar = 74 μm. See also Figure 5.
Video 2. Representative example of a well stabilized movie from a mouse anesthetized with isoflurane and ketamine/xylazine. Maximum intensity Z projection of a 10 min time series. Green = CD8α; White = EpCAM. Scale bar = 74 μm. See also Figure 5.
After anesthesia onset, shave the abdominal area with clippers or with hair removal lotion, followed by cleaning with 70% isopropanol.
Place the mouse ventral side up on heating pad and place the snout of the animal in isoflurane facemask. We place a thin steel plate over the ATC2000 heating pad, connected with thermal paste, to enable the use of a magnetic isoflurane mask. Alternatively, you can tape the isoflurane mask directly onto the heating pad.
Turn on isoflurane flow to face mask. Observe the breathing pattern of the animal and maintain a stable plane of anesthesia by delivering between 0.5% and 2% isoflurane. Labored breathing indicates the animal has been anesthetized too deeply.
Secure the limbs of the mouse with laboratory tape.
Insert a Vaseline-coated rectal thermometer and secure it by taping it to the tail. Set the feedback loop of the animal temperature controller to 37 °C.
Verify that a sufficient anesthesia level has been reached with a toe pinch (Figure 6A).
Position the ring assembly above the center of the abdomen and adjust it such that it is parallel to the front lens of the objective (Figures 3B and 6H). You might need to reposition the mouse to ensure that the ring has unobstructed access to the area. It is much safer to reposition the intact animal before incisions. Move the ring out of the way for surgery.
Make an approximately 2 cm midline incision in the skin with surgical scissors (Figure 6B). Locate the linea alba and make a 1.5 cm incision along it in the peritoneum to expose the small intestine, taking care to avoid inadvertently introducing hair in the surgical area or cutting through blood vessels (Figure 6C).
Using two wetted cotton tipped applicators, delicately move the intestine and position the antimesenteric side upwards. Look for areas that are free of food matter. Take care not to accidentally twist the gut and pinch the blood supply. Locate the stomach and find the piece of intestine distal to it. This will be the distal duodenum or jejunum. Exteriorize a small loop through the opening in the peritoneum (Figure 6D). Notice the blood vessels on both sides of the intestine and avoid cutting or damaging them. Using a cautery set to medium power, gently cauterize the antimesenteric surface of the intestine (Figure 6E). With small scissors, make an approximately 1 cm long opening along the cauterized edge (Figure 6F). Use cotton tipped applicators to gently open and flatten the gut, revealing the mucosal surface of the villi (Figure 6G). You may also use forceps but grab only the edges of the tissue.
Work quickly to avoid drying the tissue. If necessary, you can add a drop of PBS onto the mucosal surface to keep it moist. However, it is best not to disturb the mucus layer over the villi, because this will increase the amount of tissue movement.
If you notice any bleeding, cauterize the wounded area immediately. If excess blood has pooled near the region of interest, add a drop of PBS and use gauze or a cotton tipped applicator to remove the excess. It is important to minimize blood loss because any blood on the imaged area will reduce the fluorescence intensity, but, as above, washing the tissue can damage the mucus and cause additional tissue motion.
Hold the suction ring in your dominant hand and lower it onto the exposed mucosal surface. Control the waste vacuum valve with your other hand and adjust the pressure to the lowest possible setting that achieves a complete seal against the tissue. You should see that the tissue is flattened against the coverslip (Figure 6H). Because the required pressure depends on how well the intestine seals to the ring, it is less important to keep the measured pressure consistent than ensuring the tissue is stable and undamaged in each preparation.
Figure 6. Overview of the surgical procedure. (A) Secure the animal and verify adequate anesthesia. (B) Make a midline incision taking care not to damage skin blood vessels (arrow). (C) Open peritoneum along linea alba and avoid major vessels. (D) Gently exteriorize the gut and find appropriate section free of food. (E) Cauterize the antimesenteric side. (F) Cut along the cauterized area to open the intestine. (G) Gently stretch and flatten mucosal surface. (H) Position suction ring above the exposed intestine and gently lower it onto the tissue while adjusting suction force to adhere the tissue to the coverslip.
Add a drop of PBS on top of the coverslip for immersion and lower your objective to make contact with the fluid. With a long working distance objective, you will typically need to move the objective away from the ring to find the focus.
Using the 25× (0.95) objective and a green fluorescence filter cube, look through the eyepiece and observe the preparation under epifluorescence illumination. Erythrocytes in capillaries of the villi should be traveling at a high speed; you might see an occasional bigger immune cell moving through the blood stream. If the tissue is moving, gradually increase the pressure with the fine adjustment. Alternatively, inspect other areas within the vacuum ring to find a more stable location. You can further increase the stability of the preparation by gently lifting the ring by a few millimeters to isolate the attached tissue from the mouse’s respiration (Move the objective out of the way before attempting this.). If you can see erythrocytes stopped in capillaries or moving in a backwards and forward motion, your vacuum pressure is too high, or you raised the preparation too much and the blood vessels have become pinched. Good preparations should show minimal movement of the villi and robust perfusion through the capillaries.
Start image acquisition in live mode and adjust the gain and laser power as necessary to utilize the full dynamic range without saturation. Image in simultaneous mode after calibrating the detectors’ gains and wavelengths to avoid bleed through. Choosing optimal parameters for intravital imaging involves balancing the need for high acquisition speed to track moving cells, high sensitivity to detect dimly labeled cells, and large field-of-view to make the most of every labor-intensive experiment. For more details on acquisition setup for intravital imaging, please see the review (McArdle et al., 2016).
Set the top and bottom of Z stack to encompass the top of the villus, the subepithelial area, and lamina propria. The basal lamina can be seen by reflection microscopy (Figure 4, blue arrows). If you use EpCAM labeling, the transition between the epithelium and lamina propria is visible as a distinct change in tissue texture (Figure 4, blue dotted lines showing some examples). Autofluorescent or non-specifically labeled macrophages are characteristic cells in the lamina propria that serve as a useful landmark (Figure 4, orange arrows).
Adjust the step size to 2.8 μm. Set the timelapse imaging to acquire Z stack every 30 s or faster, trimming the Z stack if needed. Acquire data for a minimum of 10 min per location. Save the data as .lif file.
Monitor the mouse plane of anesthesia throughout the experiment by observing the breathing pattern every 5–10 min and adjusting the amount of vaporized isoflurane as needed. Do a pinch test between the acquisitions to verify if the animal is fully anesthetized.
After completion of the imaging, euthanize the animal according to your approved protocol.
Clean the ring with PBS and 70% alcohol and remove the coverslip and any remaining vacuum grease.
Image processing
The suction ring often provides enough stability to the tissue to proceed with analysis without further motion removal. However, if necessary, small motion artifacts can be minimized after acquisition in Fiji (Schindelin et al., 2012). Whether the mechanical stabilization is sufficient for cell tracking is determined visually. Loosely, post-processing is necessary if there was gross tissue motion between Z steps or between sequential time steps of > 1/2 of a typical cell diameter (Figure 5).
Open the 1–3 channel .lif file in Fiji as a hyperstack using the Bioformats importer. Delete any Z steps that are entirely outside of the detectable tissue or region of interest because blank images without meaningful signal disrupt the alignment algorithm.
Change the color of each channel to either red, green, or blue, and then flatten the multichannel image to an RGB image. (This step can be skipped if there is only one channel.)
If each villus shows independent motion that ought to be corrected separately, create small crops of each villus. Each cropped region should be just large enough to capture the entire villus through all Z stacks and time points. Process all further steps independently for each region.
Apply the MSTHyperStackReg2 plugin (Ray et al., 2016). This software first aligns each Z step of each time point individually. Next, a maximum intensity Z projection is created to register the time series, and the calculated transform for each time step is applied to each Z position. At each stage, the entire sequence (either in Z or in T) is registered using minimum weighted spanning trees to choose a reference image and to ensure that outliers (images with particularly extreme motion artifacts) do not degrade the quality of the global alignment. This algorithm corrects lateral tissue movement in X and Y but does not correct vertical drift (Z motion).
i. First, perform coarse registration. We recommend using rigid body alignment using the approximate with a large graph width (the larger of half the number of Z steps per stack or half the number of time points) and allowing it to automatically choose the anchor image and add a border (Figure 7A). The automatically produced maximum intensity image can be used to visually check the alignment. Save the aligned Z stack as an intermediate step. High quality images with small non-linear tissue distortions can be successfully corrected using affine transformations, though affine transforms often produce very bad results from noisy or sparse images.
ii. Next, perform fine alignment on the results. We recommend using rigid body transforms, the exact method, a small (i.e., 5) graph width, automatic anchor selection, and no additional border (Figure 7A). This step can take a long time (hours), depending on the dimensions of your image and the graph width.
iii. The improvement in image quality using image registration is illustrated in Video 3 by comparison to Video 2.
Video 3. Improvement in image quality using image registration. The 3D data from Supplementary Video 2 was processed with MISTICA image alignment, which minimized jitter and drift. Maximum intensity Z projection of a 10 min time series. Green = CD8α; White = EpCAM. Scale bar = 74 μm.
In Fiji, adjust the image properties to set the voxel dimension and time step. Save the final result as a .tif hyperstack.
Immune surveillance of the epithelium can be measured by calculating the area covered by all labeled cells during a set amount of time in Fiji.
Load the raw .lif file or the aligned .tif hyperstack into Fiji.
Use the reflection signal or the EpCAM channel to identify the epithelial layer. At the top of each finger-like villus, there is a region approximately 10 μm deep that is nearly exclusively epithelium. IEL often migrate along the basement membrane separating the SI epithelium from the lamina propria. Create a substack of the volume encompassing the epithelium at the top of the villus and the basement membrane and trim each video to a set amount of time (e.g., 10 min).
Using the polygon tool, draw a region of interest (ROI) encircling each villus and save each region to the ROI manager (Figure 7B).
Split the channels. On the CD8α channel, use a median filter (2D, radius 2 pixels) to remove pixel noise.
Create a maximum intensity Z projection and then a maximum intensity T projection to combine all timepoints and Z locations into a single image, which has the location of every cell overlaid in the 10 min movie (Figure 7B).
Using the threshold tool, determine the intensity threshold that separates cells from background non-specific signal (Figure 7B). Ideally, this can be determined by performing one preliminary experiment with a labeled isotype control antibody. In the absence of this negative control sample, set the threshold to be just under the intensity of the dimmest labeled IEL. Use the same threshold for all samples by pressing the Set button and typing the determined threshold. Apply the threshold to binarize the image.
In Fiji, set the measurements to include area and area fraction; uncheck limit to threshold. Measure each ROI defining a villus. The results window will report the total area of the villus and the fraction that was visited by a cell in the time period (Figure 7C).
The movement of the cells can be displayed with a rainbow time projection (Figure 7D). Use the maximum intensity Z projection time series from step 2e and apply the Fiji plugin Temporal-Color Code (Miura, 2022) to show each time step as a unique color. Fast cells will appear as rainbow streaks while immobile cells appear white (all colors overlaid).
Figure 7. Examples of image processing steps and results. A. Screenshots of MSTHyperStackReg plugin parameters for an image with 85 Z-steps and 40 timepoints. Left: First round (approximate) settings, with graph width half of the Z stack. Right: second, more precise round of alignment. B. Screenshots of intermediate results during the calculation of villi coverage by intestinal intraepithelial lymphocytes (IEL). Left: Each villus is outlined using the EpCAM channel. Middle: median smoothing is applied to the CD8α channel and a maximum intensity projection over all Z steps and all time points is applied to overlay the position of every IEL. Right: a threshold is applied to find the area visited by an IEL at least once during the 10 min movie and the villi outlines are overlaid (yellow). C. In each villus, the percentage of the area visited by an IEL is calculated. D. Alternatively, IEL movement can be displayed as a rainbow time projection. E. CD8α-AF488 IEL (green) were tracked in Imaris. The tracks are shown as orange lines. EpCAM-AF647 (purple) marks the small intestinal (SI) epithelium. White arrows show a few of the many examples of artifacts that are visible in both the AF488 and AF647 channels. The yellow arrow shows a non-specifically labeled cell. F. Cell displacement, total movement distance, and speed were calculated from the Imaris tracks. G. The migration of each cell is shown as a spider plot. Scale bars = 100 μm.
Movement statistics, such as mean speed, track length, and displacement demonstrate cell activation and patrolling behavior. Track the IEL in 3D in Imaris to quantify cell motility.
In Imaris, open the raw .lif files or tifs aligned in Fiji. If you are working with processed tifs, it is a good idea to check the voxel size and time step information in Image Properties.
If necessary, correct slow lateral and axial drift affecting the entire imaged volume in Imaris before cell tracking.
i. Use the Spots function to track bright, stable features, such as immobile cells, or autofluorescent or reflective debris. Please see below (step 3c) for details on the Spots function. Choose 3–10 spots that span the entire volume under analysis for the entire time series.
ii. In the Edit Tracks tab, select Correct Drift. Choose the options for correcting the image and objects, using translational and rotational drift, and include the entire result.
Tracking the movement of IEL can be performed semi-automatically with the Spots wizard (Figure 7E). The exact parameters need to be optimized for each project, but the overall process is similar in all cases. Detect round objects in your chosen channel (CD8α) and then apply quality, size, and intensity filters to remove debris and macrophages that have picked up the antibody. The most sensitive parameter is usually the expected object diameter; be sure to choose the typical diameter of the fluorescent spot, which may be smaller than the cell diameter if the antibodies have been internalized or if the cells are polarized. Add object filters to remove debris that is bright in multiple channels. Imaris then performs tracking of the segmented objects over time. For well-stabilized movies, the autoregressive motion algorithm usually produces the best results, although the Brownian motion algorithm can work in some cases. At this stage, adding track filters to remove stationary objects can improve performance.
There will nearly always be mistakes in the automatic tracking for intravital imaging data: single tracks inappropriately split, tracks jumping between neighboring cells, or non-IEL objects being included. Surface-labeled IEL can be distinguished from other objects by their shape (hollow, round), speed (greater than local tissue motion), and directionality (movement along basal lamina, as opposed to transverse). If multiple channels were imaged, autofluorescent objects can be seen in all channels, whereas antibody signals are usually much brighter in the proper channel (Figure 7E). Correct the centroid spots and connecting tracks manually in the Edit tab of the Spots object. It is important to use the same level of care and attention on all samples; manually correcting some images but not others can bias the final results. For further information on how to perform tracking in Imaris, please refer to Bitplane’s extensive online tutorial library.
In the Statistics tab, export the track speed, track length, and track displacement metrics to a csv file. (Figure 7F). Use the export data for plotting to export the cell positions at each time point in a convenient three-column format.
Spider graphs are an informative way to visualize cell migration. In these graphs, each cell starts at position (0, 0) and its total path is drawn in a random color, demonstrating overall cell motility and directionality.
i. The spider plots can be generated from the exported cell position data using a simple script or stand-alone application found on GitHub (github.com/saramcardle/IEL) (Figure 7G).
ii. Alternatively, a spider graph can be generated in Imaris. First, select all the IEL tracks and duplicate the spots. In the new spots object, select all of the tracks and then apply the Translate tracks function in the Tools tab. This will move all of the tracks to originate at the same location. The newly adjusted position of each cell can be exported to a spreadsheet for plotting.
Limitations of image analysis
The cell-by-cell statistics produced here inherently have high variance due to natural biological variability (see plots in Figure 7). Additionally, each experiment only yields a small number of tracked cells (~20–40). Therefore, it is only possible to detect large differences between groups.
Even with tissue stabilization and drift correction, some tissue movement is usually still present. This tissue movement defines the lower limit of reliable quantification for cell speed and travel distance. To measure this for each dataset, track some autofluorescent spots that should not move during image acquisition, such as macrophages and food debris, using the Imaris spots wizard. Calculate their average speed and travel distance. The results show the sensitivity of your measurements; differences between cell groups smaller than typical gross tissue movement should be ignored, and preparations with unusually large tissue movement should be discarded.
Sometimes, it can be difficult to distinguish antibody-labeled IEL from non-specific signals or autofluorescent objects. It is important to optimize the antibody concentration and injection time for your specific experimental protocol. Additionally, it can be helpful to perform a preliminary experiment using an isotype control antibody to visualize the intensity, location, and shape of artifacts as a negative control.
Please plan your experiments to minimize batch effects. If comparing two (or more) groups of mice, always image a mouse from each group back-to-back using the same batch of antibodies. Alternate which group is chosen to be run first each day, so that there is no systemic bias in the length of antibody incubation time or fasting time.
i. For projects comparing the activity of different cell populations, rather than different mouse conditions, it is possible to image two cell populations simultaneously with two different fluorophore-labeled antibodies. In this case, the movement characteristics of one population can be normalized to that of the other population in each mouse individually. This is an elegant way to minimize the differences in tissue motion in each preparation.
Notes
It is vital to consult your Institutional Animal Care Committee or equivalent body to ensure that all procedures were reviewed and approved according to the governing laws.
Intravital microscopy is a complex procedure. As each microscopy system is unique, it is best to seek advice from core facility staff or experienced users on how to establish the best parameters for imaging. Novice users should first master the handing of the microscope using an immobile object, such as a euthanized mouse injected with an antibody targeting the cells of interest. We encourage using untreated littermate control animals to understand the nature of autofluorescence in the tissue. Animals that received isotype control antibodies can serve as valuable controls for the specificity of antibodies targeting cells of interest. By comparing the images obtained from these kinds of samples, novice users can develop understanding of proper labeling and learn to distinguish it from artifacts and autofluorescence, which is often very strong in the lamina propria. For additional information on intravital microscopy in the gut, we refer readers to previous studies by Sujino et al. (2016), Chen et al. (2019), and Koike et al. (2021).
The suction ring assembly can be purchased from Zera Development Company; please see https://www.zeradevelopment.com for contact information. For the best performance of the vacuum circuit, make it as short as possible and seal all the leaks. Use Teflon tape to create a tight seal between metal parts of the system. Use large-bore vacuum tubing for connections between parts and minimize the volume of vacuum trap. Alternatively, the vacuum trap can be placed between the vacuum filter and the gauge assembly.
Acknowledgments
We wish to thank Dr. Grzegorz Chodaczek for sharing his expertise in intravital microscopy and for the development of suction ring techniques at La Jolla Institute for Immunology.
Funding: National Institutes of Health grants P01 DK46763, R01 AI61516, and MIST U01 AI125955 (MK); Chan-Zuckerberg Initiative Imaging Scientist Grant 2019‐198153 (SM).
This protocol is derived from the original research paper (Seo et al., 2022; DOI: 10.1126/sciimmunol.abm6931).
Competing interests
The authors have no conflicts or competing interests.
References
Ahl, D., Eriksson, O., Sedin, J., Seignez, C., Schwan, E., Kreuger, J., Christoffersson, G. and Phillipson, M. (2019). Turning Up the Heat: Local Temperature Control During in vivo Imaging of Immune Cells. Front Immunol 10: 2036.
Chen, Y., Koike, Y., Chi, L., Ahmed, A., Miyake, H., Li, B., Lee, C., Delgado-Olguin, P. and Pierro, A. (2019). Formula feeding and immature gut microcirculation promote intestinal hypoxia, leading to necrotizing enterocolitis. Dis Model Mech 12(12).
Cheroutre, H., Lambolez, F. and Mucida, D. (2011). The light and dark sides of intestinal intraepithelial lymphocytes. Nat Rev Immunol 11(7): 445-456.
Edelblum, K. L., Shen, L., Weber, C. R., Marchiando, A. M., Clay, B. S., Wang, Y., Prinz, I., Malissen, B., Sperling, A. I. and Turner, J. R. (2012). Dynamic migration of γδ intraepithelial lymphocytes requires occludin.Proc Nat Acad Sci U S A 109(18): 7097-7102.
Hoytema van Konijnenburg, D. P., Reis, B. S., Pedicord, V. A., Farache, J., Victora, G. D. and Mucida, D. (2017). Intestinal Epithelial and Intraepithelial T Cell Crosstalk Mediates a Dynamic Response to Infection. Cell 171(4): 783-794.e13.
Koike, Y., Li, B., Chen, Y., Ganji, N., Alganabi, M., Miyake, H., Lee, C., Hock, A., Wu, R., Uchida, K., et al. (2021). Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy. Bio Protoc 11(5): e3937.
Looney, M. R., Thornton, E. E., Sen, D., Lamm, W. J., Glenny, R. W. and Krummel, M. F. (2011). Stabilized imaging of immune surveillance in the mouse lung. Nat Methods 8(1): 91-96.
McArdle, S., Mikulski, Z. and Ley, K. (2016). Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis. J Exp Med 213(7): 1117-1131.
Miura, K. (2022). Temporal-Color Coder plugin for Fiji. Shell. Fiji. Retrieved from https://github.com/fiji/fiji/blob/847ee410deedda9ba1de673820a5fa63446aa2e1/plugins/Scripts/Image/Hyperstacks/Temporal-Color_Code.ijm (Original work published 2011)
Mouse Room Conditions. (n.d.). The Jackson Laboratory. Retrieved November 19, 2022, from https://www.jax.org/jax-mice-and-services/customer-support/technical-support/breeding-and-husbandry-support/mouse-room-conditions
Ray, N., McArdle, S., Ley, K. and Acton, S. T. (2016). MISTICA: Minimum Spanning Tree-Based Coarse Image Alignment for Microscopy Image Sequences. IEEE J Biomed Health Inform 20(6): 1575-1584.
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.
Seo, G.-Y., Shui, J.-W., Takahashi, D., Song, C., Wang, Q., Kim, K., Mikulski, Z., Chandra, S., Giles, D. A., Zahner, S., et al. (2018). LIGHT-HVEM Signaling in Innate Lymphoid Cell Subsets Protects Against Enteric Bacterial Infection. Cell Host Microbe 24(2): 249-260.e4.
Seo, G. Y., Takahashi, D., Wang, Q., Mikulski, Z., Chen, A., Chou, T. F., Marcovecchio, P., McArdle, S., Sethi, A., Shui, J. W., et al. (2022). Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection. Sci Immunol 7(73): eabm6931.
Sujino, T., London, M., Hoytema van Konijnenburg, D. P., Rendon, T., Buch, T., Silva, H. M., Lafaille, J. J., Reis, B. S. and Mucida, D. (2016). Tissue adaptation of regulatory and intraepithelial CD4+ T cells controls gut inflammation. Science 352(6293): 1581-1586.
Sumida, H., Lu, E., Chen, H., Yang, Q., Mackie, K. and Cyster, J. G. (2017). GPR55 regulates intraepithelial lymphocyte migration dynamics and susceptibility to intestinal damage. Sci Immunol 2(18): eaao1135.
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4,721 | https://bio-protocol.org/en/bpdetail?id=4721&type=0 | # Bio-Protocol Content
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Simplifying Barley Leaf Rust Research: An Easy and Reproducible Infection Protocol for Puccinia hordei on a Small Laboratory Scale
CS Caroline I. Skoppek §
JS Jana Streubel *
(*contributed equally to this work, § Technical contact)
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4721 Views: 621
Reviewed by: Zhibing Lai Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in Molecular Plant Pathology Feb 2022
Abstract
Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.
Key features
• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation
• Plant age–independent protocol
• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility
• No specialized equipment needed
• Includes simple spore harvesting method
• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)
• Protocol is also applicable for a detached leaf assay
Graphical overview
Keywords: Puccinia hordei Rust infection Biotrophic fungi Barley Golden Promise Wheat germ agglutinin Alexa Fluor 488 staining Resistance assessment
Background
In 2050, agriculture will have to feed more than nine billion people (FAO, 2009). This is an enormous challenge, especially in times of climate change, shortage of arable land, and spread of plant diseases. To secure future food supply, the development of high-yielding and stress-tolerant crops is urgently needed. Barley (Hordeum vulgare) is among the most important cereals in the world, needed as fodder and to brew beverages. However, infections with biotrophic fungal pathogens, such as rust or powdery mildew, severely threaten its annual yield, and existing resistance strategies can easily be overcome (König et al., 2012; Dinh et al., 2020). There is an ongoing arms race between pathogens and their host plants that necessitates the constant development of new and more durable resistance mechanisms. In the past decade, biotechnological breakthroughs such as the use of programmable DNA-binding proteins have revolutionized the precise targeting of genes to study their function (Miladinovic et al., 2021). In the future, this will allow the development of plants with potentially new resistance mechanisms. Consequently, reliable plant–pathogen infection protocols are a critical prerequisite to evaluate the infection phenotypes of the resulting plants in the lab. Working with biotrophic fungal pathogens can be particularly challenging because they require a living host and cannot be easily cultivated on plates. Here, we describe a comprehensive step-by-step protocol for reproducible rust infection assays in barley on a small lab scale. The protocol was established for Puccinia hordei infection of the barley cultivar Golden Promise (Skoppek et al., 2022). It includes a pre-propagation and easy harvesting protocol to produce a sufficient number of fresh spores for an infection trial. The detailed process information, uniform spore dispersal, and ease of handling allow a rapid establishment of the method. The equipment required is commonly available and inexpensive. In addition, we demonstrate that our infection protocol can easily be combined with macroscopic and microscopic evaluation methods to assess a certain resistance phenotype. Finally, the protocol can be easily transferred to other host plants and to other fungal pathogens, such as other rust fungi or powdery mildew.
Materials and reagents
Biological materials
Barley Golden Promise seeds
Urediniospores of leaf rust (Puccinia hordei; Ph) isolate I-80
Reagents and solutions for infection procedure
Tween 20 (Carl Roth, catalog number: 9127.1)
Sodium hypochlorite solution (NaClO + H2O) (Carl Roth, catalog number: 9062.3)
6% NaClO solution (see Recipe 1)
0.01% Tween 20 solution (see Recipe 2)
Optional: Reagents and solutions for fungal staining and microscopy
Wheat germ agglutinin, Alexa fluor 488TM conjugate (WGA AF488) (Thermo Fisher, catalog number: W11261)
Potassium hydroxide (KOH) (Carl Roth, catalog number: 6751.1)
Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)
Potassium chloride (KCl) (AppliChem, catalog number: A1039.1000)
Disodium hydrogen orthophosphate (Na2HPO4) (Carl Roth, catalog number: 6751.1)
Potassium dihydrogen phosphate (KH2PO4) (Duchefa, catalog number: P0574.1000)
Acetic acid (CH3COOH) 100% (Carl Roth, catalog number: 3738.1)
Glycerol (C3H8O3) 86% (Carl Roth, catalog number: 4043.3)
Bleaching solution (see Recipe 3)
1 M KOH (see Recipe 4)
10× phosphate buffered saline (PBS) buffer pH 7.4 (see Recipe 5)
1× phosphate buffered saline (PBS) (see Recipe 6)
WGA AF488 stock solution (see Recipe 7)
WGA AF488 staining solution (see Recipe 8)
30% Glycerol solution (see Recipe 9)
Recipes
6% NaClO Solution
Reagent Final concentration Amount
NaClO (12%) 6% 50 mL
ddH2O n/a Ad 100 mL
Caution: Dilute under fume hood, wear protective gear. Store at 4 °C in the dark (use brown bottle or wrap bottle in aluminum foil)
0.01% Tween 20 Solution
Reagent Final concentration Amount
Tween 20 (100%) 0.01% 20 μL
ddH2O n/a 199.98 mL
Autoclave and store at room temperature (RT)
(Optional) Recipes for staining:
Bleaching solution
Reagent Final concentration Amount
Ethanol (absolute) 75% 75 mL
CH3COOH 100% 15% 15 mL
ddH2O n/a Ad 100 mL
Caution: Dilute under fume hood, wear protective gear. Store at RT
1 M KOH
Reagent Final concentration Amount
KOH 1 M 5,611 g
ddH2O n/a Ad 100 mL
Store at RT.
10× PBS Buffer
Reagent Final concentration Amount
NaCl 1.4 M 40.91g
Na2HPO4·2H2O 10 mM 0.89 g
KCl 27 mM 1.013 g
KH2PO4 18 mM 1.23 g
Dissolved in ddH2O n/a 500 mL
Adjust to pH 7.4 with 10% KOH. For 1× PBS, dilute 10× PBS 1:10 with ddH2O (Recipe 6)
1× PBS Buffer
Reagent Final concentration Amount
10× PBS Buffer 1× 10 mL
ddH2O n/a Ad 100 mL
Store at RT.
WGA AF488 stock solution
Reagent Final concentration
WGA AF488 1 mg/mL in H2O
Prepare aliquots and store at -20 °C. Protect from light.
WGA AF488 staining solution
Reagent Final concentration Amount
WGA AF488 stock solution 10 μg/mL in 1× PBS 10 μL/mL
Always prepare fresh before use.
30% glycerol solution
Reagent Final concentration Amount
Glycerol (86%) 30% 34,88 mL
ddH2O n/a Ad 100 mL
Store at RT
Laboratory supplies
Pots, 9–10 (maximum) cm diameter
Soil (Einheitserde classic; Profisubstrat, e.g., 814510; Meyer-Shop.de)
Metal trays (50 cm × 29.5 cm)
1.5 mL reaction tubes (Sarstedt, catalog number: 72.690.550)
Medical clay (Drug store)
Black ceramic tile (Hardware store)
Small spatula
Razor blade (Wilkinson)
Pipe cleaners, cotton, flexible, 6 mm diameter, 15–20 cm length (Carl Roth, catalog number: YC35.1 or Amazon e.g., VAUEN Cotton pipe cleaners)
Plastic cups (Plastikbecher.de, PP Becher 560/500 g natur A’50)
Parafilm (Carl Roth, catalog number: H666.1)
Lighter
Filtered tips 1 mL (Sarstedt, Biosphere plus, catalog number: 70.3050.255)
Silica beads (Carl Roth, catalog number: 1779.2)
Flexible tube for vacuum pump (0.8 cm diameter)
Scissor
Exsiccator with lid
Disposable gloves
Small plastic labels
2.0 mL reaction tubes (Sarstedt, catalog number: 72.695.500)
Object slides (Carl Roth, catalog number: 0656.1)
Cover slides (Carl Roth, catalog number: 1871.2)
Equipment
Precision balance (e.g., Sartorius)
Poly klima cabinet, true daylight (Poly klima GmbH, PK 520)
Vacuum pump (Welch, model: 2522C-02A)
Airbrush (GANZTON SP180K airbrush system, e.g., Amazon)
Water bath
4 °C refrigerator
-80 °C freezer
Scanner
Fluorescence microscope (Nikon, Nikon Ti Eclipse)
Software and datasets
NIS Element Software (Nikon)
Procedure
Pre-propagation of Puccinia hordei (Ph) on barley plants
Preparation of plants for pre-propagation
A pre-propagation step is recommended to generate fresh spores.
Note: A pre-propagation step is recommended if a new rust isolate will be established in the lab, if the rust spores have been stored at -80 °C for more than four months, or if the number of freshly stored spores is not sufficient for the planned infection experiment. In the pre-propagation step, a susceptible cultivar should be used to generate a sufficient number of fresh spores for upcoming infection experiments. For new rust isolates, the pre-propagation step allows to estimate the infection strength of the new isolate and, if needed, can be the basis for adapting parameters of the planned infection experiment (for example the number of spores required for a sufficient infection). For Ph isolate I-80, the pre-propagation is usually performed with the barley cultivar Großklappige, but from our experience, Golden Promise is also suitable.
Depending on the number of spores required for subsequent infection trials, select the number of seeds. In our hands, 50 plants for pre-propagation are sufficient to yield spores for infection of 1,000 plants.
If necessary, sterilize seeds before putting them into the soil.
Note: We prefer to sterilize our seeds with 6% NaClO prior to sowing them into soil. This reduces the risk of transmitting any pests by the seeds that might occur due to pre-harvest infections in the greenhouse or during storage. It is very important to keep the plants free of any secondary infection, as this will contaminate the propagated rust spores and might influence the outcome of the infection, for example due to priming of the plants’ resistance mechanisms. However, if the seeds were produced and stored under clean conditions, this step is not required.
Fill 10 pots [9–10 cm (maximum) diameter] with soil.
Plant five seeds per pot.
Note: The pots have a small diameter (maximum 10 cm); five plants per pot are easy to infect and handle. However, it is also possible to add up to 10 plants, but this makes the handling of the individual plants a bit more difficult.
Grow the plants under controlled conditions either in the greenhouse, phytochambers, or phytocabinet (day/night temperature 18 °C/16 °C, light 16 h/8 h).
Crucial: It is very important that the plants show NO secondary infections with other pathogens or pests.
In this protocol: use 10-day-old plants (beginning 2nd leaf stage) for infection.
Note: In principle, it is possible to infect older plants. The limiting factor here is the size of the plastic cups used to cover the pots. For older plants, bigger plastic cups have to be used.
Assembling plastic cup covers for pots
Note: An elementary feature of this protocol is the homemade plastic cup covers, which are placed over each individual pot. This generates a high humidity that is required for the efficient germination of the spores in the first 48 h after infection. The Poly klima cabinet PK 520 that was used in this protocol has a certain size limitation. Therefore, this method is more suitable than covering all the plants on the metal tray with one single cover. In addition, the individual pot covers allow an easy watering of the plants from the bottom by simply pouring water in the metal tray. For this, the covers do not need to be lifted and the humidity stays stable in the first 48 h after infection. Furthermore, we recommend reusing the pot covers. Therefore, after finishing the infection experiments, we spray the pots with 70% EtOH, rinse them in tap water to remove traces of soil or spores, and let them dry before the next use. However, if more space is available, a big plastic cover can be used to cover the whole tray, but it is very important to ensure that the cover is tight enough to keep a sufficient humidity.
One cover is needed for one pot.
One cover is assembled from two plastic cups (Figure 1A).
With the help of scissors, cut off the bottom of one of the two cups (this new opening will later be inserted into the soil, Figure 1B).
Place the two regular openings of the two cups together and close them using parafilm (Figure 1C).
Prepare as many pot covers as needed for the propagation or infection experiment (the maximum number of pots in the PK 520 is 54).
Figure 1. Preparation of pot covers. A. Take two plastic cups. B. Cut the bottom of one plastic cup. C. Connect and seal the two original openings of the plastic cups by using parafilm. The pot cover now has one opened and one closed side.
Spore preparation and infection
If the spores were frozen at -80 °C, follow steps b–s. For fresh spores, follow steps c and f–s.
Preheat water bath to 45 °C.
Weigh 6 mg of spores per 50 plants into a 1.5 mL reaction tube using a precision balance.
Note: In our experience, the use of 6 mg of Ph I-80 spores per 50 plants leads to a very strong infection pattern.
Activate the spores for 10 min by placing the tube in a water bath at 45 °C. Ensure that no water runs into the tube.
Remove reaction tube from water bath and store spores in the dark at RT for 1 h.
In the meantime, assemble the airbrush under the fume hood and prepare 0.01% Tween 20 solution according to the Recipe 2 (Figure 2A).
Spray the plants uniformly with Tween 20 solution using the airbrush (0.3 mm nozzle, 0.3 mm needle, spray from a distance of at least 10 cm; Figure 2B and 2C).
Notes:
i. It is important to avoid drop formation on the leaves. The Airbrush is able to apply a very fine film on the leaf surface. The Tween 20 solution ensures a better adhesion of the spores on the surface by reducing the surface tension.
ii. It is also possible to apply the spores dissolved in mineral oil or engineered fluids (e.g., Soltrol or Novec 7100) with an airbrush as shown for stripe rust in Sørensen et al. (2016). In our hands, the use of mineral oil caused spore clumps and the spores did not germinate well. In addition, the mineral oil or engineered fluid can cause unwanted reactions (burns) on the leaf surface.
Figure 2. Spraying leaves with the airbrush. A. Assembled GANZTON airbrush set with compressor (0.3 mm nozzle and 0.3 mm needle). B. Spraying the barley leaves. C. Slightly moisturized leaves after spraying.
Prepare the required material for the infection procedure (Figure 3A and 3B). Bend the pipe cleaner at the tip (approximately 1 cm) to form a slight angle (Figure 3A).
Add the medical clay to the spores in a ratio of 3:1 (Figure 3C) and mix well by shaking the reaction tube vigorously (Figure 3D).
Pour the spore-clay mix onto a black tile (Figure 3E).
Carefully dip the bent pipe cleaner into the spore clay mix and spread the mixture evenly over the primary leaf, moving the pipe cleaner over the leaf from top to bottom and back again with a little pressure (Figure 3F). If a secondary leaf is present, you can infect it too.
Note: For pre-propagation, the number of spores applied does not need to be as precisely defined as for a phenotyping experiment. So, it is sufficient here to gently dip the pipe cleaner into the spore medical clay mix but ensure that enough spores are available to infect all plants.
Repeat the procedure until all plants are infected.
Spray the inside of the prepared cover cups with 0.01% Tween 20 solution using the airbrush to moisturize the inside of the cover cups before putting them over the plants (Figure 3G).
Crucial: This is a very important step, to ensure a moisturized environment for the spores to germinate.
Carefully place the now moisturized cover cups over the plants and press them into the soil (Figure 3H).
Place the covered pots on a metal tray and make sure that the pots have a firm stand. Incubate them in a Poly klima PK520 cabinet or other phytocabinet.
Incubate the plants in the dark, at 20 °C/18 °C for 16 h/8 h.
Twenty-four hours after infection: turn on the light and further incubate the plants in a 16:8 h day/night rhythm at 20 °C/18 °C.
Water the plants regularly and make sure that the soil in the pots is always slightly moisturized.
Forty-eight hours after infection: remove the plastic cups.
Four days after infection, the first small lighter spots should be visible on the leaves.
Seven days after infection, clear symptoms are visible.
Figure 3. Infection procedure. A. Material needed for infection procedure. B. Image of the size and packaging of the cotton pipe cleaner. C. The medical clay is added to the spores in an approximately 3:1 ratio. D. The clay is mixed with the spores by shaking the tube. E. The spore clay mix is poured on a black tile and picked up with the pipe cleaner. F. The barley plant is infected by carefully rubbing the pipe cleaner over the surface of the adaxial side of the leaf. G. The inner side of the pot covers is sprayed with Tween 20 solution. H. The moisturized cover cups are placed over the plants and fixed by pressing them slightly into the soil.
Spore harvesting and storage
After 12–14 days, the emerging pre-propagated spores are mature for harvesting; harvesting can be done on several consecutive days. We recommend using the described spore hoover to harvest as much clean spores as possible; this will reduce the spilling of the spores in the room and avoid contamination with soil.
Prepare the equipment for the vacuum spore hoover.
Carefully heat the tip of a 1 mL filter tip by using a lighter. Press scissors or forceps on the warm tip to flatten the opening (Figure 4A).
Cut the flattened tip by another 3 mm to re-open (Figure 4A).
Prepare 3–5 of these tips for spore harvesting.
Note: The prepared tips are used to easily harvest, dry, and store the pre-propagated spores. In the harvesting procedure, we typically exchange the tips every 8–10 leaves. With this procedure, it is possible to achieve between 3 and 10 mg of spores per tip, depending on the infection strength in the infected leaves. To remove the spores from the tips, simply cut the tip open and pour the spores into a reaction tube.
We recommend marking each tip with a number. This will help to identify the batch of harvested spores after storage.
Weigh the prepared tips before harvesting using a precision balance.
Write down the weight of each tip and the harvesting date.
Assemble the spore hoover: attach a long rubber tube (inner diameter 8 mm) to the vacuum pump and attach the prepared filter tip to the other opening (Figure 4B).
Start the vacuum pump (500–700 mbar), carefully hold the tip of a leaf, and harvest the mature spores by gently running the hoover over the leaf surface. Adjust the vacuum if it is too strong or not strong enough (Figure 4C; Video 1).
We recommend harvesting 8–10 nicely infected leaves in one tip and then changing to the next tip.
For changing the tip or finishing the harvesting procedure, turn off the vacuum pump and carefully remove the filtered tip. It is recommended to store or transport the tips upright in a reaction tube rack.
Carefully place the filled tips on the precision balance and weigh again; the difference in weight determined before harvesting (steps g–h) gives the number of harvested spores. Note the number of harvested spores for each tip.
Drying of the spores: place the tips in an exsiccator filled with silica beads (Figure 4D), close the lid, seal with parafilm, and store in the dark at 4 °C for 48 h (Figure 4E). Instead of an exsiccator, any other jar or vessel that can be tightly closed can be used.
After the 48 h drying period of the spores, place two silica beads behind the filter of the tip and seal the opening with parafilm (Figure 4F). Also seal the tip of the tip with parafilm.
Note: It is important that the harvested spores are not exposed to moisture during storage, because this can cause clumps. Therefore, silica beads should be added to the tip.
The prepared spores can be directly used for the planned infection experiments or stored at -80 °C for long-term storage.
Note: The spores can be stored in a refrigerator at 4 °C for maximum 14 d before they should be used in an infection experiment or transferred to a -80 °C freezer for long-term storage.
Figure 4. Spore harvesting procedure. A. Prepare the 1 mL filter tips. Carefully heat and flatten the tip. Cut the flattened part of the tip to generate a flattened opening. B. Attach a tube with an inner diameter of approximately 8 mm to the vacuum pump and insert the tip at the end of the tube. C. Start the vacuum (between 500 and 700 mbar is normally sufficient). Carefully move the spore hoover over the leaf surface to collect the spores in the filter tip. D. For drying, store the collected spores in the tips in an exsiccator filled with silica beads. E. Tightly close the lid of the exsiccator with parafilm during storage at 4 °C. F. 1 mL filter tip with harvested spores and silica beads, closed with parafilm.
Video 1. Spore hoover. Application of the spore hoover to harvest rust spores.
Infection experiment for phenotyping of mutants or cultivars
Sow the plants required for the infection experiment.
Notes:
i. With the standard protocol, 270 plants can be analyzed per experimental series (five plants per pot, nine pots per metal tray, six metal trays distributed on the three levels of the Poly klima PK250 cabinet). This number can be increased if more than five plants per pot are sown.
ii. For experiments with plants that need to be analyzed with additional methods, like PCR, it is recommended to attach small labels to the individual plants to identify them later.
After 10 days, the plants should be of good size for infections as described before.
Note: Infections of younger or older plants are possible; however, the number of spores should be adjusted.
Make sure that the plants have not acquired infections or damage from predators in the rearing process, as this may affect the results of the infection experiment.
Make sure that sufficient plastic cup covers are available.
Spore preparation
We recommend using 10 mg of Ph I-80 spores per 100 plants.
Weigh the required number of fresh, dried spores into a 1.5 mL reaction tube.
Add medical clay in the ratio of 3:1 and mix by shaking vigorously.
Note: If there are not enough fresh spores to perform the experiment, it is possible to use a proportion of frozen spores from the -80 °C. From our experience, a spore mix in the ratio 1:5 of frozen and fresh spores can be used. The frozen spores must be activated before infection, as described in A3. Depending on the barley genotype or Ph isolate, adjust the number of spores if necessary.
Infection procedure
Spray the plants with 0.01% Tween 20 solution using the airbrush as described in A3.
Carefully pour the spore-clay mix onto a black tile (Figure 5A).
Divide the spore pile in subfractions. Calculate the required subfractions according to the metal trays, pots, and plants. For example, if you use six metal trays, prepare six subfractions; if there are nine pots on each metal tray, further split one of the six piles into nine smaller piles. If there are five plants in one pot, divide one pile in five smaller piles (Figure 5B). Each of these small piles represents one plant in the pot.
Bend the pipe cleaner at the thick end and shape it into a slight angle.
With the bent end, pick up one of the five spore piles with some pressure (Figure 5C) and circular movements and apply it to the first plant previously wetted with Tween 20.
Figure 5. Preparation of spores for infection. A. Pour the spore clay mix on a black tile. B. Divide the spore-clay mix into subfractions, according to the number of trays (six), the number of pots (nine), and the number of plants in each pot (five), by using a razor blade. C. Pick up one pile per plant using the bent end of the pipe cleaner. Note that for picture B and C, only clay was used to visualize the different steps of the subfractions.
Hold the leaf carefully with one hand and move the pipe cleaner from the tip of the leaf to the stem and back.
Crucial: Exchange the pipe cleaner if it gets wet; otherwise, the spores will clump and stick to the pipe cleaner.
Repeat steps e and f until all plants are infected.
Spray the inside of the plastic pot covers with the 0.01% Tween 20 solution using the airbrush and carefully place the covers over the infected plants.
Place the covered pots on a metal tray and make sure that the pots have a firm stand. Incubate them in a Poly klima PK520 cabinet with day and night settings of 20 °C/18 °C and 16/8 h light.
Incubate the plants for 24 h in the dark; then, turn on the light.
Forty-eight hours after infection, remove the plastic cups.
Four days after infection, light spots should be visible on the leaves.
Seven days after infection, clear symptoms are visible.
Scoring can be done at 10 and 13 days post-inoculation (dpi) for example, according to Levine and Cherewick (1952).
In this protocol, the leaf samples were harvested at 10 dpi.
Carefully separate the infected primary leaves close to the stem and fix them on a black cardboard (DIN A4) at the top and bottom of the leaf with a thin strip of tape.
Carefully place the cardboard with the leaf samples on a scanner and perform a scan with the highest possible resolution (Figure 6).
Figure 6. Example scan of infected barley plants from one infection experiment (raw scan). Eleven individual plants of a susceptible barley line infected with Puccinia hordei I-80 are shown at 10 dpi.
Histological analysis of fungal growth via WGA AF488 staining
Sample harvesting and staining
Harvest samples at the desired time point. For analyzing the fungal development via microscopy, harvesting time points between 2 and 6 dpi are recommended, depending on the cultivar and isolate used.
Perform WGA AF488 staining as described in Redkar et al. (2018), with the following modifications for barley:
Prepare the solutions described above as Optional Recipes for staining.
Harvest approximately 2 cm × 2 cm leaf segments per plant.
Bleach the leaves until they are completely decolorized. Instead of 100% ethanol, use the bleaching solution (Recipe 3, Figure 7A). The samples can be stored in this solution until needed.
Treat the leaves with 1 M KOH (Recipe 4). KOH treatment should be reduced to 1 h at RT.
Note: Depending on the genotype and age of the leaf, this process might be optimized.
Crucial: After KOH treatment the samples will get very soft; handle them with care.
A vacuum infiltration step is not necessary.
Wash the samples at least three times with water.
Wash the samples once with 1× PBS (for 10× PBS stock, see Recipe 5; for 1× PBS see Recipe 6).
Dilute the WGA AF488 stock in sterile ddH2O (1 mg/mL; see Recipe 7). Prepare aliquots and store at -20 °C in the dark (e.g., wrapped in aluminum foil).
Samples should be incubated in WGA AF488 staining solution (10 μL per 1 mL 1× PBS; see Recipe 8) for 24 h in the dark (e.g., wrapped in aluminum foil or stored in a closed drawer). Remove the staining solution and wash with 1× PBS. Store the sample in 1× PBS.
Note: The WGA AF488 staining is light-sensitive; therefore, always protect your samples from the light. For example, avoid direct sunlight during handling and store the samples in a drawer or cover them with aluminum foil or black paper.
Histological analysis
Carefully transfer the samples stored in 1× PBS onto an object slide with the bottom side of the leaf facing down.
Add a few drops of 30% glycerol solution (Recipe 9) to the slide before carefully placing the coverslip without air bubbles (Figure 7B). Protect from light.
Note: The staining of the fungal structures on the leaf samples is stable for several months if the slides are stored in the dark, e.g., in a drawer, wrapped in aluminum foil, or placed in a folder for microscopy slides.
Figure 7. Preparation and microscopy of infected leaf samples. A. Leaf samples in bleaching solution, harvested at 6 dpi. B. Leaf samples on the object slide after staining with WGA AF488. C. Histological analysis of infected leaf sample at 6 dpi with 10× magnification using the channel for GFP. D. Merged picture of the brightfield and GFP channel at 10× magnification.
Analyze the fungal development using a fluorescence microscope (in this protocol, Nikon Ti fluorescence microscope).
Note: For microscopy, the samples should be harvested between 2 and 6 dpi; otherwise, the infection sites in susceptible genotypes are usually too large to determine their size at 10× magnification. In our hands, 4 dpi is most suitable.
It is possible to calculate the size of the infection sites using the object count function of the NIS Elements software.
Note: The calculation with the NIS software is based on a one-point threshold definition. With the GFP filter and 10× magnification, the hue of a characteristic fluorescence signal in the respective infection site is selected to define the threshold for object detection. Based on this calibration, the software calculates the fluorescing area in μm2. For very detailed images, it is advisable to examine the samples with a confocal microscope.
Validation of protocol
The protocol was used for the infection analysis presented in Skoppek et al. (2022).
General notes and troubleshooting
General notes
Always work very clean. Exchange used material if you change the fungal pathogen to avoid cross contamination.
If you are using a different fungal isolate or plant genotype than described here, always pre-define the settings again.
You can also transfer this protocol to stripe rust or to powdery mildew as well as to other plants.
The protocol is also applicable for a detached leaf assay.
Acknowledgments
This work was funded by university core funding only. We thank Gwendolin Wehner and Frank Ordon from the Julius Kühn Institute in Quedlinburg for Ph isolate I-80. We thank Jens Boch for general support.
This protocol was derived from Skoppek et al. (2022). The staining method was adapted for barley from Redkar et al. (2018).
Competing interests
The authors declare to have no competing interests.
References
Dinh, H. X., Singh, D., Periyannan, S., Park, R. F. and Pourkheirandish, M. (2020). Molecular genetics of leaf rust resistance in wheat and barley. Theor Appl Genet 133(7): 2035-2050.
FAO (Food and Agriculture Organization of the United Nations). (2009). How to Feed the World in 2050. Rome. https://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf
König, J., Kopahnke, D., Steffenson, B. J., Przulj, N., Romeis, T., Röder, M. S., Ordon, F. and Perovic, D. (2012). Genetic mapping of a leaf rust resistance gene in the former Yugoslavian barley landrace MBR1012.Mol Breed 30: 1253-1264.
Levine, M. N. and Cherewick, W. J. (1952). Studies on dwarf leaf rust of barley. USDA Tech Bull.
Miladinovic, D., Antunes, D., Yildirim, K., Bakhsh, A., Cvejić, S., Kondić-Špika, A., Marjanovic Jeromela, A., Opsahl-Sorteberg, H. G., Zambounis, A. and Hilioti, Z. (2021). Targeted plant improvement through genome editing: from laboratory to field.Plant Cell Rep 40: 935-951.
Redkar, A., Jaeger, E. and Doehlemann, G. (2018). Visualization of growth and morphology of fungal hyphae in planta using WGA-AF488 and Propidium Iodide co-staining.Bio-101: e2942.
Skoppek, C. I., Punt, W., Heinrichs, M., Ordon, F., Wehner, G., Boch, J. and Streubel, J. (2022). The barley HvSTP13GR mutant triggers resistance against biotrophic fungi.Mol Plant Pathol 23(2): 278-290.
Sørensen, C. K., Thach, T. and Hovmøller, M. S. (2016). Evaluation of spray and point inoculation methods for the phenotyping of Puccinia striiformis on wheat. Plant Dis 100(6): 1064-1070.
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Microbiology > Microbe-host interactions > Fungus
Plant Science > Plant immunity > Host-microbe interactions
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A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues
MG Mehmet Girgin
NB Nicolas Broguiere
LM Lorenzo Mattolini
ML Matthias Lutolf
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4722 Views: 1214
Reviewed by: Rajesh RanjanHélène LégerSamantha Haller
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Original Research Article:
The authors used this protocol in Stem Cell Reports May 2021
Abstract
Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering.
Key features
• A new gastruloid culture system to model post-implantation mouse embryonic development in vitro
• High-throughput formation of epiblast-like aggregates on hydrogel microwells
• Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues
Graphical overview
Keywords: Gastruloid Organoid Developmental biology Embryoid Synthetic embryos
Background
The first attempts to model mouse embryonic development in vitro begun with a coincidental observation. When teratocarcinomas from neonatal mouse testes were analyzed, scientists noticed a structural similarity to the developing mouse embryo (Stevens, 1959). Hence, they called these tumors “embryoid bodies” (Pierce et al., 1960). It was further shown that embryoid bodies retained the tumor-forming capacity to form cell types ranging from cartilage to neural tissue, a multipotency that was later attributed to the embryonal carcinoma (EC) cells (Kleinsmith and Pierce, 1964). The use of EC cells to mimic embryonic development was rapidly replaced by embryonic stem cells (ESCs) following their isolation in 1981 (Evans and Kaufman, 1981). Pioneering studies have demonstrated self-organization potential of embryoid bodies to recapitulate, to a limited extent, gastrulation-like events and antero-posterior axis determination (ten Berge et al., 2008). However, this was not accompanied by axial morphogenesis.
More recently, gastruloids have taken the extent of self-organization potential of mouse ESCs (mESCs) to demonstrate that embryoid bodies could undergo axial morphogenesis (van den Brink et al., 2014). In this model, when treated with Wnt agonist CHIR99021, small aggregates of mESCs (~300 cells) were shown to break symmetry and demonstrated polarized T/Bra expression in a reproducible way. When cultured further, gastruloids elongated and established patterning along antero-posterior, dorso-ventral, and medio-lateral axes (Beccari et al., 2018). Moreover, the multi-axial patterning was linked to spatiotemporal activation of Hox gene clusters, a phenomenon that is conserved across many species (Santini et al., 2003). Such self-organization potential of mESCs, in the absence of any extraembryonic tissue, was remarkably similar to the developing post-occipital region of the mouse embryo; however, tissues mapping to anterior brain regions were completely absent in gastruloids.
Mechanical forces and extracellular matrix composition have significant influence on the developing mouse embryo (Hiramatsu et al., 2013). When gastruloids are embedded in a basement membrane substitute, they remarkably organize into structures bearing somites and a central neural tube-like tissue (Veenvliet et al., 2020). Even under these conditions, gastruloids fail to form any anterior neural tissues corresponding to the brain regions.
The epiblast is the domain that forms the embryo proper. However, contribution from extraembryonic tissues is required since development is halted in their absence (Donnison et al., 2005; Rodriguez et al., 2005). Studies accounting for this necessity have therefore established hybrid embryoid models, combining embryonic stem cells with trophoblast stem cells (TSCs) (Harrison et al., 2017; Rivron et al., 2018) and/or extraembryonic endoderm cells (XENs) (Sozen et al., 2018). More recently, mESCs were co-cultured with transdifferentiated TSCs and XENs to generate structures that almost completely recapitulate E8.5 embryos, including brain tissues (Amadei et al., 2022; Tarazi et al., 2022). However, the complexity of the tri-culture system and necessity of special equipment to grow them until late stages remain as the major limitations in generating synthetic embryos.
Previously, it was shown that embryos lacking extraembryonic Wnt source could still break symmetry and initiate gastrulation, suggesting an autonomous developmental potential of epiblast cells (Yoon et al., 2015). Moreover, overactivation of Wnt signaling in the epiblast was shown to deplete anterior neural progenitors in the favor of mesoderm derivatives; this phenotype could be rescued by inhibition of Wnt signaling (Osteil et al., 2019). Therefore, we reasoned that a similar trade-off could be happening in conventional gastruloids, owing to overactivation of Wnt signaling by CHIR99021 treatment.
Here, we report a new model system based on aggregation of mESCs to derive post-implantation epiblast-like structures (EPI aggregates). We formulated a serum-free epiblast-induction medium comprising Activin-A (Tgf-β agonist), Fgf2 (Fgf agonist), and knockout serum replacement, which promoted the acquisition of epiblast identity, followed by their spontaneous symmetry breaking and subsequent morphogenesis without any external Wnt stimulation. Moreover, inhibition of Wnt signaling during early stages of EPI aggregate formation helped to maintain anterior neural progenitors, which then committed to generate forebrain-, midbrain-, and hindbrain-like tissues. This protocol expands the cell type repertoire that can be generated within gastruloids.
Materials and reagents
Mouse embryonic stem cell lines
SBr [SOX1-GFP; T/BRA-mCherry] mESC line (Deluz et al., 2016)
WNT [TLC-mCherry] mESC line (Ferrer-Vaquer et al., 2010; Faunes et al., 2013)
TGF-β [AR8-mCherry] mESC line (Serup et al., 2012)
Cell culture materials
Ultra-low attachment 96-well plate (Corning, catalog number: 7007)
6-well plate, tissue culture–treated (Falcon, catalog number: 38016)
6-well plate, non tissue culture–treated (Corning, catalog number: CLS3471)
PEG microwells (400 μm microwell diameter, 121 wells/array in 24-well format) (Brandenberg et al., 2020). Commercially available Gri3D® 96-well plate (400 μm microwell diameter, 121 wells/array) could be used alternatively.
10 cm plate, non tissue culture–treated (Corning, catalog number: CLS430591)
Pipettes (2, 5, 10, 25, and 50 mL)
Micropipettes (1–10, 2–20, 20–200, and 100–1,000 μL)
Pipette tips (10, 20, 200, and 1,000 μL)
Cell culture reagents
Base media and dissociation reagents
DMEM + GlutaMAX (Gibco, catalog number: 61965-026). Keep at 4 °C
DMEM/F12 + GlutaMAX (Gibco, catalog number: 31331-028). Keep at 4 °C
Neurobasal (Gibco, catalog number: 21103-049). Keep at 4 °C
Accutase (Gibco, catalog number: A11105). Keep at 4 °C
PBS (Gibco, catalog number: 10010023). Keep at room temperature
Media supplements
ESC-qualified FBS (Gibco, catalog number: 16141-079). Keep stock at -80 °C, working aliquots at -20 °C
KnockOutTM serum replacement (Thermo Fisher Scientific, catalog number: 10828010). Keep stock at -80 °C, working aliquots at -20 °C
N2 supplement (Gibco, catalog number: 17502001). Keep at -20 °C
B27 supplement (Gibco, catalog number: 17504001). Keep at -20 °C
Sodium pyruvate (Gibco, catalog number: 11360-070). Keep at 4 °C
MEM non-essential amino acids (Gibco, catalog number: 11140-035). Keep at 4 °C
2-mercaptoethanol (Gibco, catalog number: 31350-010). Keep at 4 °C
Penicillin/Streptomycin (Gibco, catalog number: 15140-122). Keep working aliquots at -20 °C
GlutaMAX (Gibco, catalog number: 35050-038). Keep at room temperature
Growth factors and small molecule inhibitors
CHIR99021 (Tocris, catalog number: 4423). Keep stock at -80 °C, working aliquots at 4 °C
PD0325901 (Selleckchem, catalog number: S1036). Keep stock at -80 °C, working aliquots at 4 °C
LIF (in-house preparation). Keep stock at -80 °C, working aliquots at 4 °C
FGF2 (Gibco, catalog number: PMG0035). Keep stock at -80 °C, working aliquots at -20 °C. Use within three months after reconstitution
ACTIVIN A (R&D systems, catalog number: 338-AC). Keep stock at -80 °C, working aliquots at -20 °C. Use within three months after reconstitution
XAV939 (Tocris, catalog number: 3748). Keep stock at -80 °C, working aliquots at -20 °C
Immunostaining materials and reagents
4% PFA (Thermo Fisher, catalog number: J19943.K2)
DAPI (Sigma, catalog number: 9542)
Glass slides
Coverslips (1.5 thickness)
Mounting medium (Vectashield, catalog number: H-1000-10)
Nail polish
Triton X-100 (Sigma, catalog number: X100)
Mouse embryonic stem cell (mESC) maintenance medium (500 mL) (see Recipes)
EPI differentiation medium (500 mL) (see Recipes)
Primary and secondary antibodies (Table 1)
Table 1. List of primary and secondary antibodies
Target Species Dilution Catalogue number Supplier
Anti-E-cadherin Rabbit 1:500 24E10 Cell Signaling Technology
Anti-Sox2 Rabbit 1:400 ab97959 Abcam
Anti-Sox1 Goat 1:50 af3369 R&D Systems
Anti-Otx2 Goat 1:25 af1979 R&D Systems
Anti-Brachyury Goat 1:300 sc-17745 (C-19) Santa Cruz
Anti-Brachyury Rabbit 1:100 ab209665 Abcam
Anti-Oct4 Mouse 1:200 sc-5270 (C-10) Santa Cruz
Anti-Nanog Rat 1:300 14-5761-80 Thermo Fisher
Anti-Cdx2 Rabbit 1:200 ab76541 Abcam
Anti- aPKC Mouse 1:100 sc-17781 (H-1) Santa Cruz
Anti-Sox17 Goat 1:200 AF1924 Abcam
Anti-Cdx2 Rabbit 1:200 ab76541 Abcam
Anti-mCherry Rat 1:400 M11217 Thermo Fisher
Phalloidin AF488 1:1,000 A12379 Thermo Fisher
Phalloidin AF635 1:1,000 A34054 Thermo Fisher
Anti-chicken Alexa Fluor 488 Donkey 1:500 703-545-155 Jackson ImmunoResearch
Anti-rat Alexa Fluor 568 Goat 1:500 A-21247 Thermo Fisher
Anti-goat Alexa Fluor 568 Donkey 1:500 A-11057 Thermo Fisher
Anti-goat Alexa Fluor 647 Donkey 1:500 A-21447 Thermo Fisher
Anti-rabbit Alexa Fluor 488 Donkey 1:500 A-21206 Thermo Fisher
Anti-rabbit Alexa Fluor 568 Donkey 1:500 A-10042 Thermo Fisher
Anti-rabbit Alexa Fluor 647 Donkey 1:500 A-31573 Thermo Fisher
Anti-mouse Alexa Fluor 568 Donkey 1:50 A-10037 Thermo Fisher
Anti-mouse Alexa Fluor 647 Donkey 1:50 A-31571 Thermo Fisher
Equipment
Incubator with regulated temperature and humidity
Centrifuge
Biological safety cabinet
4 °C fridge, -20 °C freezer, and -80 °C freezer
Eclipse inverted microscope (Nikon, catalog number: TS100)
Pipettes (single and multichannel)
Sterile tips
Hemocytometer and Countess 3 automated cell counter (catalog number: AMQAX2000)
Zeiss LSM700 inverted confocal microscope
Nikon Eclipse Ti inverted microscope
EC Plan-Neofluar 10×/0.30 and Plan-Apochromat 20×/0.80 air objectives
Software
Zen software (2009)
NIS elements (version 4.11.0)
Fiji ImageJ (version 2.0.0-rc-69/1.52n)
GraphPad Prism software (version 8.4.2)
RStudio (version 1.3.1056)
Procedure
Passaging of mouse embryonic stem cells
In a Falcon tube, prepare 4.5 mL of mESC maintenance medium (without CHIR99021, PD0325901, and LIF) to collect cells in. In a separate tube, prepare 3 mL of s2iL medium (see Recipe 1).
Check mESCs under microscope. The colonies should be round and not touching each other (Figure 1).
Figure 1. Representative immunofluorescence image of mouse embryonic stem cells (mESCs) grown in s2iL medium stained for SOX2 (green) and F-Actin (red) (left panel), and KLF4 (green) and TFCP2L1 (red) (right panel). Scale bars = 200 µm.
Remove medium from the mESCs and wash with 2 mL of PBS (without calcium and magnesium).
Add 550 μL of room-temperature accutase for dissociation. Incubate for 2–3 min at room temperature. Gently tap the plate to lift up colonies.
Pipette up and down 10–15 times to dissociate colonies into single cells. Make sure cells are single by checking under the microscope.
Place the cell suspension in previously prepared 4.5 mL of mESC maintenance medium.
Spin down at 200× g (1,000 rpm) for 5 min.
Remove supernatant and resuspend in 1 mL of s2iL medium.
Count cells with hemocytometer or automated cell counter.
Seed 50,000–60,000 cells per well of 6-well plate in s2iL medium. Distribute cells equally by shaking the plate back and forth and left and right.
Place the plate back in the incubator and culture for two or three days at 37 °C with 5% CO2 and 21% O2.
Preparing EPI aggregates on PEG microwells
Carefully remove PBS from PEG microwells (400 μm well diameter, 121 wells per array) without touching the hydrogel.
Add 50 μL of EPI differentiation medium on top of the array and place the microwells in the incubator for at least 30 min to equilibrate.
Meanwhile, remove medium from the mESCs and wash with 2 mL of PBS (without calcium and magnesium).
Add 550 μL of room-temperature accutase. Incubate for 2–3 min at room temperature. Gently tap the plate to lift up colonies.
Pipette up and down 10–15 times to dissociate colonies into single cells.
Place the cell suspension in previously prepared 4.5 mL of mESC maintenance medium.
Spin down at 200× g (1,000 rpm) for 5 min.
Remove supernatant and resuspend in 10 mL of PBS.
Spin down at 200× g (1,000 rpm) for 5 min.
Remove supernatant and resuspend in 10 mL of PBS.
Spin down at 200× g (1,000 rpm) for 5 min.
Resuspend in appropriate volume of EPI differentiation medium to obtain a cell suspension of 484,000 cells/mL.
Carefully remove the 50 μL of EPI differentiation medium from the microwell arrays and add 35 μL of cell suspension, to have 100–150 cells per each well of the microwell array.
Do not shake or swirl the plate as it will disturb the equal segregation of cells. Incubate at 37 °C for 30 min for cells to sediment.
Slowly add 1 mL of EPI differentiation medium or EPI+XAV differentiation medium (see Recipe 2) from the notch. Do not dispense liquid directly on the microwell array, as it will cause cells to lift up.
Place the plate back in the incubator and culture for at least 72 h at 37 °C with 5% CO2 and 21% O2. Cells should sediment at the bottom of microwells and form clumps within an hour of seeding (Figure 2).
Figure 2. Representative images showing mouse embryonic stem cells (mESCs) seeded at indicated numbers at 0 h and resulting in EPI or EPI+XAV aggregates at 72 h. Scale bar = 200 μm.
Transferring EPI aggregates into 96-well plates
At approximately 75 h of culture, carefully remove the EPI differentiation medium from the microwells. Dispense 1 mL of fresh N2B27 directly on the microwell array at different locations to lift EPI aggregates up.
Collect EPI aggregates in non-tissue culture–treated 10 cm dishes. Repeat the collection with 1 mL of N2B27 five times. Add 5 mL of N2B27 directly to the 10 cm dish to add up to 10 mL.
Observe EPI aggregates under the microscope. It is advised to do the picking under sterile conditions, under the cell culture hood.
Pick EPI aggregates under the microscope in 10 μL and dispense directly in a well of ultra-low attachment 96-well plate filled with 180 μL of N2B27. Make sure that transferred EPI aggregates are not damaged and maintain smooth edges.
Repeat this step until the 96-well plate is completely filled. It usually takes 30 min to 1 h to fill a full plate.
Place the plate in the incubator.
Every 24 h until 168 h, change 150 μL of medium with fresh N2B27 using a multichannel pipette (Figure 3).
Figure 3. Montage of EPI or EPI+XAV aggregates formed from 100 cells/well shown at 144 h and 168 h after segmentation. T/Bra expression is shown in red. Sox1 expression is shown in green. Note the Sox1-positive domain localized on the opposite end of T/Bra-positive domain in EPI+XAV aggregates.
Preparing gastruloids
Follow steps B1–B11.
Resuspend in appropriate volume of N2B27 medium to obtain a cell suspension of 7,500 cells/mL. For example, 37,500 cells should be added in 5 mL of N2B27 to prepare a full 96-well plate of gastruloids.
Add 40 μL of the cell suspension per well of a 96-well plate using a multichannel pipette to target 300 cells/well. Place the plate in the incubator for 48 h at 37 °C with 5% CO2 and 21% O2.
At 48 h, prepare 15 mL of N2B27 with 3 μM of CHIR99021. Add 150 μL per well. Note that the final CHIR99021 concentration is 2.36 μM. Place the plate back in the incubator.
At 72 h, gently flush gastruloids to lift up shed cells. Wait 1 min for the main aggregate to sediment. Carefully remove 150 μL of medium and add exact volume of fresh N2B27 medium.
Repeat step D5 every 24 h until 168 h (Figure 4).
Figure 4. Montage of a full 96-well plate of gastruloids at 168 h after segmentation. T/Bra expression is shown in red. Note the autofluorescent signal over the darker, anterior regions. Scale bars = 200 μm.
Immunostaining and imaging
Collect EPI aggregates and gastruloids from 96-well plates with a cut 1,000 μL pipette tip and transfer to low attachment 6-well plates in 3 mL of PBS/well. Incubate for 10 min at room temperature.
Transfer into a new well containing 3 mL of 4% PFA and fix for 2 h at 4 °C.
After fixation, transfer into new wells to do three serial PBS washes (3 mL) of 20 min at room temperature. It is very important to coat the pipette tips with coating solution (PBS + 10% FBS) before transferring fixed aggregates, to prevent sticking on the walls of the tip.
Transfer into a new well containing 3 mL of blocking solution (PBS + 10% FBS + 0.3% Triton X-100) and incubate for 1 h at room temperature.
Transfer to low attachment 24-well plates in 300 μL of blocking solution containing primary antibodies and DAPI. List of primary antibodies used can be found in Table 1. Incubate for at least 24 h at 4 °C on a shaker. Cover the plate with aluminum foil to preserve fluorescence intensity of the reporters.
Next day, transfer back to low attachment 6-well plate and wash away primary antibodies by three serial PBS washes (3 mL) of 20 min at room temperature.
Transfer to low attachment 24-well plates in 300 μL of blocking solution containing secondary antibodies (Table 1) and DAPI. Incubate for at least 24 h at 4 °C on a shaker. Cover the plate with aluminum foil to preserve fluorescence intensity of the reporters.
Next day, transfer back to low attachment 6-well plate and wash away secondary antibodies by three serial PBS washes (3 mL) of 20 min at room temperature.
Carefully aspirate aggregates in 100 μL and place them on glass slide. Remove excess PBS around without touching the aggregates. Add 20–30 μL of mounting medium dropwise. Place the coverslip on top and seal with nail polish. Keep the mounted samples in the dark until imaging.
Image with Zeiss LSM700 inverted confocal microscope with EC Plan-Neofluar 10×/0.30 or Plan-Apochromat 20×/0.80 air objectives (Figure 5).
Figure 5. Representative immunofluorescence images of gastruloids, EPI, and EPI+XAV aggregates at 168h stained for SOX1 (top) or SOX17 (bottom). Scale bars: 200 μm.
Preparation of EPI aggregates for bulk RNA sequencing
EPI aggregates were lysed with 200 µL TRIzol, followed by addition of 70 µL of chloroform to trigger phase separation. Then, the aqueous phase was collected.
The extraction process was repeated a second time, and an equal volume of isopropanol was added to precipitate the RNA, which was collected by centrifugation at 20,000× g for 30 min.
The pellet was washed with 15 mM sodium acetate in aqueous 70% ethanol, followed by salt-free 70% ethanol, before picking up in RNase-free water.
Data analysis
Image analysis
Live imaging of EPI aggregates was performed with a Nikon Eclipse Ti inverted microscope, objective 10×, 0.3 N.A., using an Andor/iXon DU-888 camera (pixel size 1.2265 μm), equipped with an incubation chamber at 37 °C, 5% CO2. The images were analyzed with ImageJ using custom ActionBar (Mutterer, 2017) and BIOP basics (BIOP Basics ActionBar, c4science) plugins.
For measuring reporter activity in EPI aggregates and gastruloids, brightfield, GFP (for SOX1), and mCherry (for T/BRA-mCherry, TLC-mCherry, AR8-mCherry) channels were acquired. Thresholding and segmentation were performed sequentially for each channel by the custom script (Guiet et al., 2022; DOI:10.5281/zenodo.7409423). The coverage index was calculated by dividing the area of the object identified in mCherry channel to the brightfield area.
For morphology measurements, a custom script was used (Guiet et al., 2021; doi:10.5281/zenodo.4544370). Brightfield images were thresholded and segmented. Maximum inscribed circle function was used to fit circles in the identified object. Axial length was determined by connecting centers of the fit circles. Elongation index was calculated by dividing axial length to the diameter of the maximum inscribed circle (Figure 6).
GraphPad prism was used to analyze the data and calculate significance. In all experiments, the data were collected from three independent experiments, and at least 24 Epi aggregates or gastruloids per experiment were analyzed. Number of data points and statistical tests performed can be found on the legends of the figures in the original paper (Girgin et al., 2021).
Figure 6. Representative post-analysis images of EPI and EPI+XAV aggregates at 168 h showing T/Bra (red) and Sox1 (green) expression domains and calculated coverage indices. Black lines and circles indicate axial length and inscribed circles, respectively. Elongation index is calculated by dividing the axial length with the diameter of largest inscribed circle. Scale bars = 200 μm.
Bulk RNA sequencing analysis
RNA quantity and quality were assessed on NanoDrop, qubit, and Agilent TapeStation 4200 profiling, and showed absorbance ratios 260/280 of 1.85 ± 0.12 and RNA integrity numbers of 9.9 ± 0.2 (average ± SD), supporting good purity and absence of degradation. TruSeq stranded mRNA LT libraries were prepared according to Illumina protocol 15031047 Rev. E, starting from 300 ng of RNA, quantified by qubit DNA HS, profiled on TapeStation 4200, and sequenced on an Illumina HiSeq 4000 at a targeted depth of 36 Mreads/sample and paired-end read length of 81,8i,8i,81. The reads were trimmed for adapters with bcl2fastq v2.20.0, aligned to the mouse genome mm10 with STAR 2.7.0e, and a count matrix was assembled using the cellranger v4.0 curation of ENSEMBL annotations. In the manuscript, “gene expression” refers to natural logarithm of counts per million for bulk RNA-seq data. The data was collected from four independent experiments. Cell type signatures were allocated based on previous reports (Pijuan-Sala et al, 2019). All RNA-seq datasets produced in this study are publicly available in the Gene Expression Omnibus (GEO) database under accession code GEO: GSE171210.
Notes
The passage number of mESCs should be taken into consideration when generating EPI aggregates or gastruloids. Mouse ESCs kept in culture for more than 15–20 passages might lead to inefficient generation of EPI aggregates or gastruloids.
The quality of mESC culture is critical to successfully generate EPI aggregates. Different cell lines might need different seeding densities and passaging frequency. Make sure that mESC cultures are not confluent and colonies do not fuse. In such cases, passage them at lower density and delay EPI aggregate preparation.
When seeding on PEG microwells, an overestimation of 1.5–2× of the desired cell number per well is recommended. During the seeding process, some cells will sediment into grooves and gaps between the microwells and will therefore not contribute to the aggregate.
For making EPI aggregates and gastruloids, different cell lines might need titration of starting cell numbers. When testing a new cell line, a range of 100–300 cells and 150–600 cells should be tested for EPI aggregates and gastruloids, respectively. For gastruloids, testing of final CHIR99021 concentrations (2–5 μM) is important to achieve the most efficient elongation.
Do not use FGF2 and ACTIVIN-A proteins reconstituted more than three months ago.
When transferring EPI aggregates into a 96-well plate, make sure the aggregates do not spend more than 1 h outside of the incubator. If transfer is taking too long, have 15 min incubation periods in between.
Make sure that transferred aggregates have smooth edges and a size of 200–220 μm diameter. Too small or too big aggregates will not elongate properly.
During daily medium exchanges, gently flush the EPI aggregates and gastruloids to lift off shed cells and remove them. Accumulation of shed cells have a negative impact on optimal development.
The quality of N2B27 medium is crucial for proper differentiation and axial elongation. Make sure to use fresh N2 and B27 supplements and do not use complete N2B27 medium older than three weeks.
Recipes
Mouse embryonic stem cell (mESC) maintenance medium (500 mL)
434 mL of DMEM + GlutaMAX
50 mL of ESC-qualified FBS. Final concentration: 10%
5 mL of sodium pyruvate. Final concentration: 1 mM
5 mL of MEM non-essential amino acids. Final concentration: 1×
1 mL of 2-mercaptoethanol. Final concentration: 0.1 mM
5 mL of penicillin/streptomycin. Final concentration: 50 U/mL
CHIR99021. Final concentration: 3 μM
PD0325901. Final concentration: 1 μM
LIF (in-house preparation). Final concentration: 0.1 mg/mL
Prepare base medium (steps a–f) and use within a month. Add CHIR99021, PD0325901, and LIF fresh on the day of culture to complete s2iL medium.
EPI differentiation medium (500 mL)
237 mL of DMEM/F12 + GlutaMAX
237 mL of neurobasal
2.5 mL of N2 supplement. Final concentration: 0.5×
5 mL of B27 supplement. Final concentration: 0.5×
2.5 mL of GlutaMAX. Final concentration: 0.5×
5 mL of sodium pyruvate. Final concentration: 1 mM
5 mL of MEM non-essential amino acids. Final concentration: 1×
1 mL of 2-mercaptoethanol. Final concentration: 0.1 mM
5 mL of penicillin/streptomycin. Final concentration: 50 U/mL
FGF2. Final concentration: 12 ng/mL
ACTIVIN A. Final concentration: 20 ng/mL
KnockOutTM serum replacement. Final concentration: 1%
Prepare N2B27 medium (steps a–i) and use within three weeks. Add FGF2, ACTIVIN A, and KnockOut serum replacement fresh on the day of culture to complete EPI differentiation medium. To make EPI+XAV medium, add 10 μM XAV939.
Acknowledgments
We thank Giuliana Rossi and Alfonso Martinez-Arias for useful feedback on the original manuscript. We thank Sylke Hoehnel and Nathalie Brandenberg (SUN biosciences) for providing PEG microwell plates and technical support. We thank members of the Lutolf laboratory for discussions and sharing materials. We thank Romain Guiet and Olivier Burri for providing the image analysis codes, and Arne Seitz and other members of Bioimaging and Optics Facility (EPFL) for microscopy support. We thank all personnel of the Histology Core Facility for their technical support. This work was funded by EPFL. This protocol was adapted from previous work (Girgin et al., 2021). This work was funded by a Sinergia grant (CRSII5_189956) from the Swiss National Science Foundation, the National Center of Competence in Research (NCCR) Bio-Inspired Materials and EPFL.
Competing interests
The authors declare no competing interests.
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Stem Cell > Embryonic stem cell > Cell differentiation
Developmental Biology > Morphogenesis > Organogenesis
Cell Biology > Cell imaging > Live-cell imaging
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Original Research Article:
The authors used this protocol in eLIFE Dec 2022
Abstract
Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts.
Key features
• This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments.
• Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers.
• The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft.
Graphical overview
Keywords: Microtubules αβ-tubulin heterodimer A- and B-microtubule lattices Multi-seams Kinesin motor-domain decoration Purified tubulin Xenopus egg cytoplasmic extracts Single- and dual-axis cryo-electron electron tomograms Segmented subtomogram averaging
Background
Microtubules are essential components of the cytoskeleton, built from the αβ-tubulin heterodimer that shares both heterotypic (A-type; α-β, β-α) and homotypic (B-type; α-α, ββ-) lateral interactions within their shaft. While the B-type lattice predominates in microtubules assembled in vitro, multi-seams of the A-type occur frequently in microtubules assembled from purified tubulin (Kikkawa et al., 1994; Sosa and Milligan, 1996; Sosa et al., 1997; Dias and Milligan, 1999; des Georges et al., 2008; Debs et al., 2020). Conversely, very little is known concerning the organization of the αβ-tubulin heterodimer within cytoplasmic microtubules. Using a segmented subtomogram averaging (SSTA) strategy on microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts, we found that both the location and number of seams vary within individual microtubules, leaving holes of one to a few subunits size inside their shaft (Guyomar et al., 2022). This observation has consequences for our understanding of microtubule dynamics, since it implies that tubulin can engage unique lateral interactions without longitudinal ones at the microtubule growing end, and provides a molecular basis to the recent observation that tubulin exchange does not happen uniquely at microtubules ends, but also in their shaft (Théry and Blanchoin, 2021).
By contrast to single-particle analysis (SPA)- and full-length subtomogram averaging (STA)-based approaches, where several thousands of images are averaged to obtain one to a few structures supposedly representatives of the whole sample, SSTA addresses the structural heterogeneity of individual microtubule lattices, but with no aim to gather high-resolution information on the αβ-tubulin heterodimer. Ideally, localization of changes in lattice types and holes within individual microtubules would be better described without averaging; however, we found that the imaging artifacts inherent to cryo-electron tomography, as well as denaturation of the specimen at the air–water interfaces, limit their characterization in the raw data (Guyomar et al., 2022). While those limitations do not depend on the instrument used, high-end cryo-electron microscopes equipped with modern direct electron detection cameras may produce cryo-electron tomograms with a better signal-to-noise ratio that should permit a reduction in segment size, and hence a finer sampling of the structural heterogeneity of the microtubule lattice.
At the specimen level, SSTA requires decoration of the microtubules with a protein that binds every αβ-tubulin heterodimer, such as kinesin motor-domains. While this is easily performed in open systems such as microtubules assembled in vitro from purified components or in Xenopus egg cytoplasmic extracts, it requires removing the membrane with detergents for whole cells (Kikkawa et al., 1994; McIntosh et al., 2009). Hence, new strategies will have to be envisioned to overcome this limitation and explore the structural heterogeneity of microtubules in intact cells.
Equipment
MacBook Pro (16 inches, 2021), Apple M1 Max 3.22 GHz, LPDDR5 RAM 64 Go, SSD 4 To, macOS version 13.0.1 (Apple Inc.)
Notes:
Since IMOD and PEET are multi-platform, the procedure can be performed on computers running macOS, Windows, or Linux operating systems.
We recommend at least one CPU with four physical cores and 16 GB of RAM.
Extended keyboard with numeric keypad (Apple Inc.).
Note: If using a MacBook Pro without external keyboard, we recommend using a numerical keypad to enter values in the graphical user interfaces of IMOD and PEET.
M1 wireless three-button mouse (Satechi)
Software and datasets
IMOD (v4.12.30, 2022-10-13), University of Colorado, Boulder (https://bio3d.colorado.edu/imod/) (Kremer et al., 1996; Mastronarde, 1997).
PEET (v1.16.0 alpha, 2022-10-13), University of Colorado, Boulder (https://bio3d.colorado.edu/PEET/) (Nicastro et al., 2006; Heumann et al., 2011).
Cryo-electron tomogram of microtubules assembled in vitro from purified porcine brain tubulin in the presence of GMPCPP (https://ftp.ebi.ac.uk/empiar/world_availability/11264/data/reconstructed_tomogram/GMPCPP_tomoFig5_bin4.mrc) (Guyomar et al., 2022). Other datasets are available for microtubules assembled in the presence of GMPCPP (EMPIAR-11264), GTP (EMPIAR-11253), and in Xenopus egg cytoplasmic extracts (EMPIAR-11263).
Procedure
Preparation of the working directory
Create a directory named tomogram.
Download the tomogram GMPCPP_tomoFig5_bin4.mrc deposited on the EMPIAR database (access code 11264, see the link above) and save it to the tomogram folder.
Modeling the protofilament path with 3dmod
Open a terminal window and enter “cd your_path/tomogram.”
Enter “3dmod GMPCPP_tomoFig5_bin4.mrc MT_Model.mod.” The 3dmod Information Window opens (Figure 1A) together with the ZaP window displaying the middle slice (151) of the tomogram (Figure 1B).
Select the Model mode in the 3dmod Information Window (Figure 1A, red circle).
Note: Regularly save the model during the modeling of the protofilament and microtubule paths using the short-cut key S.
Figure 1. Tomogram visualization. (A) 3dmod Information Window. Selection of the Model mode (red circle). (B) 3dmod ZaP Window.
On the main menu, select Image > Slicer (Figure 2A), click on the Checkerboard button to activate high-resolution drawing (red circle), on the Centering button (blue circle) to associate the Slicer and ZaP windows, enter 50 in the Thick: Img text box, and press Enter.
In the ZaP window, scroll up to Slice 189 and click in the middle of the microtubule at the position marked by a yellow cross in Figure 2B (arrow). The corresponding microtubule segment is presented in the Slicer Window (Figure 2C).
In the Slicer Window, position the X rotation slider to 90.0, the Z rotation slider to -57.4, and adjust the zoom text box to 10.00 with the up arrow of the slicer (Figure 2D). The microtubule is displayed in cross section and its protofilaments are well individualized.
Notes:
The appropriate zoom level in the Slicer may vary depending on the screen resolution. A 10× zoom is appropriate for a MacBook Pro retina display, which translates to a 5× zoom on a classical display.
If the microtubule is not oriented parallel to the plane of the tomogram, which happens frequently in thick ice layers, adjust the X rotation slicer to maximize the contrast of the protofilaments in cross-section.
We advise modeling consistently a microtubule from the bottom towards the top of the tomogram, especially with microtubules showing a high degree of curvature in the XY plane.
Figure 2. Microtubule selection. (A) Tomogram visualized in the Slicer. (B) Selection of the microtubule (arrow). (C) Microtubule in (B) visualized in the Slicer averaged over 50 slices. (D) Microtubule oriented in cross-section after rotation of 90° around the X-axis and -57.4° around the Z-axis.
On the main menu, select Edit > Object > Type... In the window that opens (Figure 3A), click on Object type: Open (red circle) and adjust the Sphere radius for points to 3 (blue circle).
In the Slicer (Figure 3B) unselect the Centering button (red circle) and click on the top protofilament with the middle button of the mouse. This creates a new contour at this position (green circle) and in the 3dmod Information Window (Figure 3C: Object 1, Contour 1, Point 1). Increase the thickness of the point by adjusting the Thick: Mod to 3.0 (Figure 3B, blue circle).
Figure 3. Creation of a protofilament model. (A) Configuration of the Object type menu. (B) Creation of the first point. (C) Information window showing a first contour and a first point for Object 1.
Press and maintain the Page Up key of the keyboard to scroll along the microtubule axis until View axis position indicates 106 (Figure 4A, red circle).
Note: On the MacBook Pro keyboard, the Page Up and Down can be accessed by pressing the function key together with the up and down arrow keys.
Figure 4. Modeling the protofilament path. (A) Microtubule cross-section at View axis position 106. (B) Addition of Point 2 on the same protofilament as in Figure 3B. (C) 3dmod Information Window showing a second point in Contour 1, Object 1.
Create a new point by clicking with the middle mouse button on the protofilament (Figure 4B). This creates a new point 2 for Object 1, Contour 1 (Figure 4C).
Notes:
During this procedure, it may be necessary to readjust the X and Z rotation sliders to keep the microtubule in cross-section.
Points can be repositioned by clicking with the right button of the mouse on the image.
X, Y, and Z coordinates may slightly vary with respect to the one shown in the Figures and those listed in Table 1.
Table 1. Protofilament model points
Point 1 2 3 4 5 6 7 8 9 10 11
X 1002 936 867 802 733 664 597 538 470 405 342
Y 541 586 630 672 718 761 806 845 889 931 972
Z 171 175 176 174 171 163 154 148 142 137 134
View 27 106 187 264 347 428 508 578 659 736 811
The X, Y, Z, and View axis position (View) are indicated for each point in the model.
Continue adding points at View axis positions listed in Table 1 up to the end of the microtubule, following the above procedure. Here, 11 points were added (Figure 5).
Note: During this procedure, recenter the microtubule cross-section with the left mouse key.
Figure 5. Microtubule cross-sections at positions listed in Table 1
Notes:
Note that the protofilaments gradually rotate anticlockwise, since they have a ~0.7° left-handed skew. Note also that setting the X-axis slider to -90° instead of +90° would have produced a clockwise rotation of the protofilaments.
If the protofilament rotates up to the edge of the microtubule cross-section where it cannot be individualized anymore, add a new contour (Edit > Contour > New), and click on another protofilament. Several contours may be necessary to model a long microtubule with skewed protofilaments.
Modeling the microtubule center path
In the Slicer Window, select the Centering button (Figure 6A, red circle), and in the Information Window, select point 1 in Contour 1 of Object 1 (Figure 6B).
In the main menu, select Edit > Object > New. Object 2 (in cyan) is created in the Information Window (Figure 6C).
Select Object 1 in the Information Window with the arrow button (Figure 1D, red circle), and in the main menu select Edit > Contour > Copy...
In the Copy Contour menu that opens, select Copy to object # 2 (red circle) and All contours in object (blue circle), then click on Apply (Figure 6E).
In the Edit object menu (Figure 6F), increase the Sphere radius for points to 12 (red circle). Note the increase of the sphere diameter in the Slicer (Figure 6G).
Notes:
Another method to set the sphere diameter is to select Edit > Point > Size... and in the Surf/Cont/Pt: menu that opens, select Set with mouse wheel. The sphere diameter can be precisely adjusted using the mouse wheel.
The ‘Thick: Mod’ can be increased to 5 to improve the visibility of Object 1 Points (green circle).
Figure 6. Microtubule center modeling. (A) Selection of the Centering button. (B) Selection of Point 1 in Object 1 Contour 1. (C) Selection of Object 2. (D) Selection of Object 1. (E) Copy Contours of Object 1 to Object 2. (F) Increase of the sphere radius of Point 1 in Object 1. (G) Colocalization of Points 1 of Objects 1 (green circle) and 2 (cyan circle). (H) Centering of Point 1 of Object 1 with the right mouse button.
In the Information Window, select Contour 1 and point 1 of Object 1 (as in Figure 6B).
In the Slicer, right-click in the middle of the microtubule to center the point. Adjust the position of the point with the right click if necessary (Figure 6H).
Repeat this operation for every point in Object 1 Contour 1.
Note: If several contours have been created, repeat the operation for all contours.
Save the model (short key S).
Adding points spaced every ~8 nm in the model
In the terminal, enter “addModPts MT_Model.mod 10 T.” A new file named “MT_Model_PtsAdded.mod” is created with points spaced every ~8 nm along the microtubule. Note the total number of points created as indicated in the terminal window (here, 81 points).
Notes:
The voxel size of the tomogram is 8.3 Å. This can be verified using the command “header GMPCPP_tomoFig5_bin4.mrc.” The command “addModPts MT_model.mod 10 T” places points every 10 × 0.83 nm along the microtubule, i.e., every ~8 nm, which corresponds to the kinesin motor-domain repeat along protofilaments.
To visualize the new model, select File > Open Model and select the file MT_model_PtsAdded.mod (Figure 7).
Figure 7. Addition of points spaced every ~8 nm on the model. The points of Object 1 (green circles) follow the microtubule center path, and those of Object 2 (cyan circles) follow the protofilament path.
In the terminal, enter “modTwist2EM MT_Model_PtsAdded.mod 10 1.” Two new files are created: a model file named “MT_Model_PtsAdded_Twisted.mod” and a motive list named “MT_Model_PtsAddedRefP10_initMOTL.csv.”
Notes:
To list the files present in the working directory, enter “ls” on the terminal.
The two numerical values in the modTwist2EM command are the point (10) and contour (1) numbers. The sub-volume extracted at this position will be used as the initial reference in the subtomogram averaging procedure. Hence, this reference point can be chosen based on the quality of the cross-section of the microtubule at any location along the microtubule.
In the main menu, select 3dmod > Quit 3dmod.
Subtomogram average of the full-length microtubule
In the terminal, enter “etomo” and click on Subvolume Averaging (PEET) (Figure 8A).
Figure 8. Configuration of the Setup tab of Etomo. (A) Front page of Etomo. (B) Starting PEET menu. (C) Setup tab.
In the Starting PEET panel that opens, enter “MT” in the Base name: textbox and click on OK.
Configure the Setup tab of the PEET interface as in Figure 8C:
Volume Size (Voxels): enter 54 in the X, Y, and Z text boxes.
Note: The volume size can be specified as the diameter of the microtubule (in pixels) plus twice the maximum search distance at any iteration.
Missing Wedge Compensation: Select Enabled. Let the Edge shift and Weight groups default values to 1 and 8, respectively, and the Number of Tilt Axes to 1.
Note: For dual-axis tomograms, click on 2 or more and see General note 2.
Masking: Select Cylinder and set the following parameters: Height: 54, Inner radius: 10, Outer radius: 26, Blur mask by: 2.
Particle Y Axis: Select Particle model points.
Initial Motive List > Select User supplied csv files.
Volume Table: Click on Insert.
Open a file chooser by selecting the Folder icon under Volume and select the “GMPCPP_tomoFig5_bin4.mrc” file.
Open a file chooser under Model and select the “MT_Model_PtsAdded_Twisted.mod” file.
Open a file chooser under Initial MOTL and select the “MT_Model_PtsAddedRefP10_initMOTL.csv” file.
Under Tilt Range, set the min to -53.14 and the max to 58.29.
Note: These angles can be read from the tomogram.tlt file generated during 3D reconstruction of the tomogram. If present, click on the => button under Vol # (the background of the line turns cyan) and click on Read tilt file, then select the tomogram.tlt file. This populates the Tilt Range with the minimal and maximal tilt angles of the series.
Reference: Enter 10 in the Particle text box and let the default In Volume value to 1.
Note: Resize the MT-Etomo panel if necessary.
Configure the Run tab as in Figure 9:
Figure 9. Configuration of the Run tab of Etomo. (A) Iteration runs. (B) Rotations used to adjust the subvolumes with the reference.
Parallel Processing: in the # CPUs Used text box, allocate the maximum as indicated in the Max text box (here, 10).
Particles per CPU: divide the number of particles by the number of CPUs on your computer (here, 8).
In the Iteration table, unselect Remove duplicates, Bandpass filtering, and select Strict search limit.
Iteration Table: Enter Max = 12 and Step = 4 in the Phi, Theta, and Psi columns. Fix the Search distance to 5, the Low-pass Filter Cutoff to 0.1, the Low-pass Filter Sigma to 0.05, and the Ref Threshold to 60.
Notes:
i. Since we have selected the Particle Y axis as Particle model points in the Setup tab, Phi turns around the microtubule (Y) axis, Theta turns in plane, and Psi turns out off plane (Figure 9B).
ii. Phi has been set to 12° (i.e., 24° since the search occurs in both directions) to keep the angular search restricted to an interval between two protofilaments. This number might be adjusted using 360°/2N, where N is the protofilament number.
Select line 1 by clicking on the => button, click on the Dup button to duplicate the line, and change the Max and Step values by dividing by 2 those of the first run.
Decrease the Search Distance by one.
Reiterate steps (e–f) to implement 4 Runs.
Spherical Sampling for Theta and Psi: Select None.
Number of Particles to Average: Set the Start and Incr to 40, the End to 80, and the Additional numbers to 81.
Notes:
i. Using this strategy, three maps will be calculated with 40, 80, and 81 particles.
ii. Adjust these numbers to the total number of particles generated by the addModPts command if needed.
Configure the More Options table (Figure 10)
Figure 10. Configuration of the More Options tab of Etomo
Alignment: Select the Fast rotational matching and Use absolute value of cross-correlation checkboxes.
Processing: Select the Align averages to have their Y axes vertical.
Note: The Masking during c <N> averaging can be left selected but is not relevant to this procedure.
Subtomogram averaging
Select the Run tab and press Run.
Note: With the current configuration (see Equipment section), the process takes ~9 min to complete.
At the end of the process, click on Open averages in 3dmod. The 3dmod Information Window opens (Figure 11A), together with the 3dmod ZaP Window displaying the middle Y section of the 40 particles subtomogram average (MT_AvgVol_40P40.mrc, Figure 11B), the 3dmod Model View that displays the isosurface (Figure 11C), and the Isosurface control panel (Figure 11D).
Note: Click on Open References in 3dmod to check that the inner and outer masks were properly positioned and oriented.
Figure 11. Subtomogram average of the full-length microtubule. (A) 3dmod Information Window. (B) 3dmod ZaP window displaying the intermediate 40 particles subtomogram average. (C) Isosurface of (B). (D) Isosurface control panel.
Click on the 3d Model View window and, in the main menu, select Edit > Controls... In the MV Controls panel that opens, move the Near clipping slider to 510 (Figure 12A).
Figure 12. Inspection of the subtomogram average of the full-length microtubule. (A) MV Controls menu. (B) Selection of the 81 particle subtomogram average (MT_AvgVol_81.mrc) in the ZaP window. (C) Visualization of the 81 particle subtomogram average showing a B-type lattice organization. (D) Rotation of the map by 180° around the Y-axis. (E) Visualization of the 81 particle subtomogram average showing two seams of the A-lattice type and the middle protofilament with thinner densities with respect to the adjacent protofilaments.
Select the 3dmod ZaP window and click twice on the right arrow to display the final map MT_AVgVol_4P81.mrc (Figure 12B). The isosurface view displays kinesin molecules that are slightly offset longitudinally from one protofilament to the next (Figure 12C), indicating that the underlying αβ-tubulin heterodimers share homotypic lateral interactions of the B-type.
Select the MV controls panel, enter 180 in the Y-axis textbox, and press Enter (Figure 12D). The kinesin molecules display large longitudinal offsets between the three front protofilaments (Figure 12E), indicating that the underlying αβ-tubulin heterodimers share heterotypic lateral interactions of the A-type. In addition, the kinesin densities of the central protofilament are less well defined than those of the two adjacent protofilaments, suggestive of a transition in lattice-type in this region.
Select the Etomo panel and click on File > Exit, then answer Yes to the question “There are still 3dmod programs running. Do you wish to end these programs?”
Segmented subtomogram averaging
In the terminal, enter “splitIntoNSegments 4 MT_Model_PtsAdded_Twisted.mod MT_model_PtsAddedRefP10_initMOTL.csv.” This command creates five directories numbered segment1 to segment5. As indicated in the terminal, segment1 to segment4 contain 20 particles each, and segment5 contains one particle.
Note: The directory segment5 can be deleted using the command “rm -r segment5.”
Enter “cd segment1.
Enter “etomo” and click on Subvolume Averaging (PEET) (see Figure 8A).
In the Starting PEET window, select the Copy project from and click on the folder icon on the right (Figure 13A, red circle).
In the Copy project from window, click on the folder icon with an arrow (Figure 13B, red circle) to move up one level, and double-click on the MT.epe file.
In the Starting PEET window (Figure 13A), rename the Base name MT_S1 and press OK. This opens the Etomo panel for MT_S1 (Figure 13C).
In the volume Table, click on the Model file chooser (Figure 13C, red circle), double-click on the folder segment1, then double-click on the file “MT_model_PtsAdded_Twisted.mod.”
Click on the Initial MOTL folder icon (Figure 13C, blue circle), double-click on the folder segment1, then double-click on the file “MT_model_PtsAddedRefP10_initMOTL.csv.”
Notes:
i. If you choose a reference point other than 10 for the full-length microtubule, adjust the Reference: Particle number to 10, which is half the number of particles in the segments.
ii. See General note 2.
Select the Run tab (Figure 14) and change the number of Particles per CPU to 2.
Note: Adjust the value with respect to the total number of CPU on your computer.
In the Iteration Table, change the Ref Threshold to 18 in each row.
Note: If only one row is present, add the three other rows as in Figure 9B.
In the Number of Particles to Average, change the Start, Incr., and End values to 20, erase the value present in the Additional numbers textbox and press Run.
Note: With these settings, only one map will be calculated.
In the terminal, enter “cd ../segment2.”
Repeat steps c-k used for segment1 using its MT_S1.epe file as a template. Modify the Base name accordingly (MT_S2 to MT_S4); in the Setup tab, select the corresponding model and initial motive list files, and press Run.
Figure 13. Preparation of the first segment. (A) Starting PEET menu. (B) Selection of the MT.epe file of the full-length microtubule. (C) PEET interface of segment 1.
Figure 14. Configuration of the ‘Run’ tab of segment1
Inspect the subtomogram averages of segment1–4, as done for the full-length microtubule (see §6, Figure 12). The three first segments display a A-lattice type organization in the three front protofilaments observed in the Y = 180° view (Figure 15A–15C), while the fourth segment displays a B-type lattice organization in the same region (Figure 15D), implicating that a hole of at least one monomer (or a larger odd number) exists at the transition region between segments 3 and 4 (visualized in the raw data, see Figure 5 of Guyomar et al., 2022).
Note: An additional seam is visible on the right in all subvolumes and can be visualized more clearly by turning the maps around Y by 90°.
Figure 15. Segmented subtomogram averaging. (A) Segment 1. (B) Segment 2. (C) Segment 3. (D) Segment 4. Views turned by 180° around the Y-axis are displayed in (A–D). The kinesin motor domain densities in the three front protofilaments of segments (1–3) are shifted longitudinally by ~49.2 Å indicating that the underlying αβ-tubulin molecules share heterotypic interactions of the A-type. Conversely, they are shifted longitudinally by ~9.2 Å in segment 4 indicating that the underlying αβ-tubulin molecules share homotypic interactions of the B-type.
Validation of protocol
This protocol allowed us to analyze 134 microtubules that were divided into 938 segments (see Table 1 in Guyomar et al. 2022). A total of 172 lattice-type transitions were observed within individual microtubules assembled from purified tubulin in the presence of GTP (24 microtubules, 195 segments, 119 transitions), GMPCPP (31 microtubules, 238 segments, 37 transitions), and Xenopus egg cytoplasmic extracts in the presence of 5% DMSO (64 microtubules, 419 segments, 6 transitions) and RanQ69L (15 microtubules, 86 segments, 2 transitions).
General notes and troubleshooting
General notes
A detailed tutorial on subtomogram averaging of full-length microtubules decorated with kinesin motor domains can be found at the following URL (session Aligning and Averaging Filaments: Microtubules): https://bio3d.colorado.edu/RML_2017/2017_IMOD_PEET_Workshop. A tutorial video is also available at the following URL: https://www.youtube.com/watch?v=aC8gEurMhO8.
When performing subtomogram averaging on dual-axis cryo-electron tomograms, a missing prism mask must be created. A suitable mask can be generated using program dualAxisMask if the two tilt files, tomogram_a.tlt and tomogram_b.tlt, are present in the working directory. The general format of the command to enter in the terminal is “dualAxisMask szMask dirname basename edgeShift.” In the example of the tutorial, this could have been “dualAxisMask 54. tomogram 2.” This command creates a file named “tomogram_DualAxisMask.mrc.” Alternatively, this mask file can be generated using the command “multiTiltMask.” After creating the mask, on the Setup tab of Etomo’s PEET interface, select Number of Tilt Axis: 2 or more. A new column named Missing Wedge Mask appears in the Volume Table. Click on the folder icon and select the tomogram_DualAxisMask.mrc file.
After copying the project from ../../MT, the Volume, Model, and Initial MOTL will default to those of the project you copied from. Additionally, the browsing directory will typically also default to the source directory, so when you open up a file chooser, you will see files in the parent directory. Therefore, make sure you are choosing the newly generated model and motive list in the appropriate segment <N> directory and not the parent.
Acknowledgments
This work was financially supported by the French National Research Agency (ANR-16-CE11-0017 and ANR-18-CE13-0001) to D.C. Cryo-electron microscopy data were acquired on the Microscopy Rennes imaging center platform (Biosit, Rennes, France), member of the national infrastructure France-BioImaging (FBI) supported by the French National Research Agency (ANR-10-INBS-04). This protocol was adapted from Guyomar et al. (2022). We thank Céline Callens (team MiToS, IGDR) and Sophie Chat (team QCPS, IGDR) for testing the protocol and for their useful feedback.
Competing interests
The authors declare no conflicts of interests.
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Théry, M. and Blanchoin, L. (2021). Microtubule self-repair. Curr Opin Cell Biol 68: 144‑154.
Supplementary information
The sessions to reconstruct the full-length microtubule and the 4 segments are provided as a zipped supplementary file. Download and decompress this file, then place the "GMPCPP_tomoFig5_bin4.mrc" file downloaded onto the EMPIAR database into the tomogram folder. ETomo sessions can be launched using "etomo *.epe", where * replaces the file name.
The original tilt series and the reconstructed cryo-electron tomograms used in Guyomar et al. (2022) have been deposited onto the EMPIAR database (access codes 11253, 11263, 11264), see Supplementary Table 2 in Guyomar et al. (2022). This includes all the models used to segment the 134 microtubules analyzed in this study. Subtomogram averages of the microtubules presented in the Figures in Guyomar et al. (2022) have been deposited on the EMDB database, see Supplementary Table 1 in Guyomar et al. (2022).
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© 2023 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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4,724 | https://bio-protocol.org/en/bpdetail?id=4724&type=0 | # Bio-Protocol Content
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Peer-reviewed
HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice
LH Laura V. Heeb
BT Betül Taskoparan
AK Antonios Katsoulas
MB Michal Beffinger
PC Pierre-Alain Clavien
SK Sebastian Kobold
AG Anurag Gupta *
JB Johannes vom Berg *
(*contributed equally to this work)
Published: Vol 13, Iss 14, Jul 20, 2023
DOI: 10.21769/BioProtoc.4724 Views: 927
Reviewed by: Durai SellegounderEhsan KheradpezhouhOlga Bielska
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Original Research Article:
The authors used this protocol in Science Translational Medicine Sep 2021
Abstract
The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.
Keywords: CRISPR/Cas9 HDR template Mouse transgenesis Knockout PD-L1 Restriction site Frameshift
Background
Programmed cell death ligand 1 (PD-L1), also known as CD274, is known to control adaptive immune responses during various pathological conditions such as autoimmune diseases, infections, and cancer (Francisco et al., 2010; Jubel et al., 2020). In particular, higher expression of PD-L1 on antigen-presenting cells as well as on cancer cells is known to engage with PD-1 on activated CD8+ T cells, thereby inhibiting their cancer response (Han et al., 2020). Thus, the study of the role of PD-L1 in cancer development and progression was and still is of utmost importance. For this, loss-of-function mouse mutants are invaluable tools whose generation had been time and resource intense for decades.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) is an RNA-guided nuclease system that has been adapted to be a potent gene editing tool. It has quickly evolved to be the method of choice for targeted gene editing and genome-wide screens. It is superior to previously used methodologies such as homologous recombination in embryonic stem cells or use of ZNF (zinc fingers) (Lee et al., 2010; Meyer et al., 2010; Söllü et al., 2010) and TALENs (transcription activator-like effector nucleases) (Cermak et al., 2011; Zhang et al., 2011). Compared to ZNF and TALENs, it relies on DNA–RNA heteroduplex formation rather than protein–DNA interaction (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013). Here, we show a protocol using the CRISPR/Cas9 system to specifically knockout PD-L1 in the C57BL/6 mouse. After the CRISPR/Cas9-mediated dsDNA break, cell-intrinsic DNA repair mechanisms such as non-homologous end joining (NHEJ) and homology-directed repair (HDR) will take place. During NHEJ-based repair, the CRISPR/Cas9-induced double-strand break is repaired by random insertion or deletion of nucleotides at the cut site, which should in theory result in two out of three cases to a frameshift and premature translational stop. This method enables simple and fast generation of loss-of-function alleles and has been rapidly adapted to generate mouse mutants but does not generate precise gene edits. Compared to NHEJ, which leads to random heterogenous outcomes and requires sequencing of the targeted locus when screening founder animals, HDR is template based and more precise, allowing the insertion of specific sequences into the DNA break (Miyaoka et al., 2016; Yang et al., 2020). Here, we show the targeted deletion of exon 3 by insertion of a HindIII restriction site that leads to a defined stop-codon by homologous recombination. This leads to a functional knockout of PD-L1 and can be easily screened by PCR amplification of the targeted locus and a subsequent restriction digest. This protocol can be adapted to target PD-L1 in other mouse strains or cell lines and, most importantly, to target other genes of interest.
Materials and reagents
Flat PCR caps, 8–250 strips (Thermo Fisher Scientific, catalog number: AB0784)
Qiagen DNeasy blood & tissue kit (Qiagen, catalog number: 69504)
Qiagen Taq PCR core kit (Qiagen, catalog number: 201225).
Note: Alternatively, high fidelity polymerases such as Pfu (Promega, catalog number: M7741), Phusion (NEB, catalog number: M0530), or Q5 (NEB, catalog number: M0491) can be used to decrease probability of amplification errors, which may increase the accuracy of Sanger sequencing.
Oligos and primers (Table 1) (iDT)
Streptococcus Pyogenes Cas9, 2× NLS SpCas9 (New England Biolabs, catalog number: M0646)
TE buffer, RNase free (Invitrogen, catalog number: 12090-015)
Agarose, LE, analytical grade (Promega, catalog number: V3125)
DNA dye (gel loading dye Purple 6×) (New England BioLabs, catalog number: B7024S)
Restriction enzyme (HindIII) (New England Biolabs, catalog number: R0104T)
Restriction enzyme buffer (2.1) (New England Biolabs, catalog number: R0104T)
DNA ladder (Mass Ruler Low Range DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0311)
Fluorescently labeled antibody against PD-L1 (clone 10F.9G2, PE-Dazzle594 conjugate) (BioLegend, catalog number: 124324)
Fluorescent reagent to discriminate cell viability (Zombie Aqua) (BioLegend, catalog number: 423101)
Erythrocyte lysis buffer (RBC lysis buffer) (BioLegend, catalog number: 420302)
Recombinant murine interferon γ (IFNγ) (PeproTech, catalog number 315-05)
ddH2O
Microinjection buffer (see Recipes): Tris-base (Biosolve, catalog number: 20092391), HCL (Merck Millipore, catalog number: 109057), EDTA (Invitrogen, catalog number: 15575-038)
TAE buffer (see Recipes): Tris-base (Biosolve, catalog number: 20092391), acetic acid (Sigma-Aldrich, catalog number: 33209), EDTA (Invitrogen, catalog number: 15575-038)
Equipment
Biometra PCR thermocycler (Bio-Rad, model: C1000 Touch Thermal Cycler)
Centrifuge (Vaudaux-eppendorf, catalog number: 5418/0005108)
NanoDrop (DeNoVix DS-11 + spectrophotometer)
Gel chamber (Bio-Rad, Sub-Cell GT)
Machine to run gel (BioRad Power-Pac Basic)
Machine to image gel (Quantum ST4, 1120, Skylight Xpress)
Flow cytometer (LSR Fortessa, BD)
Software
CLC Genomics Workbench (version 22, Qiagen, https:///www.qiagen.com/)
Ensembl (http://www.ensembl.org/index.html)
CRISPOR (http://crispor.tefor.net/)
Note: For all three points, various alternatives exist: snapgene (https://www.snapgene.com/) for point one; for point two, we suggest UCSC genome browser (https://genome.ucsc.edu/); for point three, chopchop (http://chopchop.cbu.uib.no/)
FlowJo (version 10, BD)
Procedure
Design of crisprRNA targeting exon 3 of CD274 and 4-base pair HindIII restriction site insert
Search CD274 on an appropriate genome browser (e.g., www.ensembl.org or www.genome.ucsc.edu) and download gene sequence. Check the gene for transcript variants, exons/introns, and corresponding protein domains. Cd274 has one protein-coding transcript (Ensembl Gene: CD274 ENSMUSG00000016496; Ensembl Transcript: CD274-201), which has seven exons.
Open CD274 sequence on CLC Genomic Workbench and annotate regions of interest [(e.g., exons of targeted transcript with corresponding open reading frames (ORF)].
Choose the location you want to target (i.e., exon 3) (Figure 1).
Copy the sequence you want to target into an appropriate guideRNA design tool (e.g., www.crispor.tefor.net). Choose guide sequence length, the protospacer adjacent motif (PAM site), the Cas9 protein you want to use (i.e., 20bp-NGG–Sp Cas9), and the latest assembly of the respective Mus musculus reference genome (i.e., UCSC Dec. 2011 mm10=C57BL/6J). PAM is a short DNA sequence (2–6 bp, depending on the Cas enzyme) following the DNA region targeted for cleavage by CRISPR/Cas9 and, in case of the Streptococcus pyogenes (Sp) Cas9 nuclease, can be found 3–4 nucleotides downstream of the predicted cut site. While the guide sequence defines the crRNA or guideRNA–DNA interaction, it is essential for the DNA–protein interaction.
Choose the best guide sequence to use (see guide sequence “guide_61/fw_ex3” in Table 1 and Figure 1).
Note: The goal is to get a double-strand break that leads to homologous recombination, so that a ssDNA donor repair template containing a 4-base pair HindIII restriction site with 57 bp (left) and 50 bp (right) homology arms on either side can be inserted. This will lead to a +1 frameshift and a stop codon 75 bp downstream of the restriction site (Figure 2).
Figure 1. Genomic landscape of potential targets in exon 2 and 3 of wildtype PD-L1 with possible guide sequences. Sequence of exon 2 and 3 from CD274 with corresponding open reading frames (ORF) (+1) as depicted in CLC Genomics Workbench (dark blue). Exons are depicted in blue and start codons for ORF+1 are depicted in yellow and purple (ATG1 and ATG2, respectively). Possible guide sequences for targeting crRNA are depicted in lilac. Alternative stop codon (TGA ORF+2), 72 bp downstream of guide_61/fw_ex3-induced cutting site (in between A and C, on position 57 and 58 of exon 3), is depicted in red.
Table 1. Oligos and primers
Name Application Sequence (5′–3′) Length (bp) Manufacturer
guide_61/fw_ex3 crRNA GTATGGCAGCAACGTCACGA 20 Alt-R CRISPR, iDT
tracrRNA tracrRNA Sequence according to manufacturer 67 Alt-R CRISPR, iDT
HDR template HindIII +1fs ssDNA homology-directed repair donor template CGTTTACTATCACGGCTCCAAAGGACTTGTACGTGGTGGAGTATGGCAGCAACGTCAAGCTTGGAGTGCAGATTCCCTGTAGAACGGGAGCTGGACCTGCTTGCGTTAGTG 111 Megamer, iDT
guideRNA_61/fw_Left Fw Primer CCCCGCCCCATGAAGTTATT 20 Microsynth
guideRNA_61/fw_Right Rv Primer TGCAGCTTGACGTCTGTGAT 20 Microsynth
Figure 2. In vitro digestion of candidate guideRNAs to test cutting efficiency. Target DNA was digested for 20 min at 37 °C with a cocktail consisting of crRNA, tracrRNA, and Cas9 enzyme. Digestion products were run on a gel and cutting efficiency was assessed based on the resulting bands. Uncut amplicons (white arrows) show a band at 400 bp and the digested products (blue arrows) show a band at 200 bp.
Annotate chosen guide sequence in CLC Genomics (Figure 1).
Annotate ssDNA donor repair template. See sequence “HDR template HindIII +1fs” in Table 1 and Section C.
Order lyophilized crRNA and the universal tracrRNA. Note that tracrRNA does not confer target specificity, so it can be ordered in bulk and combined with different, target-specific crRNAs.
Test guideRNA by in vitro digest
Extract DNA from murine cells or biopsy with an appropriate DNA extraction kit (e.g., Qiagen DNeasy blood & tissue kit).
Amplify DNA target site from extracted DNA template with the Qiagen Taq PCR core kit and appropriate forward and reverse primers (Table 1). Location of primers and sequence of expected 414 bp amplicon is shown in Figure 3. See PCR reagents in Table 2 and conditions in Table 3.
Figure 3. Induction of +1 frameshift and premature translational stop by homology-directed repair (HDR) of ssDNA template. (A) Overview of exon 3 (dark blue) and 414 bp amplicon (yellow) spanning the homology repair-directed template insert (HDR template HindIII +1fs, dark green, homology arms depicted in light green) carrying a HindIII restriction site (orange). Alternative stop codon [stop (ORF +2)] on position 138–141 of wildtype PD-L1 exon 3 is depicted in red. Primers spanning the amplicon are depicted in purple. (B) Sequence of 111 bp HDR template Hind III +1fs as depicted in CLC Genomics Workbench. 57 bp (left) and 50 bp (right) homology arms span the 4 bp (AGCT) HindIII restriction site insert. Insertion of 4 bp HindIII restriction site replacing 3 bp (CGA) of wildtype PD-L1 leads to a +1 frameshift and a premature stop of translation due to the alternative stop codon in ORF +2.
Table 2. Concentrations and volumes for target DNA PCR amplification
Reagent Concentration Volume (25 μL total/reaction)
guideRna168forwLe 10 μM 1.25
guideRNA168forwRi 10 μM 1.25
dNTPs 200 μM 0.5
10× buffer 2.5
Taq polymerase 2.5 U 0.125
ddH2O 18.375
DNA 1
Table 3. PCR Conditions
Step Time (s) Temperature (°C)
1 60 98
2 10 98
3 30 65
4 30 72
Go to step 2 35×
5 120 72
6 Up until use 4
Dilute lyophilized crRNA and tracrRNA (usually 2 nmol) to 100 μM stock concentration in TE buffer. Then, further dilute to 10 μM working concentration in TE buffer.
Note: Working with RNA requires RNase-free working conditions, as RNA is not very stable and can be degraded by RNases very quickly. RNA samples should always be stored on ice and only diluted in RNase-free reagents. The crRNA and tracrRNA can be diluted and aliquoted in TE buffer and stored at -80 °C for up to a year.
Per reaction, in a PCR reaction tube, incubate 3.68 μL of crRNA with 1.84 μL of tracrRNA in 5 μL of microinjection buffer at 78 °C for 10 min on a PCR heating block, followed by 37 °C for 30 min (Mix 1).
Note: For uncut control, include one reaction to which you do not add crRNA. Replace this volume (3.68 μL) with ddH2O.
Remove from heating block and slowly cool down to room temperature (RT) for 15 min.
Take 10.7 μL of Mix 1 and add 0.5 μL of Cas9 (20 μM) and 38.8 μL of ddH2O. Pipette up and down a couple of times and incubate for 10 min at RT (Mix 2).
Take 15 μL of Mix 2 and add 2 μL of Cas9 10× buffer and 400–500 ng of target DNA and fill up with ddH2O to 20 μL.
Note: With a final volume of 20 μL, maximum 3 μL of DNA target and 15 μL of Mix 2 can be added. When the concentration of the target DNA is low and e.g., 6 μL needs to be added to reach 400–500 ng, increase the Cas9 10× buffer to e.g., 3 μL, Mix 2 to 22.5 μL, and final volume to 30 μL.
Incubate at 37 °C for 60 min and load onto 2% agarose gel (2 g of agarose/mL of TAE buffer).
Add loading dye to the samples and load DNA ladder and samples onto the gel. Run the gel at 100 V until bands have separated well.
Note: The in vitro digestion allows you to evaluate the potential of your guide for in vivo cutting efficiency. You get an idea of the cutting efficiency by comparing the intensity of the uncut amplicon to the two cutting products. When a guide already does not cut the target amplicon in an in vitro digest, the probability of efficient cutting in vivo might also be low. Likewise, when comparing two guides for the same DNA target site, a combination of specificity score and cutting efficiency will help to choose the optimal guide. Expected band sizes are approximately 400 bp for the uncut amplicon and 200 bp for the cut product (Figure 2).
Design ssDNA oligo as HDR donor template to integrate defined frameshift and enzyme restriction site
Choose appropriate guide sequence to target locus of interest.
Note: For a complete loss-of-function phenotype, a good guide sequence does not target a locus in front of an alternative start site (ATG), a locus including SNPs, or a locus close to the C-terminal site. Furthermore, it is important to not target non-constitutive exons (exons not present in all isoforms of the gene) (Doench, 2018). We targeted exon 3, as these guide sequences generally show higher specificity and efficacy than guides targeting exon 2. We chose guide_61/fw_ex3, as this crRNA has the highest specificity and efficacy score and less off targets than guide_109/fw_ex3 depicted by CRISPOR (Table 4).
Table 4. Potential crRNAs with respective target loci, sequence and specificity, and efficacy scores according to CRISPOR. The most promising crRNAs (*) have been tested by in vitro digestion (Figure 2). Two asterisks (**) mark the crRNA chosen for zygote microinjection.
crRNA Target Sequence MIT CFD Doench ‘16 Mor.-Mateos
guide_29/fw_ex2 Exon 2 CAAAACATGAGGATATTTGC 68 79 44 12
guide_63/fw_ex2 Exon 2 CAGCCTGCTGTCACTTGCTA 68 82 46 56
guide_64/fw_ex2* Exon 2 AGCCTGCTGTCACTTGCTAC 79 91 48 36
guide_22/fw_ex3* Exon 3 GTTTACTATCACGGCTCCAA 91 96 63 41
guide_61/fw_ex3** Exon 3 GTATGGCAGCAACGTCACGA 96 96 66 44
guide_109/fw_ex3 Exon 3 GCTGGACCTGCTTGCGTTAG 94 97 42 66
guide_95/rev_ex3 Exon 3 AGTACACCACTAACGCAAGC 93 97 60 9
Design ssDNA donor repair template:
To induce homology-directed repair and generate a +1 frameshift, design a ssDNA template that spans the targeted locus and cutting site with two homologous arms of 57 bp (left) and 50 bp (right).
Note: The double-strand break will be between position 17 and 18 of the protospacer (guide) sequence. This might differ according to the type of Cas9 and guideRNA used. We used Sp Cas9 that has a cutting site between the third and the fourth bp upstream of the PAM site of guide_61/fw_ex3 (TGG).
Add an insert of 1 or 4 bp in between the two homology arms to induce a frameshift (see “HDR template HindIII +1fs” in Table 1 and Figure 3).
Note: We designed the insert and homology arms in a way that 3 bp of the wildtype sequence (CGA) get replaced by a 4 bp HindIII restriction site (AGCT), thereby conveniently replacing the A of the start codon in exon 3 with the T of the restriction site.
Add homology arms with 57 bp (left) and 50 bp (right) homologous sequences.
Order ssDNA donor template.
Preparation of microinjection mix with CRISPR/Cas9 ribonucleoprotein particle (RNP) plus HDR oligo repair template
Resuspend lyophilized crRNA and tracrRNA in 1× microinjection buffer to a final concentration of 10 μM:
Mix a total of 1.84 μL of crRNA and tracrRNA with 5 μL of 10× injection buffer and 0.5 μL of Streptococcus pyogenes Cas9 protein (20 μM) and subsequently incubate for 15 min at 37 °C.
For knock-in mouse production, add 500 ng of ssDNA donor template (HDR template HindIII +1fs) after incubation.
Dilute the mix with ddH2O to a final volume of 50 μL.
Spin the final mix down at 21,000× g for 3 min at RT.
Keep injection mix at RT during the injection procedure.
Notes:
The microinjection procedure was performed at the transgenesis core of the University of Zürich, Switzerland and cannot be performed with simple laboratory equipment. A short description of the procedure is found in section E. For a more detailed description of the reproductive biology and the microinjection procedure, please refer to the transgenesis core of your research facility.
As a quality control measure after the microinjection procedure, the enzymatic activity of the injection mix (can be stored at -80 °C) can be tested by adding target DNA and essentially performing an in vitro digestion as described in section B.
Zygote microinjection, embryo culture, and retransfer into pseudopregnant foster animals
Microinjection was performed at the transgenesis core of the University of Zürich, Institute of Laboratory Animal Science under license of the cantonal veterinary office in accordance with federal law. C57BL/6 mice at 3–4 weeks of age (Charles River Laboratories, Germany) were super ovulated by intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (Folligon, MSD Animal Health GmbH, Luzern, Switzerland) followed 48 h later by injection of 5 IU of human chorionic gonadotropin (Pregnyl, MSD Animal Health GmbH, Luzern, Switzerland). Mouse zygotes were obtained by mating C57BL/6J stud males with superovulated C57BL/6 females. Zygote microinjections (pronuclear injection into the male pronucleus), embryo culture o/n, and retransfer of 2-cell stage embryos into pseudopregnant foster animals via surgical embryo transfer were performed according to standard mouse transgenesis protocols (e.g., Harms et al., 2014; Quadros et al., 2018).
Screening of founders by PCR followed by HindIII digest of the amplicon
Take biopsies of the resulting founder pups after zygote implantation.
Amplify the DNA target site by PCR with Qiagen Taq PCR core kit (Table 2 and Table 3). This leads to a 414 (mutated) or 413 (wild type) bp amplicon.
Note: As a negative control, DNA template in PCR mix can be substituted with 1 μL of ddH2O.
After amplification, carefully open the PCR strip and add 0.5 μL of HindIII restriction enzyme/reaction, close reaction tubes again, and incubate at 37 °C in the PCR machine overnight. Cave: confirm on the NEB table (https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers) the activity of HindIII in your Taq buffer. Depending on your Taq, you might need to add HindIII reaction buffer to ensure proper activity.
Prepare 1.5% agar gel (1.5 g of agarose/mL TAE buffer).
Load DNA ladder and samples with added loading dye onto the gel.
Run at 100 V until bands of standard size have separated well.
Note: In vitro digestion of amplicon with inserted HindIII restriction site leads to cutting of the 414 bp amplicon into approximately 200 bp fragments. If the donor template has integrated successfully, we expect one band at 200 bp, while the unintegrated wildtype band stays at 413 bp due to the missing HindIII restriction site. Therefore, amplicons derived from wildtype mice will be visible on the gel as one band at 413 bp. Amplicons derived from homozygous mutants will show one band at 200 bp, while heterozygous mutants will show both bands at 200 bp and 413 bp, respectively (Figure 4).
Figure 4. PCR validation of PD-L1 knockout founder pups. Target gene was amplified and subsequently digested with HindIII restriction enzyme. Numbers 1–19 represent the different founder pups. The left side shows DNA ladder with depicted sizes. 400 bp bands show wildtype alleles, while 200 bp bands represent cut alleles that integrated the ssDNA donor template with HindIII restriction site. Subsequent digestion with HindIII restriction enzyme after PCR cuts the alleles harboring the de novo integrated HindIII 200 bp fragments. Heterozygous pups show two bands; homozygous pups show one band. Only pup number 10 has both mutated alleles.
Confirm frameshift via Sanger sequencing
Prepare your samples for sanger sequencing:
Isolate DNA from founder pups and amplify it to get amplicon as described in step F2.
Note: Sanger sequencing methods are most precise when DNA is approximately 300–1,000 bp. Choose your primers accordingly.
Check quality of DNA template by UV absorption using a NanoDrop.
Note: Good-quality DNA will have an A260/A280 ratio of 1.7–2.0.
Dilute the DNA template to desired concentration according to the protocol of your sequencing provider.
Note: A general rule is 1.5 ng/μL per 100 bp. As our amplicon is 414 bp long, 6–7.5 ng/μL should suffice.
Dilute your primers according to the protocol of your sequencing provider.
Note: The general rule is 4 μM for premixed primers and 10 μM for separate primers.
Send the DNA amplicons and primers in for sequencing.
Import sequencing files into CLC Genomics Workbench.
Align sequencing files with the sequence of the amplicon and check for conflicts (Figure 5).
Figure 5. Confirmation of +1 frameshift in founder pups via Sanger Sequencing. The alignment of the sequencing data to the amplicon shows a clean incorporation of the insertion template with the HindIII restriction site AGCT into the genome of founder pup number 10 (10_premix.scf).
Backcross founder pups with clean insertions to C57BL/6 mice
Backcross founders once to wild type to minimize potential off-target effects:
Mate positive founders with C57BL/6 wildtype mice.
Perform routine genotyping as described in section F and choose heterozygous mice for subsequent het × het crossing to yield homozygous mutants (Figure 6).
Figure 6. Routine genotyping by PCR and HindIII digestion. C- shows negative control (PCR and HindIII restriction reaction without DNA template); -/- shows homozygous mutant with 200 bp fragments; +/- shows heterozygous mutant with both 200 bp and 400 bp fragments; +/+ shows wildtype gene with 400 bp fragments; L is the DNA ladder.
Optional: confirm phenotypic knockout in homozygous PD-L1 mutants by flow cytometry
Collect blood.
Perform lysis of erythrocytes using RBC lysis buffer according to manufacturer’s instructions.
Resuspend cells in medium containing 10 ng/mL murine IFNγ.
Note: IFNγ stimulation leads to a strong upregulation of PD-L1 on the surface of cells.
Stain the cells with Zombie Aqua and primary fluorophore-conjugated antibodies.
Note: In general, it is enough to only stain the cells with a viability dye and an anti-PD-L1 antibody. However, it is recommended to also stain the cells for immune cell markers such as CD45, CD3, and CD11b to discriminate between populations. General protocols for surface staining can be found on the websites of flow cytometry antibody providers.
Acquire cells on flow cytometer.
Gate on cells of interest in FlowJo (Figure 7A).
Check PD-L1 expression (Figure 7B).
Figure 7. Confirmation of phenotypic knockout of PD-L1. Mouse peripheral blood mononuclear cells (PBMCs) were stimulated overnight with 10 ng/mL IFNγ and consequently surface-stained for PD-L1. (A) Gating strategy. (B) Representative overlay of histograms of PD-L1 expression of CD45+ immune cells as gated in A), showing PD-L1-/- cells in black (-/-) and wildtype cells in red (+/+).
Notes
The described procedure can be adapted in various ways. For example, instead of introducing a restriction enzyme recognition site, an already existing restriction enzyme recognition site in the open reading frame of interest may also be destroyed by an indel by simply leaving away the HDR template. Such a simple non-homology directed repair event may lead to a frameshift, resulting in a loss of protein expression as well. However, screening for loss of restriction sites does not guarantee a particular frameshift and can even lead to in-frame insertions or deletions. The method described here circumvents this problem by introducing a novel restriction site together with a particular frameshift. This allows for convenient screening for these defined, desired frameshifts at minimal trade off with regards to targeting efficiency, since the insert is very small.
Most importantly, this method can be adapted to other genes of interest, targeted insertion of larger genomic regions, as well as to generation of defined mutants in cell lines, where targeting both alleles is more crucial than for mouse transgenesis.
Recipes
Buffers and solutions
1× microinjection buffer
10 mM tris-HCl (pH 7.5) and 0.1 mM EDTA. Tris-HCL and EDTA can be used again up to six months after preparation; the microinjection buffer should be mixed fresh every time.
50× TAE (Tris-acetate-EDTA) buffer
Dissolve 242 g of Tris base in 700 mL of ddH2O. Add 57.1 mL of 100% acetic acid and 100 mL of 0.5 M EDTA (pH 8.0). The pH should be approximately 8.5; if not, adjust pH. Adjust the solution to a final volume of 1 L. After preparation, solution can be stored and used at RT for up to six months.
Acknowledgments
The authors thank Dalila & Mark Ormiston, Monika Tarnowska and Celil Sert for excellent technical assistance in pronuclear injection and all reproductive techniques connected to it.
This work was supported through grants of the Novartis Foundation for Medical-Biological Research (16C231), Swiss Life Jubiläums Stiftung (1283-2021), Swiss National Science Foundation (NRP79, 407940_206465) and Swiss Cancer Research (KFS-3852-02-2016, KFS-4146-02-2017, KFS-5306-02-2021) to J.v.B. and grant from Edoardo R., Giovanni, Giuseppe und Chiarina Sassella-Stiftung to AG. S.K. is supported by the Marie-Sklodowska-Curie Program Training Network for Optimizing Adoptive T Cell Therapy of Cancer funded by the H2020 Program of the European Union (Grant 955575); by the Hector Foundation; by the International Doctoral Program i-Target: Immunotargeting of Cancer funded by the Elite Network of Bavaria; by Melanoma Research Alliance Grants 409510; by the Else Kröner-Fresenius-Stiftung; by the German Cancer Aid (to S.K.); by the Ernst-Jung-Stiftung; by the LMU Munich’s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative by the Go-Bio initiative; by the m4 Award of the Bavarian Ministry of Economical Affairs, by the Bundesministerium für Bildung und Forschung; by the European Research Council Grant 756017, ARMOR-T and the ERC proof-of-concept Grant 101100460; by the German Research Foundation (DFG) (KO5055-2-1 and 510821390 ) by the SFB-TRR 338/1 2021–452881907; by the Wilhelm-Sander-Stiftung, by the Fritz-Bender Foundation and by the Deutsche José-Carreras Leukämie Stiftung.
The described mouse line had been generated on a collaborative basis by S.B. an J.v.B. in 2016 and employed in Schneider et al. (2021), doi: 10.1126/scitranslmed.abc8188.
Competing interests
J.v.B. and M.B. are shareholders and part time employees of InCephalo AG. J.v.B. is an inventor of patents related to immunotherapy of cancer and has received licensing fees for these, speaker fees from Bristol Meyer Squibb and research support by Boehringer Ingelheim Animal Health for work not related to this manuscript. S.K. is inventor of several patents in the field of immuno-oncology. S.K. received license fees from TCR2 Inc and Carina Biotech. S.K. received research support from TCR2 Inc., Arcus Bioscience, Plectonic GmBH and Tabby Therapeutics for work unrelated to the manuscript. Beyond this, the authors have no relevant financial interest to disclose.
Ethical considerations
Microinjection was performed at the transgenesis core of the University of Zürich, Institute of Laboratory Animal Science under license of the cantonal veterinary office (No. 177-G) in accordance with Swiss federal law.
References
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Schneider, M. A., Heeb, L., Beffinger, M. M., Pantelyushin, S., Linecker, M., Roth, L., Lehmann, K., Ungethüm, U., Kobold, S., Graf, R., et al. (2021). Attenuation of peripheral serotonin inhibits tumor growth and enhances immune checkpoint blockade therapy in murine tumor models. Sci Transl Med. 13(611): eabc8188.
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VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry
OC Olivia M.S. Carmo
MD Matthew W.A. Dixon
Published: Vol 13, Iss 15, Aug 5, 2023
DOI: 10.21769/BioProtoc.4725 Views: 339
Reviewed by: Kathrin Sutter Anonymous reviewer(s)
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Original Research Article:
The authors used this protocol in PLOS Pathogens Aug 2022
Abstract
Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite’s pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.
Keywords: Malaria Flow cytometry Protein transport Surface proteins Live cell
Background
The most virulent form of malaria is caused by the protozoan parasite Plasmodium falciparum, killing over 600,000 people annually (Weiss et al., 2019). During the asexual blood stage, the parasite invades the red blood cell (RBC) and feeds on intracellular hemoglobin; however, while circulating in the host bloodstream, the infected RBC is at risk of elimination if it transits the host’s splenic sinuses. To avoid splenic clearance, the parasite remodels the host cell by exporting proteins into the RBC cytoplasm (Marti et al., 2004; Sargeant et al., 2006; Boddey et al., 2013; Heiber et al., 2013). During this process, a key modification is the presentation of the variant antigen P. falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected RBCs (Smith et al., 1995). The antigen EMP1 is encoded by the var gene family where each parasite expressing one of approximately 60 variants at any time. These variants act as adhesins to a range of cellular ligands, expressed on a range of endothelial cells in the capillaries throughout the body, allowing the infected RBC to sequester within the host’s vasculature. The cytoadhesion of infected RBCs can lead to fatal complications associated with cerebral and placental malaria (Storm and Craig, 2014; Jensen et al., 2020; Sahu et al., 2021). EMP1 transport to the surface remains poorly understood as the parasite, with the parasite building its own de novo trafficking system, which is highly divergent from classical eukaryotic trafficking machinery. For these reasons, EMP1 trafficking is of high interest from both clinical and basic biology perspectives.
In our recent studies, we identified parasite proteins that are exported into the host cell and affect the trafficking and presentation of the antigen EMP1 (McHugh et al., 2020; Carmo et al., 2022). The majority of EMP1 is trafficked to the host cell surface 16–20 h post invasion, and mid-trophozoite stage-infected RBCs (20–32 h post invasion) are used to study EMP1 presentation at the host cell surface (Kriek et al., 2003). To determine if EMP1 is present at the surface of infected RBCs, we employ two complementary assays. These include: 1) trypsin cleavage assay, where surface-exposed EMP1 is shaved from the surface by trypsin and the membrane-embedded domain is then detected by immunoblotting (Cooke et al., 2006; Carmo et al., 2022); and 2) binding assays, in which the infected RBCs are passed through a channel coated with ligand and the number of cells adhering is quantified (McHugh et al., 2015 and 2020; Carmo et al., 2022). These assays are useful; however, they have their limitations. For example, the trypsin assay is semi-quantitative at best, while defects in adhesion underflow can be multifactorial (not due to EMP1 surface presentation alone).
In an alternative approach outlined here, antibodies specific to the ectodomain of EMP1 are used to label live cells, which are then quantitated by flow cytometry, to measure the number of labeled cells and their relative intensity of surface-exposed EMP1 (first established in Smith et al., 1995; Beeson et al., 2004; Elliott et al., 2005) (Figure 1). Of the techniques assaying EMP1 surface presentation published to date, this is the most time- and resource-efficient method. In addition, the assay is implemented in a 96-well plate format, making it easily scalable. This technique can also be adjusted to quantitate surface presentation of surface proteins in other cellular contexts where an antibody is available for the ectodomain of the protein of interest.
Figure 1. Cell labeling and analysis flowchart. (A) Live cell labeling of the ectodomain of the VAR2CSA, an EMP1 variant available on the surface of P. falciparum infected red blood cells (RBCs). The VAR2CSA variant is associated with adhesion to chondroitin sulphate A in the placenta, causing placental malaria. A zoom-in window depicts the VAR2CSA antigen (blue) with a list of the live cell labeling steps: incubations with three antisera, the final of which is conjugated to an Alexa Fluor 488, and finally a DNA stain. (B) The labeled cells are analyzed by flow cytometry. (C) Major populations can be separated by plotting the Pacific blue height (cells with DNA) against FITC height (cells labeled with Alexa Fluor 488), where each dot is a single cell and high densities of cells are indicated by blue through to red hues. Schematics of RBCs indicate the type of cell gated in each quartile. Uninfected RBCs do not have DNA; therefore, Q4 represents uninfected cells. Q1 represents infected cells that do not have VAR2CSA ectodomains available to the antisera. Q2 indicates infected cells that have VAR2CSA ectodomains available to the antisera. These data are used to compare the Q2 populations between cell lines to determine if mutations to P. falciparum cell lines affect VAR2CSA translocation to the surface of the infected RBC.
Materials and reagents
Storage notes: All primary antibodies are stored at -20 °C. Secondary antibodies and DNA stains are stored at 4 °C unless otherwise noted. All reagents for tissue culture were prepared in a class II biosafety cabinet while practicing aseptic technique, including the use of sterile pipette tips.
96-well plate, v-well, polystyrene with lid, sterile (Sarstedt, catalog number: 3 82.1583.001)
Sterile 35 mm × 10 mm tissue culture dish (Sigma, Corning, catalog number: 430165)
BSA 30% (w/v) (CSL Immulab, catalog number: 06701305)
Rabbit anti-VAR2CSA antibody recognizing an epitope on the ectodomain of VAR2CSA [Duffy and Rogerson labs, University of Melbourne, code: R1945 (Reeder et al., 1999)]
Monoclonal anti-rabbit IgG (γ-chain specific) antibody produced in mouse (Sigma, catalog number: R1008-2ML)
Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen, Life Technologies, catalog number: A11029)
Alexa Fluor 647 goat anti-mouse IgG (H+L) (Invitrogen, Life Technologies, catalog number: A21236)
Hoechst 33342 (Invitrogen, catalog number: H3570)
SYTO 61 (Life Technologies, catalog number: S11343) to measure parasitemia. SYTO 61 is diluted from 5 mM to 100 μM in DMSO and stored in 50 μL aliquots at -20 °C
Dimethyl sulfoxide (DMSO) (ChemSupply, catalog number: DA013-500M)
RPMI 1640 medium with GlutaMAX supplement and HEPES (Life Technologies, Gibco, catalog number: 72400120)
Hypoxanthine (Sigma, catalog number: H9636-5G) made to 200 mM in 1 M NaOH (ChemSupply, catalog number: SL178-500G), filter sterilized, and stored in 1 mL aliquots at -20 °C
Gentamicin 10 mg/mL in deionized water (Sigma, catalog number: G1397)
D-glucose (ChemSupply, catalog number: GA018-500G)
D-sorbitol (ChemSupply, catalog number: SL151-500G), made to 5% w/v in MilliQ water, filter sterilized, and stored at 4 °C
Albumax II (Life Technologies, catalog number: 11021045) dissolved in RPMI 1640 medium with GlutaMAX supplement and HEPES at 5%, filter sterilized, and stored in 25 mL aliquots at -20 °C
Media solution (910 mM D-glucose, 0.45 mg/mL gentamicin), stored in 5 mL aliquots at -20 °C
Pooled sera from any blood type (Lifeblood Australia), heat inactivated for 1.5 h, filter sterilized, and stored in 25 mL aliquots at -20 °C
Red blood cells (Lifeblood Australia), O+
Malaria mix (1% O2, 5% CO2, and 94% N2) (Coregas, catalog number: 388150)
Giemsa’s stain improved solution R66, Gurr for microscopical staining (VWR chemicals, catalog number: 350864X)
Methanol (ChemSupply, catalog number: MA004)
Triton X-100 (BioXtra, Sigma, catalog number: T9284)
Sodium hypochlorite 8-14% (Ajax Finechem, catalog number: AJA485-5L)
Complete culture media (CCM) stored at 4 °C, used at 37 °C (see Recipes)
Equipment
Incubator (LabQuip Sciences, SEM Equipment, model: 18FD)
Multichannel pipettes, 8-channel, 20–200 μL (Socorex, model: 855) and 5–100 µL (Bohit, model: m100)
Class II biosafety cabinet (Laftech, EuroClone, model: safemate 1.2 vision)
Centrifuge (Hettich, model: Rotina 420)
Flow cytometer [BD Biosciences, model: FACSCanto II Flow Cytometer System with an integrated BD High Throughput Sampler (HTS)]. The following filter sets were used for fluorophore detection: Alexa Fluor 647 and SYTO-61 (APC, 660/20 nm), Alexa Fluor 488 (FITC, 530/30 nm), and Hoechst 33342 (Pacific Blue, 450/50 nm)
Software
FACSDiva 8.0.1 (BD Biosciences, https://www.bdbiosciences.com/en-au/products/software/instrument-software/bd-facsdiva-software)
FlowJo v10 (BD Biosciences, https://www.flowjo.com/solutions/flowjo)
Prism 9 (Dotmatics, GraphPad, https://www.graphpad.com/scientific-software/prism/)
Procedure
All sample preparation is performed under sterile conditions. Centrifugation steps are at either 528× g for 5 min (5 acc/1 dec) (A3–B1) or 528× g for 90 s (9 acc/9 dec) for 96-well plates. Slow deceleration is used when centrifugation is performed in Falcon tubes to reduce the disturbance of the infected RBCs pellet.
Prepare malaria parasite cell culture
Maintain parasite-infected RBC culture at 5% hematocrit (percentage by volume of RBCs in culture) with O+ RBCs in CCM at 37 °C in a low oxygen environment (malaria mix). As an example, a 35 mm × 10 mm tissue culture dish would contain 250 μL of RBCs and 4.75 mL of CCM, yielding a 5% hematocrit culture. O+ RBCs are used as they are compatible with pooled sera from any blood type, avoiding RBC agglutination.
Parasitemia (the percentage of infected RBCs relative to total RBCs) should be kept at ≤ 5% and the CCM replaced every 24–48 h. Monitor the parasitemia via thin blood smears and Giemsa staining.
Thin blood smears and Giemsa staining: Deposit 1–2 μL of RBCs at one end of a glass microscope slide. Use another glass slide held at a 45° angle to smear the cells across the first slide. Allow to air dry; then, fix the cells by immersing the slide in methanol for at least 5 s. Allow the slide to air dry and incubate the fixed slide in 10% Giemsa stain diluted in water for 5 min. Remove the slide from the stain and wash off excess stain with tap water. Blot the slide dry with paper towels; then, add immersion oil and visualize the cells with a 100× objective on a light microscope. Cell fixation and staining can be done in a non-sterile environment. Parasites will be stained dark purple and RBCs light purple. Use a physical cell counter to count ≥500 cells to determine the parasitemia, or percentage of cells infected with a parasite, prior to an experiment.
Synchronize parasite cultures using the osmolyte D-sorbitol. Parasite-infected RBCs from 20 h post invasion (hpi) are susceptible to hypotonic lysis when incubated in a 5% solution of D-Sorbitol (Ginsburg et al., 1983; Counihan et al., 2017). This physiology is leveraged to synchronize cultures, as ring-stage parasites survive sorbitol treatment.
Harvest the culture by centrifugation at 528× g for 5 min (5 acc/1 dec), aspirate the media, and resuspend the pellet in 5% D-sorbitol (w/v) to obtain a 5% hematocrit solution (the original volume of the culture). Incubate the D-sorbitol-treated culture in a water bath at 37 °C for 5–10 min. Harvest the sorbitol-treated culture, aspirate the supernatant, resuspend the pellet in CCM to 5% hematocrit, and return the culture to a fresh culturing dish or flask. One sorbitol synchronization will retain parasites that are ~0–20 hpi. Sorbitol-synchronize the parasites again 8 h later to narrow the age range to ~8–20 hpi. For the protocol outlined here, parasites must be synchronized to ~20–32 hpi (i.e., two synchronizations, 12 h apart).
Transgenic parasites are maintained in the presence of selection reagent. To generate transgenic parasite cell lines, follow the detailed protocols and recipes outlined in Rug and Maier (2013). Our transgenic parasite cell lines were generated in a CS2 background, an isolate expressing the EMP1 variant VAR2CSA (Duffy et al., 2006). It is recommended to use the parent cell line as a positive control, as we do in this protocol.
CS2 is a clonal parasite line that expresses var2csa as a dominant var gene transcript, allowing us to use an antibody for the encoded antigen (the EMP1 variant var2csa) to assess antigen trafficking to the host cell surface (Elliott et al., 2005; Duffy et al., 2006).
Dilute and aliquot cells
The infected RBC culture should be at 5% hematocrit with a parasitemia between 3% and 5%. Harvest mid-trophozoite stage cultures (~20–32 hpi) by centrifugation at 528× g for 5 min (5 acc/1 dec).
Aspirate the spent media leaving the RBC pellet undisturbed. Resuspend the RBCs in 1% BSA/PBS and dilute as required to obtain a solution containing a 3%–5% parasitemia and 2%–4% hematocrit.
Load 20 μL of diluted cells into a 96-well plate in duplicate or triplicate per condition and cell line. At a minimum, you will require a no-primary control (wells incubated with the BSA diluent and that receive the second and third antibody treatments), totalling ≥ 6 wells per cell line if performed in triplicate.
Add 100 μL of wash buffer (1% BSA/PBS) to each well. Alternatively, add 100 µL of wash buffer prior to loading cells in step B3. We recommend a multichannel pipette for all subsequent washing steps.
Centrifuge the plate at 528× g for 90 s (9 acc/9 dec) to pellet the cells, then aspirate the media using an aspirating tip. We find that the accuracy of aspiration is improved when a sterile pipette tip (no filter) is mounted on the end of the aspirator tip. Touch the tip to the wall of each well to aspirate the supernatant.
Cell staining
Note: All antisera are diluted in wash buffer (1% BSA/PBS).
Resuspend each well with 10 μL of either rabbit polyclonal anti-VAR2CSA antibody (1:100) or 1% BSA/PBS as a control and incubate for 30 min at 37 °C.
Add 100 μL of wash buffer (1% BSA/PBS), centrifuge the plate, and aspirate the supernatants.
Resuspend each well in 100 μL of wash buffer (1% BSA/PBS), centrifuge the plate, and aspirate the supernatants.
Resuspend each well with 10 μL of mouse anti-rabbit IgG (1:100) and incubate for 30 min at 37 °C.
Repeat steps C2 and C3 (wash steps).
Resuspend each well with 10–20 μL of goat anti-mouse antibody conjugated to a fluorophore (1:100) and incubate for 30 min at 37 °C.
For cell lines expressing GFP, we use Alexa Fluor 647 goat anti-mouse IgG (H+L) and Hoechst 33342 DNA stain.
For cell lines not expressing GFP, we use Alexa Fluor 488 goat anti-mouse IgG (H+L) and Hoechst 33342 or SYTO-61 nucleic acid stain.
Repeat steps C2 and C3.
Incubate the cells in the DNA or nucleic acid stain.
For Hoechst 33342 staining, add 20 μL of stain (1:2,000) and incubate for 30 min at 37 °C. Wash cells twice (step C5), then wash the cells once in 100 μL of PBS alone. After this final wash, resuspend the cells in 200 μL of PBS (0.2%–0.4% hematocrit final), and then proceed with flow cytometry measurements. We use low hematocrit for these experiments to reduce the likelihood of capillary blockage during flow cytometry.
For SYTO-61 staining, incubate cells in 20 μL of SYTO-61 (5 μM in PBS) for 15 min at room temperature (Klonis et al., 2011). Add 180 μL of PBS to dilute the wells to 0.02% hematocrit and incubate the cells for 30 min at room temperature before being measured.
Flow cytometry using FACSDiva software
The steps below refer to a non-GFP-expressing cell line, labeled with Alexa Fluor 488 tertiary antibody and Hoechst 33342. Perform these steps for one well containing a double-stained positive sample (or well), e.g., the positive control, then apply the gating strategy to all wells in the plate.
Use a side scatter height (SSC-H) vs. forward scatter area (FSC-A) plot to identify and gate the total RBC population (Figure 2A). Select this population.
Then, use a forward scatter height (FSC-H) vs. forward scatter width (FSC-W) plot to gate singlet events (Figure 2B). Select this population.
Plot Pacific blue height (DNA stain) vs. FITC height (fluorophore-conjugated tertiary antibody) (Figure 2C). Adjust the voltage of Pacific blue and FITC so that the Pacific blue-H +/- and FITC-H +/- populations can be clearly delineated. In our example plot (Figure 2C), the quadrant divide sits at approximately 103. Once the voltages are optimized, apply these acquisition paraments to all wells in the plate and all biological repeats. As visualized in Figure 1, the Q4 population represents uninfected RBCs, the Q1 population is infected but does not have labeled EMP1 on the surface, and the Q2 population is both infected and presenting EMP1 on the surface.
Note: If there is a substantial number of high-FITC events to the right of the main Q2 population, these are likely non-specific events. Consider increasing the volume of wash buffer during the wash steps or the number of washes after the final antisera incubation. Alternatively, use the secondary-only antibody controls as a guide for drawing a right boundary on the FITC+ populations.
Update the high throughput sampler (HTS) loader settings (Table 1).
Table 1. Flow cytometer HTS loader settings
Sample flow rate (μL/s) 0.5
Sample volume (μL) 20
Mixing volume (μL) 100
Mixing speed (μL/s) 200
Number of mixes 4
Wash volume (μL) 800
Collect a total of 50,000 events per well.
Export all data as FCS-3 files.
For instrument cleanup, flush the system sequentially with ≥ 400 μL of 0.1% (w/v) Triton X-100, ≥ 400 μL of 1% sodium hypochlorite, and ≥ 800 μL of ultra-pure water.
Figure 2. Flow cytometry analysis of a single well. Dots represent events detected. Blue to green to red hues indicate increasing population densities. (A) Side scatter height vs. forward scatter area to gate the major population, or total cells (infected and uninfected RBCs). (B) Doublet discrimination by plotting forward scatter height vs. forward scatter width and gating for the major population, or single cells. (C) Pacific blue height vs. FITC height to parse the single cell events into quartiles. Q2, the Pacific blue+/FITC+ population as a percentage of the single cell events, is used to compare the surface presentation of VAR2CSA between cell lines.
Data analysis
Open FCS-3 files in FlowJo and apply the same gating strategy as outlined above (Figure 2A and 2B).
After delineating the total population into each quartile (Figure 2C), export the cell frequency of parent data for Q2 (i.e., the percentage of cells in Q2 compared to the single cell gate) for all wells.
Average the Q2 frequency across the technical repeats for each cell line and condition independently.
Subtract the Q2 averaged frequency of the no-primary controls from that of the experimental samples. This operation accounts for non-specific events in Q2.
Collect at least four biological repeats and perform the analysis above.
Using Prism 9 software, enter the biological replicate values in a column table where each column is a unique parasite cell line. Perform an unpaired t-test with Welch’s correction to determine the p-value.
Plot individual values as a scatterplot with bar (mean and standard deviation).
Notes
Cell staining incubations were performed at 37 °C in standard atmospheric conditions. If practical, these incubations may be performed in malaria gas mix.
If live cell flow cytometry is not an option, the cells can be fixed after step C8. Fixation may be performed by incubating each sample with 20 μL of 4% Formaldehyde/0.0065% glutaraldehyde in PBS for 10 min, followed by three washes with 200 μL of PBS.
Recipes
Complete culture media (CCM)
Thaw then add the following reagents to 500 mL of RPMI 1640 medium with GlutaMAX and HEPES, store at 4 °C.
25 mL of pooled human sera
25 mL of 5% w/v Albumax II, dissolved in RPMI 1640 medium with GlutaMAX and HEPES
5 mL of media solution (910 mM D-glucose, 0.45 mg/mL gentamicin)
1 mL of 200 mM hypoxanthine
Acknowledgments
We thank Michael Duffy and Stephen Rogerson for providing us with critical reagents. This original protocol was based on Beeson et al. (2004) and Smith et al. (1995). We also thank Molly P. Schneider for reviewing the protocol and for her helpful suggestions. MWAD thanks the National Health and Medical Research Council (1098992) (https://www.nhmrc.gov.au) for funding this work. The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
Competing interests
The authors have declared that no competing interests exists.
Ethics
Red blood cells and serum were acquired from the Australian Red Cross Lifeblood blood service. All blood products were anonymous and individual donors could not be identified. This work was approved with the written consent of the University of Melbourne Human Research Ethics Committee (approval number 1750526.3).
References
Beeson, J. G., Mann, E. J., Elliott, S. R., Lema, V. M., Tadesse, E., Molyneux, M. E., Brown, G. V. and Rogerson, S. J. (2004). Antibodies to variant surface antigens of Plasmodium falciparum-infected erythrocytes and adhesion inhibitory antibodies are associated with placental malaria and have overlapping and distinct targets. J Infect Dis 189(3): 540-551.
Boddey, J. A., Carvalho, T. G., Hodder, A. N., Sargeant, T. J., Sleebs, B. E., Marapana, D., Lopaticki, S., Nebl, T. and Cowman, A. F. (2013). Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome. Traffic 14(5): 532-550.
Carmo, O. M. S., Shami, G. J., Cox, D., Liu, B., Blanch, A. J., Tiash, S., Tilley, L. and Dixon, M. W. A. (2022). Deletion of the Plasmodium falciparum exported protein PTP7 leads to Maurer's clefts vesiculation, host cell remodeling defects, and loss of surface presentation of EMP1. PLoS Pathog 18(8): e1009882.
Cooke, B. M., Buckingham, D. W., Glenister, F. K., Fernandez, K. M., Bannister, L. H., Marti, M., Mohandas, N. and Coppel, R. L. (2006). A Maurer’s cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells. J Cell Biol 172(6): 899-908.
Counihan, N. A., Chisholm, S. A., Bullen, H. E., Srivastava, A., Sanders, P. R., Jonsdottir, T. K., Weiss, G. E., Ghosh, S., Crabb, B. S., Creek, D. J., et al. (2017). Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate. Elife 6: e23217.
Duffy, M. F., Maier, A. G., Byrne, T. J., Marty, A. J., Elliott, S. R., O’Neill, M. T., Payne, P. D., Rogerson, S. J., Cowman, A. F., Crabb, B. S., et al. (2006). VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum. Mol Biochem Parasitol 148(2): 117-124.
Elliott, S. R., Duffy, M. F., Byrne, T. J., Beeson, J. G., Mann, E. J., Wilson, D. W., Rogerson, S. J. and Brown, G. V. (2005). Cross-reactive surface epitopes on chondroitin sulfate A-adherent Plasmodium falciparum-infected erythrocytes are associated with transcription of var2csa. Infect Immun 73(5): 2848-2856.
Ginsburg, H., Krugliak, M., Eidelman, O. and Cabantchik, Z. I. (1983). New permeability pathways induced in membranes of Plasmodium falciparum infected erythrocytes. Mol Biochem Parasitol 8(2): 177-190.
Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., et al. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.
Jensen, A. R., Adams, Y. and Hviid, L. (2020). Cerebral Plasmodium falciparum malaria: The role of PfEMP1 in its pathogenesis and immunity, and PfEMP1-based vaccines to prevent it. Immunol Rev 293(1): 230-252.
Klonis, N., Crespo-Ortiz, M. P., Bottova, I., Abu-Bakar, N., Kenny, S., Rosenthal, P. J. and Tilley, L. (2011). Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion. Proc Natl Acad Sci U S A 108(28): 11405-11410.
Kriek, N., Tilley, L., Horrocks, P., Pinches, R., Elford, B. C., Ferguson, D. J., Lingelbach, K. and Newbold, C. I. (2003). Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasite-infected erythrocytes. Mol Microbiol 50(4):1215-1227.
Marti, M., Good, R. T., Rug, M., Knuepfer, E. and Cowman, A. F. (2004). Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306(5703): 1930-1933.
McHugh, E., Batinovic, S., Hanssen, E., McMillan, P. J., Kenny, S., Griffin, M. D., Crawford, S., Trenholme, K. R., Gardiner, D. L., Dixon, M. W., et al. (2015). A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking. Mol Microbiol 98(6): 1101-1114.
McHugh, E., Carmo, O. M. S., Blanch, A., Looker, O., Liu, B., Tiash, S., Andrew, D., Batinovic, S., Low, A. J. Y., Cho, H. J., et al. (2020). Role of Plasmodium falciparum Protein GEXP07 in Maurer’s Cleft Morphology, Knob Architecture, and P. falciparum EMP1 Trafficking. mBio 11(2): e03320-19.
Reeder, J. C., Cowman, A. F., Davern, K. M., Beeson, J. G., Thompson, J. K., Rogerson, S. J. and Brown, G. V. (1999). The adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A is mediated by P. falciparum erythrocyte membrane protein 1. Proc Natl Acad Sci U S A 96(9): 5198-5202.
Rug, M. and Maier, A. G. (2013). Transfection of Plasmodium falciparum. Methods Mol Biol 923: 75-98.
Sahu, P. K., Duffy, F. J., Dankwa, S., Vishnyakova, M., Majhi, M., Pirpamer, L., Vigdorovich, V., Bage, J., Maharana, S., Mandala, W., et al. (2021). Determinants of brain swelling in pediatric and adult cerebral malaria. JCI Insight 6(18): e145823.
Sargeant, T. J., Marti, M., Caler, E., Carlton, J. M., Simpson, K., Speed, T. P. and Cowman, A. F. (2006). Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites. Genome Biol 7(2): R12.
Smith, J. D., Chitnis, C. E., Craig, A. G., Roberts, D. J., Hudson-Taylor, D. E., Peterson, D. S., Pinches, R., Newbold, C. I. and Miller, L. H. (1995). Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82(1): 101-110.
Storm, J. and Craig, A. G. (2014). Pathogenesis of cerebral malaria--inflammation and cytoadherence. Front Cell Infect Microbiol 4: 100.
Weiss, D. J., Lucas, T. C. D., Nguyen, M., Nandi, A. K., Bisanzio, D., Battle, K. E., Cameron, E., Twohig, K. A., Pfeffer, D. A., Rozier, J. A., et al. (2019). Mapping the global prevalence, incidence, and mortality of Plasmodium falciparum, 2000-17: a spatial and temporal modelling study. Lancet 394(10195): 322-331.
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4,726 | https://bio-protocol.org/en/bpdetail?id=4726&type=0 | # Bio-Protocol Content
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Engineering Agrobacterium tumefaciens with a Type III Secretion System to Express Type III Effectors
VR Vidhyavathi Raman
KM Kirankumar S. Mysore
Published: Vol 13, Iss 15, Aug 5, 2023
DOI: 10.21769/BioProtoc.4726 Views: 445
Reviewed by: Wenrong HeYao XiaoYe Xu
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Original Research Article:
The authors used this protocol in Nature Communications May 2022
Abstract
Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens–mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens.
Keywords: Type III secretion system Type III effectors Agrobacterium Triparental mating Transformation
Background
The Type III secretion system (T3SS) is a specialized complex structure that translocates Type III effectors (T3Es) into the plant cell, where they interact with the host immune system. T3SS is present in many pathogenic Gram-negative bacteria (Cornelis, 2006). Heterologous expression of the genes encoding the type III secretion apparatus is functional in Pseudomonas fluorescens (Huang et al., 1988) and Escherichia coli (Ham et al., 1998). Gene cluster coding structural and regulatory components of T3SS from P. syringae have been cloned and are available in a plasmid called pLN18 (Jamir et al., 2004). We have transferred this plasmid into Agrobacterium tumefaciens to express T3SS and deliver T3Es to suppress plant defense and enhance plant genetic transformation (Raman et al., 2022). Because of the large size, we used triparental mating to mobilize pLN18 to A. tumefaciens in the presence of helper plasmid (pRK2013; containing tra genes) containing E. coli, using the protocol by Wise et al. (2006) with some modifications. In addition, we optimized culture growth and fractionation of the liquid culture into pellet and supernatant fractions to avoid cell lysis that leads to false positive results.
Materials and reagents
1.5 mL microcentrifuge tubes (USA Scientific)
50 mL Falcon tubes (Corning, catalog number: 352070)
0.45 μm Durapore PVDF membrane Millipore filter (Steriflip-HV Sterile Centrifuge Tube Top Filter Unit) (Millipore Sigma, catalog number: SE1M003M00)
Amicon Ultra-15 centrifugal filters (Millipore Sigma, catalog number: UFC901024)
Amicon Ultra-0.5 centrifugal filters (Millipore Sigma, catalog number: UFC501024)
A. tumefaciens GV2260 (Rifampicin resistance)
E. coli HB101 (pRK2013) (Kanamycin resistance)
Donor E. coli containing pLN18 (Kanamycin and Tetracycline resistance)
Luria Bertani (LB) medium (MP Biomedicals, catalog number: 3002011)
Luria Bertani (LB) medium with agar (MP Biomedicals, catalog number: 3002201)
PhiLOV-specific antibody (gift from Dr. John M. Christie, University of Glasgow)
Bacto peptone (BD, catalog number: 211677)
Yeast extract (BD, catalog number: 212750)
Bacto agar (BD, catalog number: 214010)
NaCl (J.T. Baker, catalog number: 3624-19)
K2HPO4 (J.T. Baker, catalog number: 4012-01)
NaH2PO4 (Fisher Scientific, catalog number: S369-500)
NH4Cl (Fisher Scientific, catalog number: A661)
KCl (Fisher Scientific, catalog number: P217-500)
MgSO4·7H2O (Sigma-Aldrich, catalog number: M1880)
CaCl2 (J.T. Baker, catalog number: 1313-01)
FeSO4·7H2O (Sigma-Aldrich, catalog number: F8263)
Sucrose (Invitrogen, catalog number: 15503-022)
Rifampicin (Sigma-Aldrich, catalog number: R3501-1G)
Kanamycin sulfate (Sigma-Aldrich, catalog number: 70560-51-9)
Tetracycline (Sigma-Aldrich, catalog number: T3258)
Yeast extract peptone (YEP) medium (1 L) (see Recipes)
20× AB buffer (1 L) (see Recipes)
20× AB salts (1 L) (see Recipes)
Water agar (see Recipes)
25% sucrose (see Recipes)
Agrobacterium minimal (AB)-sucrose agar medium or AB medium (see Recipes)
0.8% NaCl solution medium (see Recipes)
Hrp-derepressing (HDM) medium (see Recipes)
Equipment
28 °C incubator with shaker (New Brunswick Scientific)
37 °C incubator with shaker (New Brunswick Scientific)
Spectrophotometer (Bio-Rad, catalog number: 170-2525)
Eppendorf tabletop centrifuge (Eppendorf, catalog number: 5424)
Eppendorf tabletop refrigerated centrifuge (Eppendorf, catalog number: 5424R)
Eppendorf centrifuge with swing-bucket rotor (Eppendorf, catalog number: 5810R)
Procedure
Mobilization of T3SS to A. tumefaciens through triparental mating
Triparental mating is used to mobilize a large plasmid from E. coli to A. tumefaciens by using another E. coli carrying helper plasmid—in this case, HB101 (pRK2013).
Streak out the recipient A. tumefaciens strain (e.g., GV2260) from the -80 °C freezer onto YEP agar plates containing antibiotic [rifampicin (Rif); 10 μg/mL] and allow it to grow in a 28 °C incubator for two days. Streak out donor (pLN18, TetR) and helper (pRK2013, KmR) plasmid-containing E. coli strains onto LB agar plates containing appropriate antibiotics [kanamycin (Km), 50 μg/mL; and tetracycline (Tet), 5 μg/mL] and allow them to grow in a 37 °C incubator for a day.
The day before triparental mating, culture a single colony of A. tumefaciens in 5 mL of YEP medium supplemented with the appropriate antibiotics [rifampicin (10 μg/mL)] and leave overnight with shaking at 250 rpm and 28 °C. Culture a single colony of E. coli strains carrying donor and helper plasmids in 5 mL of LB liquid medium supplemented with the appropriate antibiotics, shaking at 250 rpm and 37 °C overnight.
On the day of mating, from the overnight cultures, inoculate 300 μL of A. tumefaciens to 3 mL of YEP liquid medium with rifampicin (10 μg/mL) and grow for 3–5 h at 250 rpm and 28 °C; inoculate 30 μL of E. coli to 3 mL of LB liquid medium with appropriate antibiotics and grow for 3–5 h at 250 rpm and 37 °C. Grow all the cultures until A600 reaches 0.3–0.5.
Triparental mating (Figure 1 and Figure 2)
Figure 1. Experimental procedure for mobilizing pLN18 to A. tumefaciens
Figure 2. Flowchart describing timeline for the triparental mating protocol
Aliquot 500 μL each for all cultures in a 1.5 mL microcentrifuge tube separately and spin at 9,391× g for 2 min at room temperature (RT). Discard the supernatants. Wash once by resuspending the pellets in 500 μL of 0.8% NaCl and spin again.
Resuspend the pellet in 250 μL of 0.8% NaCl.
Mix 50 μL each of the three cultures in a microcentrifuge tube and spot it on a plain LB plate; allow it to dry for a few minutes and incubate the plate at 28 °C for two days.
Scrape off the mat-like growth (Figure 3) and resuspend in 500 μL of 0.8% NaCl in a 1.5 mL microcentrifuge tube. Spread 10 and 50 μL and the rest of the suspension in three separate AB (Gelvin, 2006) plates with Rif and Tet and incubate the plates at 28 °C for 2–4 days.
Figure 3. Representative image showing mat-like growth on plain LB plate
Isolate single colonies for glycerol stocks and colony PCR.
Assays using A. tumefaciens expressing T3SS and T3Es
Either electroporation or the freeze-thaw method is used to transform GV2260 (pLN18) with a broad host range plasmid containing T3E (e.g., pBBR1MCS5-AvrPto-PhiLOV). Western blot and microscopy can be used to demonstrate effector secretion and delivery, respectively. In this protocol, we use western blot hybridization to show that T3Es are expressed and delivered out of the cell.
A. tumefaciens strains expressing T3SS and T3E are streaked onto YEP agar plates along with appropriate controls and incubated at 28 °C for two days. GV2260, GV2260 (pLN18), and GV2260 (pBBR1MCS5-AvrPto-PhiLOV) are used as controls.
Inoculate two colonies into 20 mL of HDM medium (Huynh et al., 1989) in 50 mL screw-cap tubes and incubate at 28 °C for 16 h shaking at 220 rpm.
Aliquot a small amount of culture (A600 = 0.25) in a 1.5 mL microfuge tube and centrifuge in a tabletop centrifuge at 9,391× g at RT for 2 min. Use the resulting pellet for pellet fraction analysis.
Centrifuge remaining cultures at 3,220× g for 15 min at 21 °C.
Using 25 mL pipettes, carefully remove the top 15 mL of the supernatant solutions without disturbing the pellet, by lowering the pipette slowly into the supernatant.
Transfer the supernatant fraction to 50 mL tubes and filter using a 0.45 μm Durapore PVDF membrane Millipore filter with lab vacuum connection.
Transfer 13 mL of the filtrate to Amicon Ultra-15 centrifugal filters and concentrate using a swinging bucket rotor at 3,220× g for 30 min at 4 °C.
Add the remaining 12 mL of the filtrate to the same Amicon Ultra-15 centrifugal filter and concentrate at 4 °C for another 30 min at 3,220× g.
Recover the concentrated filtrate from the bottom of the filter device and again concentrate to ~30 μL using Amicon Ultra-0.5 centrifugal filters at 14,000× g for 40–60 min at 4 °C.
Recover the concentrated sample by keeping the filter device upside down in a new microcentrifuge tube and spin for 2 min at 1,000× g in a refrigerated centrifuge. Store the concentrated sample at -80 °C until analysis.
Proteins from the pellet and supernatant fractions were subjected to electrophoresis through an SDS-PAGE gel using standard protocols, and immunoblot analysis was carried out using PhiLOV-specific antibody (dilution 1:5,000) (Figure 4).
Figure 4. Western blot analysis of AvrPto expression and secretion in cell pellet and supernatant fractions
Recipes
YEP medium (1 L)
Bacto peptone 10 g
NaCl 5 g
Yeast extract 10 g
Bacto agar 15 g
Sterilize by autoclave at 121 °C for 15 min
20× AB buffer (1 L)
K2HPO4 60 g
NaH2PO4 20 g
Adjust the pH to 7.0
20× AB salts (1 L)
NH4Cl 20 g
KCl 3 g
MgSO47H2O 6 g
CaCl2 0.2 g
FeSO4·7H2O 50 mg
Water agar
Bacto agar 3.75 g in 220 mL of water
Autoclave 20× AB buffer, 20× AB salts, and water agar separately at 121 °C for 15 min
25% sucrose
12.5 g sucrose/50 mL water, mix well, and filter sterilize using 0.22 μm filter
AB-sucrose agar medium or AB medium
Add 20× AB buffer, 20× AB salts, and 25% sucrose when the water agar is warm
Water agar 220 mL
20× AB salts 12.5 mL
20× AB buffer 12.5 mL
25% sucrose 5 mL
0.8% NaCl solution medium (100 mL)
NaCl 0.8 g
Water 100 mL
Sterilize by autoclave at 121 °C for 15 min
HDM medium (1 L)
Fructose 1.801 g
(NH4)2SO4 1.004 g
MgCl2·6H2O 0.345 g
NaCl 0.099 g
1 M K2HPO4 6.6 mL
1 M KH2PO4 43.4 mL
Water 950 mL
pH 6.0 (No need to adjust the pH)
Sterilize by autoclave at 121 °C for 15 min
Acknowledgments
This work was supported by the National Science Foundation (grant # IOS-1725122 and IOS-2219792 to K.S.M.) and the Noble Research Institute, LLC. Media recipes described here are adapted from Gelvin (2006) and Huynh et al. (1989), and triparental mating procedure was modified from Wise et al. (2006). Protocols for fractionation of A. tumefaciens culture and western blot are from our published paper Raman et al. (2022). Figure 1 was generated with the help of Biorender (https://biorender.com/).
Competing interests
The authors declare no competing interests.
References
Cornelis, G. R. (2006). The type III secretion injectisome. Nat rev Microbiol 4(11): 811-825.
Gelvin, S. B. (2006). Agrobacterium virulence gene induction. Methods Mol Biol 343: 77-84.
Ham, J. H., Bauer, D. W., Fouts, D. E. and Collmer, A. (1998). A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture. Proc Natl Acad Sci U S A 95(17): 10206-10211.
Huang, H. C., Schuurink, R., Denny, T. P., Atkinson, M. M., Baker, C. J., Yucel, I., Hutcheson, S. W. and Collmer, A. (1988). Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants. J Bacteriol 170(10): 4748-4756.
Huynh, T., Dahlbeck, D. and Staskawicz, B. (1989). Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science 245(4924): 1374-1377.
Jamir, Y., Guo, M., Oh, H. S., Petnicki-Ocwieja, T., Chen, S., Tang, X., Dickman, M. B., Collmer, A. and Alfano, J. R. (2004). Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast. Plant J 37(4): 554-565.
Raman, V., Rojas, C. M., Vasudevan, B., Dunning, K., Kolape, J., Oh, S., Yun, J., Yang, L., Li, G., Pant, B. D., et al. (2022). Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation. Nat Commun 13(1): 2581.
Wise, A. A., Liu, Z. and Binns, A. N. (2006). Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol 343: 43-54.
Article Information
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© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite
Category
Plant Science > Plant transformation > Agrobacterium
Microbiology > Microbe-host interactions > Bacterium
Biological Sciences > Biological techniques > Microbiology techniques
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4,727 | https://bio-protocol.org/en/bpdetail?id=4727&type=0 | # Bio-Protocol Content
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Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS
WS Waraporn Suvannapruk
ME Max K. Edney
LF Leanne E. Fisher
JL Jeni C. Luckett
DK Dong-Hyun Kim
DS David J. Scurr
AG Amir M. Ghaemmaghami
MA Morgan R. Alexander
Published: Vol 13, Iss 15, Aug 5, 2023
DOI: 10.21769/BioProtoc.4727 Views: 452
Reviewed by: Aswad KhadilkarNihal Engin VranaVishal Nehru
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Original Research Article:
The authors used this protocol in Analytical Chemistry Jul 2022
Abstract
The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations.
Graphical overview
Keywords: PBMC isolation Monocyte Macrophage Single cell Metabolic profiling 3D OrbiSIMS
Background
Mass spectrometry (MS) analysis methods, including liquid chromatography-mass spectrometry (LC–MS) (A. Abuawad et al., 2020), liquid extraction surface analysis mass spectrometry (LESA–MS) (Eikel et al., 2011), matrix-assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry (SIMS), have been used to detect chemical and biological compounds, such as lipids, amino acids, peptides, and proteins from cells and tissue samples (Rauh, 2012; Cajka and Fiehn, 2014; Passarelli et al., 2017; Kotowska et al., 2020; Meurs et al., 2021). Classes of biomolecules, including proteins, lipids, and metabolites, are vital cellular components that have been characterized and designated to perform specific functions essential to life.
LC–MS-based metabolite analyses typically start with extraction of the metabolites from the biological samples. Analysis of metabolites using LC-MS has been limited because it requires initial liquid extraction procedures and a significant number of cells (1–6 million) to obtain a sufficient signal (Abuawad et al., 2020), leading to a lack of molecular spatial information (Tuli and Ressom, 2009).
The LESA–MS technique is a powerful tool for global, highly sensitive, and multi-analyte analysis ranging from small molecule metabolites to lipids and proteins. This technique has the limitation of involving a solvent-based approach, which allows lipid and small molecule metabolite injection into the analytical instrumentation, as well as poor spatial resolution (~1 mm), rendering single-cell level analysis impossible. For solvent extraction, sample preparation of cells or tissue for metabolomics has a unique protocol for each molecular class. Thus, solvent-extracted metabolites from cells or tissue samples are specific to that extraction protocol (Basu et al., 2018).
SIMS is a direct surface analysis technique that uses a primary ion beam that bombards the surface and generates neutral species and secondary ions (Sigmund, 1969), providing high lateral resolutions of < 100 nm and high surface sensitivity. An electrical field can be used to extract the charge species to obtain mass spectra, using the ion flight times, ion images, and depth profiles in both 2D and 3D (Walker, 2017). SIMS surface analysis has also been established for the quantification of small molecules in biological samples, such as cells and tissue samples.
Time-of-flight secondary ion MS (ToF–SIMS) is a surface analysis technique (Denbigh and Lockyer, 2015; Yoon and Lee, 2018) that provides information-rich mass, depth, and spatial resolution, along with chemical sensitivity. However, ToF–SIMS has insufficient mass accuracy and low mass resolving power for metabolite identification (Green et al., 2011; Shon et al., 2016). To overcome the pitfalls of low mass resolving power and accuracy, the 3D OrbiSIMS technique was developed, which utilizes the SIMS principle with a high mass resolving power (> 240,000 at m/z 200) and accuracy (< 2 ppm), along with the MS/MS capabilities of the OrbiTrapTM mass analyzer (Passarelli et al., 2017).
Recently, the capabilities of the 3D OrbiSIMS instrument as a new means to assess the metabolomic profiles of biological samples have been investigated. For example, Kotowska et al. (2020) used 3D OrbiSIMS imaging and depth profiling to observe a protein monolayer biochip and the depth distribution of proteins in human skin. The platform has also proved its ability to identify metabolite profiling in macrophages treated with different concentrations of the drug amiodarone (Passarelli et al., 2017). Similarly, Suvannapruk et al. (2022) used the novel technique for metabolite identification in individual cells of macrophage subsets. This method of analysis is vital for gaining new insight into metabolomic processes for identifying the metabolites of biological samples.
The development of materials with cell-instructive properties could provide an effective biomaterial-based strategy for modulating cell behavior, to minimize adverse immune responses. Rostam et al. reported that changing the surface topography and chemistry of materials can impact macrophage adhesion and polarization (H. M. Rostam et al., 2015; Hassan M. Rostam et al., 2020). In this paper, we describe for the first time the development of a 3D OrbiSIMS methodology to investigate metabolic changes derived from single anti- and pro-inflammatory macrophages in vitro. We also compare this to metabolic profiles from in vivo anti- and pro-inflammatory macrophages generated at the site of a foreign body response in a mouse model, following exposure to a catheter section coated in known macrophage-instructive surface chemistries. This method aims to directly analyse the metabolic profiles of macrophage phenotypes from in vitro and in vivo studies, with minimal sample preparation steps.
Materials and reagents
Cell culture
T75 flask (Corning 43064, catalog number: 10492371)
50 mL Falcon tube (Sigma-Aldrich, catalog number: T2318)
Pipette tips of various volumes (Fisher Scientific, catalog number: 02-707-401)
Nylon syringe filter 0.22 μm, 25 mm (Minisart®, catalog number: 17845)
Bijou tubes (Thermo Fisher Scientific, catalog number: 129B)
LS columns (Miltenyi Biotech, catalog number: 130-042-401)
Round glass slides (VWR, catalog number: 631-0149)
Glass slides (VWR, catalog number: 631-1553)
24-well plates, uncoated (CytoOne, catalog number: cc77672-7524)
Tissue-Tek Cryomold Moulds (Agar Scientific, catalog number: AGG4580)
Buffy coats from healthy volunteers provided by National Blood Services, Sheffield United Kingdom, after obtaining informed consent and following institutional ethics approval (Research Ethics Committee, Faculty of Medicine and Health Sciences, University of Nottingham; FMHS 425-1221)
Histopaque (Sigma-Aldrich, catalog number: 11191)
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537)
MidiMACSTM separator (Miltenyi Biotech, catalog number: 130-042-302)
MACS® multiStand (Miltenyi Biotech, catalog number: 130-042-303)
CD14 microbeads (Miltenyi Biotech, catalog number: 130-050-201)
Ficoll (Cytiva, catalog number: 1754402)
RPMI 1640 medium (Sigma-Aldrich, catalog number: R0883)
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F9665)
L-glutamine (Sigma-Aldrich, catalog number: G7513)
Penicillin-streptomycin (Sigma-Aldrich, catalog number: P0781)
Interferon gamma, IFN-γ (Bio Techne, catalog number: 285-IF-100)
Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Miltenyi Biotech, catalog number: 130-093-868)
Interleukin 4 (IL-4) (Miltenyi Biotech, catalog number: 130-093-919)
Macrophage colony-stimulating factor (M-CSF) (Miltenyi Biotech, catalog number: 130-096-493)
70% alcohol (any vendor)
Optimal cutting temperature compound (OCT) (Agar Scientific, catalog number: AGR1180)
Copolymer synthesis
Poly(cyclohexyl methacrylate-co-dimethylamino-ethyl methacrylate) (CHMA-DMAEMA), pro-inflammatory macrophage (M1-like)
Poly(cyclohexyl methacrylate-co-isodecyl methacrylate) (CHMA-iDMA), anti-inflammatory macrophage (M2-like)
Liquid nitrogen
Ammonium formate (Sigma-Aldrich, catalog number: 70221)
150 mM Ammonium formate (see Recipes)
MACS buffer (see Recipes)
Animal study
Clinical-grade silicon catheter 13 mm diameter (Teleflex Medical, catalog number: RUSCH170003)
Polymer synthesis
Dichloromethane (any vendor)
MED1-161 SILICONE PRIMER (Nusil)
Female BALB/c strain 19–22 g mice (Charles River)
Equipment
Stripette (Greiner Bio-One, catalog number: 760160)
Stripette gun (any vendor)
Micropipette (any vendor)
Forceps (any vendor)
Cell culture hood, class II (any vendor)
Scissors (any vendor)
Water bath (any vendor)
Centrifuge (any vender)
Automated cell counter (any vendor)
Cell culture incubator (SANYO, model: MC0-18A1C)
UV Clave (any vendor)
Refrigerator (4 °C) (any vendor)
Freezer (-80 °C) (SANYO, model: MDF-C8V1)
Cryostat CM3050 (Leica Microsystems)
Freeze dryer (any vendor)
Dip coating unit (Holmarc, model: HO-TH-01)
Vacuum oven (Thermo Fisher Scientific)
3D OrbiSIMS (IONTOF GmbH, Germany and Thermo Fisher Scientific, Germany)
Software
SurfaceLab software version 7.1 (ION-TOF, Germany), which utilizes the Thermo Fisher provided application programming interface
LIPID MAPS software (https://www.lipidmaps.org)
Procedure
Considerations before starting
Peripheral blood monocytic cells (PBMCs) isolation
PBMCs are isolated from healthy blood donor buffy coats. Here, we isolated the monocytes that were used to generate macrophages.
Macrophage polarization
Macrophages play a critical role in the modulation of the immune response. Macrophages have a spectrum of functional phenotypes that is determined by the nature of stimuli they encounter. At either ends of this spectrum there are pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. In vitro, monocytes can be polarized towards M1 and M2 phenotypes using different cytokine cocktails. Here, we investigated and compared the relationship between M1 and M2 macrophage activation methods in human primary monocyte–derived macrophages, using cytokines.
Implant sample preparation for in vivo studies
In a previous work, Rostam et al. (2020) identified polymers with immune-modulatory properties, in particular their ability to induce M1- and M2-like phenotypes in macrophages in vitro and in vivo. In this study, we coated silicon catheter segments (dimensions 2.7 mm, 5 cm length) with immune-instructive copolymers discovered in the Rostam et al. (2020) article to assess their ability to modulate macrophage phenotypes in a mouse model of foreign body response.
Ex vivo tissue sample preparation for 3D OrbiSIMS
We prepared fresh tissue sections for 3D OrbiSIMS analysis to characterize metabolomic profiling of phospholipids in tissue section samples. After 28 days of implantation, the catheter and surrounding skin segment were embedded in OCT and sectioned by cryostat.
Preparation of PBMC isolation
Arrange equipment (Figure 1A).
Place a bottle of PBS in a 37 °C water bath.
Spray the blood bag (50 mL) with 70% ethanol and place it inside the hood.
Cut the blood bag and place it into a T75 flask (Figure 1B).
Top up prewarmed PBS into a T75 flask to a final volume of 150 mL.
Gently shake the flask to mix the blood and PBS.
Add 15 mL of Ficoll into each of the four Falcon tubes (50 mL Falcon tube).
Transfer 25 mL of the blood–PBS mixture into the Falcon tubes containing Ficoll using a stripette. Angle the Falcon tube to ~45–60° and ensure the blood does not mix with the Ficoll (distinct layers are required).
Centrifuge at 1 (acceleration/deceleration) and 800× g for 30 min at room temperature (Figure 1C).
Use a stripette to remove the plasma and platelet layer (top layer).
Use a Pasteur pipette to collect the white PBMC layer from the four Falcon tubes into two new Falcon tubes.
Wash the aspirated PBMCs by adding PBS to a final volume of 50 mL.
Centrifuge at 3 (acceleration/deceleration) and 300× g for 10 min at room temperature.
After centrifugation, use a stripette to aspirate off the supernatant, leaving behind two loose pellets.
Resuspend one of the pellets in a small volume of PBS and then combine with the second pellet.
Gently pipette up and down to reduce clumping.
Wash the aspirated PBMCs again by adding PBS up to a final volume of 50 mL.
Centrifuge at 3 (acceleration/deceleration) and 200× g for 10 min at room temperature.
Aspirate off the supernatant.
Resuspend the cell pellet in PBS.
Centrifuge at 3 (acceleration/deceleration) and 350× g for 5 min at room temperature; then, aspirate the supernatant.
Prepare the MACS buffer (see Recipes).
The cells need to be resuspended and incubated with CD14+ beads. The volume of beads required depends on the required number of cells. For example, use 250 μL of beads if less than 1 × 108 cells are required, and up to 500 μL of beads if more are required.
Resuspend the cell pellet in MACS buffer (375 μL, when 3 mL of MACS buffer is used).
Add the corresponding volume of CD14+ beads (375 μL, when 3 mL of MACS buffer is used).
Mix well and incubate the cells covered with foil at 4 °C for 20 min.
Add MACS buffer into the cell suspension to a final volume of 20 mL.
Centrifuge at 3 (acceleration/deceleration) and 350× g for 5 min at room temperature.
Aspirate off the supernatant and resuspend the resulting pellet in 4 mL of MACS buffer.
Prepare the MACS columns in the hood, including a black magnet holder, purple magnets, yellow filters, MS columns, and bijou tubes (Figure 1D).
Figure 1. Processing of peripheral blood mononuclear cells (PBMC) isolation. A. Materials and reagents for PBMC isolation. B. Transfer of buffy coat into T75 flask. C. Separation of whole blood components after centrifugation into discrete layers; plasma and platelets (top yellow layer), PBMCs (middle white layer), and erythrocytes (bottom red layer). D. MACS columns for magnetic cell isolation.
Wash the columns through with 3 mL of MACS buffer each, before adding the cell suspension.
Slowly pipette the cell suspension through the columns.
Once the cells have been added, complete three wash steps using 3 mL of MACS buffer each.
To remove the cells bound to the column, detach the column from the magnet.
Add 2 mL of MACS buffer and then use the syringe component to push the cells out into a Falcon tube.
Count the cells using trypan blue, by diluting a small volume of the cell suspension 1:20 before counting.
Note: Work in a sterile environment; all materials should be sterile. Do not pour off the supernatant; always use a stripette to remove the plasma layer (top layer), to not dislodge the PBMCs layer.
Macrophage polarization using polymers
Coat a round coverslip with 200 μL of poly-lysine to promote cell attachment.
Place the coated coverslips into 24-well plates.
Make up the complete RPMI 1640 medium (see Recipes) and seed 1 × 105 isolated monocytes. Place monocytes onto the coated coverslips.
Add the different cytokines to the monocytes to generate different macrophage phenotypes:
Naïve macrophages: add complete RPMI supplemented with 10 ng of M-CSF.
M1 macrophage: add complete RPMI supplemented with 50 ng of GM-CSF and 20 ng of IFN-γ.
M2 macrophage: add complete RPMI supplemented with 50 ng of M-CSF and 20 ng of IL-4.
Incubate the cells in a humidified atmosphere of 5% CO2 at 37 °C.
On day 3 of incubation, remove 500 μL of media from each well and replace with fresh medium containing the same concentration and mix of cytokines as on day 0.
On day 6 of incubation, remove all supernatants.
Wash the cells on the coverslips with 1 mL of 150 mM ammonium formate solution three times for 30 s each to remove salts that cause unwanted signal suppression in the SIMS instrument.
Plunge the cell samples into liquid nitrogen.
Freeze-dry the samples:
Switch on power at the rear to cool the chamber (-50 °C). This step should take approximately 1 h.
When the chamber is at the required temperature, place the samples onto the shelves and close the lid.
Turn the vacuum pump on (the vacuum should be below 26.6 Pa).
Freeze-dry samples over a period of 12 h to remove water.
Collect the samples and store them in a sealed container at -80 °C until 3D OrbiSIMS analysis.
Note: We use uncoated tissue culture 24-well plates to promote cell attachment on the glass coverslip. Personal protective equipment must be worn, including protective gloves specifically designed for cryogenic handling, a closed lab coat, a face shield, and shoes when working with liquid nitrogen. Cytokines are purchased in powder form, reconstituted in stock solution, and stored at -80 °C.
Implant sample preparation for in vivo studies
Clinical-grade silicon catheters with a 2.7 mm diameter are cut to a length of 5 mm (Figure 2A and 2B).
Pierce the catheter wall with microlance needles and clamp it in a dip coating unit (Figure 2C).
Dip the catheter into Nusil MED1-161 silicone primer, using the dip coater at a dipping and withdrawing rate of 1 mm/s and a dwell time of 30 s.
Keep the catheter dry at room temperature for 2 min.
Make up the copolymer synthesis solution (see Recipes).
Coat the MED1-161 coated catheter dip into the copolymer solution using the dip coater, with a dipping and withdrawing rate of 1 mm/s and dwell time of 30 s twice. (Figure 2C).
Dry the catheter at room temperature overnight, then dry it in a vacuum at 50 °C for seven days to remove the solvent. The coated catheter segments are shown in Figure 2D.
Note: It is necessary to dry the catheter well after coating with the copolymer. The coated catheter sample should be kept at room temperature overnight, because placing it directly in the vacuum may cause popping bubbles or otherwise damage the smooth catheter surface.
Figure 2. Schematic workflow of a catheter coated with copolymer for in-vivo study. A. Clinical-grade silicone catheter. B. The catheters were cut to a length of 5 mm. C. Coating of catheter segments in immune instructive chemistries using dip coating. D. Catheter coated sample. E. Coated catheter segments are implanted in mice for 28 days.
In vivo study
In vivo studies are approved by the University of Nottingham Animal Welfare and Ethical Review Board and carried out in accordance with Home Office authorization under project license number PP5768261. Female BALB/c 19–22 g mice were used in these studies. Mice are housed in individually ventilated cages (IVCs) under a 12 h light cycle, with ad libitum access to food and water. The weight and clinical condition of the mice are monitored daily. Silicone catheter segments coated with synthesized polymers are sterilized by UV light for a period of 20 min.
Implant the catheters subcutaneously into mice for 28 days. Catheters without a coating are used as a control (Figure 2E).
Administer Carprofen 5 mg/kg subcutaneously in a single dose 1 h prior to surgery.
Anesthetize mice using 2% isoflurane. Shave an area on their flank and clean it using hydrex skin cleaner.
Insert the catheter subcutaneously using a trocar needle and displace it 1 cm from the insertion site.
Use gluture skin adhesive to seal the insertion site and allow the animal to recover.
At the end of study (day 28), mice are humanely sacrificed by CO2 euthanasia.
Dissect the tissue and catheter implantation site, extending 2 cm from the catheter center into the surrounding tissue.
Ex vivo tissue sample preparation for 3D OrbiSIMS
Prepare the catheter segment and surrounding skin for 3D OrbiSIMS analysis.
Cut the 5.5 cm × 5.5 cm fresh tissue samples into three vertical cross sections and lay them into a mould containing OCT to ensure that cross-sectional layers are facing upwards (Figure 3A).
Push the sample all the way through the OCT to avoid excessive slicing during cross-sectioning before reaching the tissue. While pushing the tissue, be careful to avoid bubbles until none of the tissue remains exposed (Figure 3B).
Place the mould into liquid nitrogen slowly and carefully to minimize splashing and rapid freezing.
After freezing, the frozen tissue samples are placed in a cryostat chamber at -20 °C.
The samples are mounted on a metal chuck with OCT and kept frozen at a cutting temperature of -20 °C (Figure 3C).
The sample on the chuck is mounted on the microtome and sectioned using a CM1850 cryostat (Leica microsystems) (Figure 3D).
The tissue sections are cut at a thickness of 10 μm and mounted on a slide (Figure 3E).
Figure 3. Schematic workflow of fresh tissue section preparation for 3D OrbiSIMS. A. The catheter segments explanted after 28 days B. Tissue embedded in OCT. C. Spread a generous amount of OCT on the metal chuck and quickly lay tissue embedded samples on it. D. Cutting the tissue section using a CM1850 cryostat. E. Mounting tissue section on the glass slide.
Tissue section slides are washed with cold DI water three times for 30 s each and cold 70% ethanol for 30 s to remove fat and lipids, which can increase the sensitivity of detection for some unwanted molecules.
Frozen tissue sections are plunged into liquid nitrogen.
The frozen samples are freeze-dried as above in Section B, step 10.
Place the slide directly in a microscope slide box cooled on dry ice.
Store the microscope slide box at -80 °C until analysis.
Note: Please wear proper personal protective equipment (PPE) when handling liquid nitrogen to prevent contact. When sectioning tissue/catheter samples, mount the sections quickly using electrostatic effects from the slide.
3D OrbiSIMS analysis
Take the cell sample slides and tissue section samples on the slides from the -80 °C freezer and warm them to room temperature without opening.
Mount the sample on the backmount holders or topmount holder and lock with a screw. Ensure the screws are tightened and samples cannot move (Figure 4A and 4B).
Using the photo box, take a photo of the sample holder before loading it into the instrument to mark the sample location.
Load the sample holder onto the transfer arm via the bayonet socket and into the 3D OrbiSIMS instrument airlock for analysis (Figure 4C).
The Orbitrap analyzer should be calibrated using silver cluster ion patterns from a silver foil.
Conduct 3D OrbiSIMS analysis using a Hybrid SIMS instrument (IONTOF, GmbH), depth profile (single beam 20 keV Ar3000+, OrbitrapTM analyzer).
Collect secondary ions using the Q Exactive HF at the 240,000 m/z and 200 mass resolution setting, in both positive and negative ion mode.
The electron flood gun operates with an energy of 21 eV and an extraction bias of 20 V for charge compensation, with an injection time of 500 ms.
For all Orbitrap data, collect mass spectral information from a mass range of 75–1125 m/z.
For in vitro analysis, select the primary ion dose for analysis to maximize the secondary ion signal. For a total ion dose per measurement of 3.95 × 1011 ions/cm2 and a duty cycle of 4.4%, maintain the pressure in the main chamber at 1.6 × 10-6 mbar using argon gas flooding and a continuous GCIB current of 230 pA over an area of 150 μm × 150 μm, with crater size of 233.1 μm × 233.1 μm.
For an ex vivo study, the total ion dose per measurement is 5.21 × 1010 ions/cm2 and a duty cycle of 4.4%. Maintain the pressure in the main chamber at 8.9 × 10-7 mbar using argon gas flooding and a continuous GCIB current of 230 pA over an area of 100 μm × 100 μm, with crater size of 180.0 μm × 180.0 μm.
Move the stage to a sample area of interest (Figure 4D and 4E).
Figure 4. Transfer sample to the stage. A. Backmount holder to fix the sample (left), cell on coverslip samples mount on the backmount holder (right). B. Topmount holder to fix the sample (left), tissue section slides mount on the topmount (right) C. 3D OrbiSIMS instrument at the University of Nottingham. D and E. The optical images of single cells and tissue section acquire within the instrument before analysis by the gas cluster ion beam.
For cell sample analysis, choose three cells with positive and negative polarity per sample.
For tissue sample analysis, choose four areas surrounding the foreign body site with both positive and negative polarity per tissue section slide.
Data analysis
Analyze the data using SurfaceLab software version 7.1.
Open data for each sample on SurfaceLab software, select the data in the .itax file, and click open.
The peak lists are created by SurfaceLab.
Search peaks: Set the minimum peak area as 1,000 to distinguish a real from a noise peak.
Discount all peaks below this number as being noise and disregard them in further data analysis.
Export peak list as secondary mass ion and secondary intensity from the software.
Import the peak lists from the OrbiTrap analysis of the single macrophage cells and tissue section sample into the LIPIDMAPS database (perform the step to generate raw data in LIPIDMAPS, as shown in Figure 5) to identify the lipids species (Figure 6A–6C).
Figure 5. Schematic workflow of steps to generate raw data to identify lipid class
Figure 6. Identification of phospholipid in single macrophages and tissue section samples. A. Negative ion mass spectrum 3D OrbiSIMS of the lipid fragments from a single cell macrophage, where 47 lipids were identified. B. In the negative ion mass spectrum 3D OrbiSIMS of the lipid fragments from a tissue section sample, 144 lipids were identified C. Venn diagram comparing the number of lipid compounds in single macrophages cells and ex vivo tissue sections by 3D OrbiSIMS measurement, illustrating that 16 lipid compounds were common to both samples, while 31 and 128 lipids were unique to single macrophage cells and tissue section samples, respectively.
Validation of protocol
For cell culture: choose three cells per macrophage phenotype and three replicate runs with positive and negative polarity by 3D OrbiSIMS. A total of eighteen cells will be consumed.
For animal studies: assign three mice for each polymer group.
For tissue sample analysis, one tissue section and four replicate areas surrounding the foreign body site are analyzed with both positive and negative polarity by 3D OrbiSIMS. In total, sixteen areas will be consumed.
Conclusion
Overall, our protocol has illustrated that the direct 3D OrbiSIMS technique can provide detailed molecular characterization both in vitro and in vivo samples, with minimal sample preparation compared to optical microscopy of stained and labelled histological samples. This molecular histology approach will help understand the immune response to medical implants and has huge potential in other fields of therapeutics and immune dysregulation.
Recipes
Complete RPMI 1640 medium
Supplement with 10% heat inactivated FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin
150 mM Ammonium formate
Dissolve 18.9 mg of ammonium formate to a final volume of 20 mL in deionized water.
MACS buffer
50 mL of PBS, 200 μL of EDTA and 250 μL of FBS
Copolymer synthesis solution in dichloromethane (5% w/v)
CHMA-DMAEMA, pro-inflammatory macrophage (M1-like)
CHMA-iDMA, anti-inflammatory macrophage (M2-like)
Acknowledgments
We acknowledge the financing of this work by the Royal Thai Government Scholarship provided by the National Metal and Materials Technology Centre (MTEC), and the National Science and Technology Development Agency (NSTDA), Thailand. This work was also supported by the Engineering and Physical Sciences Research Council (EPSRC) [grant number: EP/P029868/1] with a Strategic Equipment grant.
Competing interests
There are no conflicts of interest or competing interests.
Ethics considerations
Buffy coats from healthy volunteers provided by National Blood Services, Sheffield United Kingdom, ethical committee approval (2009/D055, Research Ethics Committee, Faculty of Medicine and Health Sciences, University of Nottingham).
Animal studies approval by the University of Nottingham Animal Welfare and Ethical Review Board, and carried out in accordance with Home Office authorization under project license number PP5768261.
References
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Biological Sciences > Biological techniques > Mass spectrometry
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Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction
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The authors used this protocol in eLIFE May 2022
Abstract
Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein–protein and protein–lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor–ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.
Keywords: Styrene Maleic Acid Lipid Particles (SMALPs) Drosophila melanogaster Affinity Purification Nicotinic Acetylcholine Receptor (nAChR) Mass Spectrometry Ligand-Receptor Interaction Native Membrane Protein Extraction
Background
Membrane proteins represent more than 60% of all drug targets; yet their insoluble behaviour makes it very difficult to study native interactions with drug molecules (Yin and Flynn, 2016; Liu et al., 2020). The development of methods to effectively extract membrane proteins and ensure their continued functionality is thus of paramount importance. The lipid bilayer surrounding membrane proteins is essential for structural integrity, stability, and ligand binding (daCosta et al., 2013). Moreover, within this lipid environment, most membrane proteins exist as part of multi-protein complexes with other membrane-embedded proteins and peripherally associated proteins. In order to understand the correct binding interactions of membrane proteins with other molecules, it is thus necessary to retain their lipid environment in a manner that does not disrupt their interacting protein partners. The requirement to keep protein and lipid associated in a native and functionally relevant state is extremely challenging, as most membrane protein extraction methods rely on the use of ionic and zwitterionic detergents for complete solubilization. Furthermore, the application of typical non-ionic and non-denaturing detergents such as Triton and Tween result in significant disruption of lipid–protein interactions that affect the native conformation of proteins (Stetsenko and Guskov, 2017).
The development of methods for extracting membrane proteins from lipid bilayers using detergents and introducing them into artificial lipid nanodiscs has facilitated a much better characterization of receptor–ligand interactions (Denisov and Sligar, 2016). The use of detergents generally employed to solubilize membrane proteins, however, leads to destabilization, aggregation, and misfolding, and their use is therefore not compatible with this type of analysis (Loo et al., 1996).
The use of small organic compounds such as styrene maleic acid lipid particles (SMALPs) allows detergent-free extraction of membrane proteins in their local lipid environment, providing a promising technique for investigating receptor–ligand interactions under native conditions (Lee et al., 2016). As a copolymer, styrene maleic acid (SMA) has a high affinity for membranes and is readily incorporated into lipid bilayers. SMA polymerizes to form a girdle, which traps disc-shaped pieces of membrane containing the membrane proteins, their interacting partners, and the lipid environment in a native state (Xue et al., 2018). The individual discs vary in size and shape, with an average size of 10 nm. It has been shown that membrane proteins with 37 transmembrane helices can be recovered in SMALP discs (Lee et al., 2016). The Piezo, a mechanosensory ion channel protein that contains 37 predicted transmembrane helices in Drosophila melanogaster, for example, has been shown to be readily incorporated into SMALP discs (Korona et al., 2022). Maintenance of the lipid environment in these nanodiscs is particularly important, since loss of lipids surrounding membrane proteins can lead to changes in measured binding affinities (Martens et al., 2018; Gault et al., 2020). To date, SMALP nanodisc technology has been applied to many different biological systems (Lee et al. 2016; Teo et al., 2019; Korona et al., 2022).
Recently, we have established a protocol that employs SMALP extractions to create nanodiscs, containing still functionally active low abundance membrane receptor protein complexes. The protocol is compatible with subsequent affinity enrichment, enabling identification of their constituent parts by mass spectrometry while retaining activity, such that native receptor–ligand interactions can also be interrogated (Korona et al., 2022). Thus, the combination of detergent-free SMALPs extraction coupled with mass spectrometry analysis provides a potential route for characterizing native membrane receptor complexes (Sobotzki et al., 2018; Kalxdorf et al., 2021).
We applied this protocol to study components of nicotinic acetylcholine receptors (nAChRs). These neurotransmitter receptors belong to a large class of insecticide targets located in synaptic plasma membranes (Ihara et al., 2020). These pentameric cys-loop ligand-gated ion channels consist of either only α-subunits or α- and β-subunits, with ligand-binding sites located between two α-subunits or between α- and β-subunits. The structural model of D. melanogaster nAChR contains 10 highly conserved subunits that assemble in various combinations to form the active receptors (Lu et al., 2022). Using our approach, we were able to elucidate which subunits assemble to form functional receptor complexes (Korona et al, 2022). We also demonstrated the ability of the five nAChRs within these discs to interact with α-Bungarotoxin (α-BTX), a small peptide toxin found in snake venom, which is known to bind and modulate the activity of this receptor.
The following protocol provides a method to study the native interaction of D. melanogaster adult head nAChRs extracted into SMALPs. This method can be readily transferred to other biological systems for the effective native extraction of membrane protein complexes and to investigate the interactions of these proteins with other molecules under native conditions. Our protocol will therefore have a major impact on future drug interaction studies that will identify potential binding to other native membrane proteins.
Materials and reagents
w1118 (D. melanogaster) [FlyBase, FBal0018186, Bloomington drosophila stock center (BDSC): 3605]
nAChRα6FSVS (D. melanogaster) (Korona et al., 2022)
Styrene maleic acid copolymer (SMA) 3:1 [supplied by Prof. Tim Dafforn, University of Birmingham, UK, personal communication https://www.birmingham.ac.uk/staff/profiles/biosciences/dafforn-tim.aspx, and commercially available, for example, SMALP 300 (Cube Biotech, catalog number: 18200)]
PierceTM quantitative fluorometric peptide kit (Thermo Scientific, catalog number: 23290)
Trypsin/Lys-C Mix (Promega, catalog number: V5073)
C-18 material (Thermo Scientific, catalog number: 84,850)
Pierce C-18 spin tips (Thermo Fisher Scientific, catalog number: 84,850)
α-Bungarotoxin (α-BTX) (Abcam, catalog number: ab120542)
Antibody
Anti-ATPase alpha 1 (Abcam, catalog number: ab2872)
Anti-mouse ECL peroxidase labeled (Merck, catalog number: GENA931-1ML)
Anti-GFP (goat monoclonal) (Abcam plc, catalog number: Ab252881)
Anti-rat IgG (goat polyclonal) (Sigma-Aldrich, catalog number: A9037)
Isotonic lysis buffer (see Recipes)
SMALP solution (see Recipes)
Coupling buffer (see Recipes)
Tris-buffer or TBS (see Recipes)
TBS-T buffer (see Recipes)
Blocking solution (see Recipes)
Laemmli buffer (see Recipes)
Sample buffer (see Recipes)
Chemical reagents
Carbachol (Insight Biotechnology Ltd, catalog number: CAS 51-83-2)
Cyanogen bromide (CNBr)-activated Sepharose affinity beads (Sigma-Aldrich, catalog number: C9 142-5G)
cOmpleteTM protease inhibitor (Merck, catalog number: 11836170001)
Marvel dried skimmed milk (5% solution)
ECL chemiluminescent detection solution (GE Healthcare, catalog number: 45-000-999)
Tween 20 (Sigma-Aldrich, catalog number: P1379-25ml)
Ammonium bicarbonate (NH4HCO3) (Sigma-Aldrich, catalog number: 09830-500G)
Bromophenol blue (Sigma-Aldrich, catalog number: 114391-5G)
Coomassie brilliant blue G250 (Sigma-Aldrich, catalog number: 1154440025)
Ethanol (Sigma-Aldrich, catalog number: 1070172511)
Acetic acid (Sigma-Aldrich, catalog number: A6283-2.5l)
Sodium acetate (NaCH3CO2) (Sigma-Aldrich, catalog number: 32319-500G-R)
Sodium hydrogen carbonate (NaHCO3) (Sigma-Aldrich, catalog number: 1063290500)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-25G)
Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: 320331-500ML)
HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) (Sigma-Aldrich, catalog number: PHR1428-1)
Aluminum sulfate-(14-18)-hydrate (Sigma-Aldrich, catalog number: 368458-500G)
Methanol (Sigma-Aldrich, catalog number: 322415-2L)
Ortho-phosphoric acid (Sigma-Aldrich, catalog number: 345245-500ML)
EDTA (Ethylenediaminetetraacetic acid) (Sigma-Aldrich, catalog number: 03695-250G)
Tris(hydroxymethyl)aminomethane (Sigma-Aldrich, catalog number: 1070897600)
Sucrose (Sigma-Aldrich, catalog number: S0389-500G)
Glycine (Sigma-Aldrich, catalog number: G7126-100G)
Acetone (Sigma-Aldrich, catalog number: 179124-500ML)
Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34851-1L)
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9760-500MG)
Iodoacetamide (IAA) (Sigma-Aldrich, catalog number: I1149-25G)
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 8170341000)
Formic acid (FA) (Sigma-Aldrich, catalog number: 695076-100ML)
Methanol (Sigma-Aldrich, catalog number: 34860-2.5L-R)
HPLC water (Sigma-Aldrich, catalog number: 270733-2.5L)
Glycerol (Sigma-Aldrich, catalog number: G5516-100ML)
Equipment
Sieve 200 mm diameter 400 micron (Endecotts, catalog numbers: 1201124)
Sieve 200 mm diameter 800 micron (Endecotts, catalog numbers: 1201125)
2 mL Dounce homogenizer (DWK Life Sciences Limited, catalog number: 357422)
Nanodrop (DeNovix, catalog number: DS-11 FX+)
Beckman coulter optimaTM Max-XP Ultracentrifuge (Beckman Coulter, High Wycombe)
V-32 Vortex Mixer (GEM Scientific, catalog number: GERT-V-32)
Speed Vac (CentriVap Benchtop Centrifugal Vacuum Concentrator with acrylic lid, catalog number: 7810030)
Beckman coulter TLA 55 55 K RPM S/N 1601300 fixed angle rotor (Beckman Coulter, High Wycombe)
Microfuge tube polypropylene (Beckman Coulter, High Wycombe, catalog number: 357448)
Mini-Protean TGX precast gels (Bio-Rad Laboratories, catalog number: 456-1084)
Nitrocellulose membrane (Bio-Rad Laboratories, catalog number: 1704158)
Trans-Blot Turbo Transfer Pack (Bio-Rad Laboratories, Inc, catalog number: 1704158)
CL-XPosure films (Thermo Scientific, catalog number: 10465145)
X-ray developer (Protec GmbH, catalog number: 1170-1-8000)
Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA)
Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific, Waltham, MA, USA)
Reverse-phase nano Easy-spray column (Thermo Fisher Scientific, Waltham, MA, USA)
Software
Proteome Discoverer 2.3 (Thermo Fisher Scientific, RRID:SCR_014477)
Procedure
Workflow overview
The procedure starts with the separation of D. melanogaster heads for downstream enrichment of cellular membranes. Soluble proteins are separated from membrane proteins by means of centrifugation. Pelleted membrane proteins are solubilized in SMA 3:1 and incubated at room temperature to form the SMALPs. After the ultracentrifugation step, the supernatant contains a heterogeneous mixture of nanodiscs with a size between 5 and 15 nm that could be further examined using transmission electron microscopy. In order to target specific membrane proteins, an affinity purification is performed. In the example we give here, the α-BTX peptide is coupled to Sepharose beads and used to purify nAChRs. If necessary, the degree of enrichment of nAChRs is analyzed using western blot. In order to be able to identify the subunits of the receptor, the samples are digested using trypsin/lys-C mix and applied to LC–MS/MS. After analysis, a comparison against a non-enriched sample is performed to determine which protein subunits are specifically enriched.
Membrane protein enrichment and incorporation in SMALPs
The starting material can be adapted to the respective question. Material can range from human to bacterial cells, and from total tissues to whole organisms such as D. melanogaster (Gulati et al., 2014; Jamshad et al., 2015; Korona et al., 2022). The extraction protocol, however, must be adapted to the corresponding starting material. In this protocol, enriched heads from D. melanogaster [w1118 and FSVS-tagged Dα6 (3xFLAG-StrepII-mVenus-StrepII)] are used to establish the protocol. D. melanogaster heads are obtained and separated according to Depner et al. (2014).
In a 50 mL Falcon tube, rapidly freeze approximately 6 g (approximately 6,000 flies; male 0.7 mg and female 1.0–1.3 mg, age dependent) of adult D. melanogaster in liquid nitrogen and vortex twice for 3 min. Cool the tube for 30 s in liquid nitrogen between vortexing.
Place two differently sized sieves into one another and transfer the flies into a liquid nitrogen–precooled 800-micron sieve. Separate heads from bodies by sieving. Collect heads in a second sieve with 400 microns in size. At this point, the heads can be stored at -80 °C for months before further extraction.
For cell lysis, add 1 mL of isotonic lysis buffer to approximately 0.8 g of separated heads. Mix the solution three times by vortexing and lyse the heads with 60 strokes in a 2 mL Dounce homogenizer with a pestle (Figure 1A).
Perform membrane protein preparation by differential centrifugation–based fractionation, as described in Depner et al. (2014). This allows for a better separation of membrane proteins from soluble proteins.
Perform a pre-cellular clearance by a centrifugation step at 200× g for 5 min. This step allows the removal of undisrupted cells, which are not broken during the extraction process.
Centrifuge the supernatants by a series of differential centrifugation steps: 1,000× g for 5 min, 3,000× g for 10 min, 5,000× g for 10 min, and 9,000× g for 15 min. Perform all steps in a 4 °C refrigerated centrifuge. After each spin, transfer the supernatant into a new centrifugation tube and use the membrane pellets for western blot analysis. By employing this differential centrifugation–based fractionation strategy, plasma membranes are partially separated from other endomembranes, increasing sensitivity and specificity of the system. With higher and higher spins, the fractions that do not contain the plasma membrane are obtained.
Western blot analysis reveals which fraction is enriched for the plasma membrane and thus should be used in downstream processing (Figure 1B).
Use fractions enriched in plasma membranes for the SMALP extraction. Resuspend membrane fractions (24–177 mg of wet pellet weight) in approximately 20–300 μL of 5% SMALP solution.
For efficient incorporation and formation of SMALPs, incubate fractions containing plasma membrane with 5% SMALP solution for 2 h at room temperature on a rocking platform. Finally, centrifuge at 100,000× g for 60 min at 4 °C and use the supernatant, which contains the SMALPs, for downstream analysis. SMALPs can be incubated on ice without precipitation, but low or rapid freezing can cause the nanodiscs to degrade.
Immunoblotting
Western blot analysis is used to investigate the enrichment of the plasma membrane proteins in fractions resulting from differential centrifugation. An anti-ATPase alpha 1 antibody is used to perform a western blot, acting as a plasma membrane marker (Figure 1B), or it can be used to determine the degree of enrichment of nAChRs.
After the centrifugation steps, load fractions on a 4%–15% SDS-PAGE and transfer onto a 0.2 μm nitrocellulose membrane.
Use 5% skimmed milk powder dissolved in TBS-T for blocking and incubate membranes for 16 h at 4 °C with the anti-ATPase alpha 1 antibody (1:1,000 concentrated in blocking solution) followed by anti-mouse ECL peroxidase labelled for 1 h. Either treat α-BTX-affinity enriched or unenriched protein samples with 1% DTT or leave untreated, boil at 60 °C for 8 min, separate by SDS-PAGE, and then transfer onto a nitrocellulose membrane.
Detect FSVS-tagged Dα6 (3xFLAG-StrepII-mVenus-StrepII) with anti-GFP (Figure 1C). This strain serves as a positive control for the successful enrichment of nAChRs using α-BTX affinity beads. Use 5% skimmed milk powder dissolved in TBS-T for blocking and incubate membranes for 16 h at 4 °C with the α-GFP antibody (1:1,000 concentrated in blocking solution) followed by anti-rat IgG antibody for 1 h.
Treat immunoblots with an ECL chemiluminescent detection solution exposed for 10 s to CL-XPosure films and visualize using an x-ray developer.
Figure 1. Overview of the protocol to enrich native plasma membrane proteins complexes and use styrene maleic acid lipid particles (SMALPs) enrichment and mass spectrometric analysis. A. In this example, D. melanogaster heads are used as a starting material. Cell membranes are first enriched using differential centrifugation. Membrane pellets are used to perform the SMALPs extraction. The plasma membrane–enriched fraction, in this case fraction 4, is mixed with SMA. The resulting nanodiscs containing the target protein of interest, in this case nAChRs, are further enriched using affinity beads coupled to α-BTX. Proteins within the discs are identified by firstly digesting to peptides using trypsin followed by LC–MS/MS. B. Western blot analysis to determine the fraction enriched in the plasma membrane using ATPase alpha 1 as marker. C. Western blot analysis to confirm the presence of the target membrane protein, in this example the fluorescent protein–tagged Dα6 nAChR subunit in enriched nanodiscs.
Coupling procedure of α-BTX to affinity beads
For this protocol, α-BTX-coupled beads are used to enrich for subunits of nAChRs. For other studies, alternative affinity enrichment strategies can be employed, such as specific antibodies used to target their corresponding membrane proteins. If ligands other than α-BTX are used, then ligand buffer solutions and coupling reactions must be optimized. Moreover, optimization of different linkers between affinity beads and ligands may be necessary if alternative ligands are employed. If antibodies or other ligands than α-BTX are used, the coupling and crosslinking reaction must be optimized. When using beads other than those described in this protocol, an optimization step should be performed.
Perform coupling of α-BTX to CNBr-activated Sepharose beads as described (Wang et al., 2003; Mulcahy et al., 2018).
Hydrate CNBr-activated Sepharose beads (0.25 g) in 1.25 mL of 1 mM HCl for 1 h at 4 °C on a rotator.
Centrifuge beads at 1,500× g for 5 min, remove the supernatant, and then wash the beads twice with 1 mL of coupling buffer.
Centrifuge beads at 1,500× g for 5 min and remove the supernatant. Resuspend α-BTX (1 mg) in 1 mL of coupling buffer and incubate together with the affinity beads at 4 °C for 16 h on a rotator.
Centrifuge beads at 1,500× g for 5 min. Remove the supernatant and keep it for measuring the coupling efficiency. At this time, the supernatant should contain no α-BTX. Determine coupling efficiency using a Pierce quantitative fluorometric peptide kit, according to the manufacturer’s instructions.
Block the beads with 1 mL of 0.2 M glycine in 80% coupling buffer at 4 °C for 16 h on a rotator.
Centrifuge the beads at 1,500× g for 5 min and wash with 1 mL of 0.1 M NaHCO3, 0.5 M NaCl, pH 8.0. Repeat this step with 1 mL of 0.1 M NaCH3CO2, 0.5 M NaCl, pH 4.0.
Wash the beads again in 1 mL of 0.1 M NaHCO3, 0.5 M NaCl, pH 8.0. After a final wash step with 1 mL coupling buffer, incubate the beads twice for 30 min in 1 mL of Tris-buffer (50 mM Tris, 150 mM NaCl, pH 8.0).
Centrifuge the beads at 1,500× g for 5 min and remove the supernatant.
Enrichment of nAChRs by α-BTX pull-down
If interacting molecules, such as toxin peptides other than α-BTX or antibodies, are used to enrich the desired membrane protein, the protocol should be adapted. The following protocol is developed with α-BTX, as this peptide has a high affinity for nAChRs (Dellisanti et al., 2007; Rahman et al., 2020).
Incubate SMALP discs (800–1000 μL of a 20–35 mg/mL protein extract, measured with a NanoDrop) with 200 μL of α-BTX-conjugated affinity beads for 16 h at 4 °C on a rotator.
Centrifuge the beads at 1,500× g for 5 min and wash two or three times, each for 10 min, with 1 mL of ice-cold TBS on a rotator at 4 °C.
Centrifuge the beads at 1,500× g for 5 min and selectively elute nAChRs twice with 100 μL of 1 M carbachol. Perform these steps on a rotator at RT.
Centrifuge the beads at 1,500× g for 5 min. The supernatant should be transferred into a new clean tube.
Add ice-cold 100% acetone to the samples at a volume of four times the sample volume and mix by vortexing. Leave proteins to precipitate for 16 h at -20 °C. An overnight precipitation using acetone allows the removal of contaminants including salts that may interfere with subsequent SDS-PAGE and analysis using western blotting. If further structural analysis of proteins is to be carried out using electron microscopy, this step may be skipped.
Centrifuge samples at 13,000× g for 15 min.
Remove the supernatant and dissolve precipitated proteins in Laemmli buffer. Heat the resulting sample at 60 °C for 8 min, load on Mini-Protean TGX precast gels, and resolve according to the manufacturer’s instructions. Perform protein staining according to Neuhoff et al. (1988). Fix gels in 40% (v/v) ethanol and 10% (v/v) acetic acid for 60 min, wash two times in water for 10 min, and stain for 16 h in Coomassie solution [0.1% (w/v) Coomassie brilliant blue G250, 5% (w/v) aluminum sulfate-(14-18)-hydrate, 10% (v/v) methanol, 2% (v/v) ortho-phosphoric acid]. Applying samples to an SDS-PAGE can be considered as an additional cleaning step.
Sample preparation for liquid chromatography–mass spectrometry (LC-MS)
Gel pieces are excised from the Coomassie stained gel lanes; proteolytic digestion, performed using a commercial available Trypsin/Lys-C mix, is performed as described (Shevchenko et al., 2006; Saveliev et al., 2013).
Immerse the gel pieces in 50 mM NH4HCO3/50% ACN and shake with a V-32 Vortex Mixer at maximum speed for 10 min. Remove the supernatant and repeat these steps with 100% ACN; finally, dry in a speed vac for 20 min.
Reduce samples with 10 mM DTT in 50 mM NH4HCO3 at 56 °C for 1 h. Remove DTT completely to avoid any inhibition effects on IAA.
Carry out alkylation with 50 mM IAA in 50 mM NH4HCO3 at room temperature without light for 45 min. Remove IAA.
Add 50 mM NH4HCO3 (fully cover the gel pieces); vortex for 5 min, centrifuge, and discard the solution.
Add 100% ACN to the gel pieces so they are completely covered with solution, shake for 10 min, and discard the solution.
Repeat these two steps (steps 4–5) and dry samples in a speed vac for 20 min. Add Trypsin/Lys-C buffer to the sample according to manufacturer’s instructions and incubate for 45 min on ice.
Next, add 30 μL of 25 mM NH4HCO3 and incubate samples at 37 °C for 16 h. Cover the gel pieces with 20 mM NH4HCO3 and shake with a V-32 Vortex Mixer at maximum speed for 10 min. Collect the supernatant containing peptides.
Next, cover the gel pieces with 50% ACN/5% FA and shake for 20 min. Again, collect the supernatant containing the peptides. Repeat this step of 50% ACN/5% FA addition and shaking for 20 min and collect the supernatant containing the peptides.
Combine all the supernatants together and dry in a speed vac until completely dry. Store samples at -20 °C.
Peptide cleanup
Peptides are desalted using C-18 stage tips according to Rappsilber et al. (2007).
Equilibrate C-18 material (three C-18 plugs are pasted in a 200 μL pipette tip) with 100 μL of methanol/0.1% FA.
Place the 200 μL pipette tip with an adaptor into a 2 mL centrifuge tube and centrifuge for 2 min at maximum speed. Remove the flowthrough.
Next, pipette 100 μL of 70% ACN and 0.1% FA to the 200 μL pipette tip and centrifuge for 2 min at maximum speed. Remove the flowthrough.
Finally, pipette 100 μL of 0.1% FA into the tip and centrifuge. Remove the flowthrough at this stage and repeat this step.
Place the 200 μL pipette tip into a new clean 2 mL tube. Resuspend the peptide pellets in 20 μL of fresh sample buffer to load the peptides onto the C-18 material.
Fully resuspend peptide samples by 15 min of vortexing. Then, carefully pipette peptides onto the C-18 material and, to ensure that the solution is in contact with the C-18 material, briefly centrifuge the 200 μL pipette tip [2–5 s at approximately 1,000 rpm (low speed)].
Incubate the C-18 stage tips for 5 min at room temperature and centrifuge at maximum 2,000× g for 5 min. Reload the flowthrough solution to make sure that as many peptides as possible bind to the C-18 material. For this, pipette again the peptide flowthrough solution onto the C-18 material and repeat the same centrifugation step.
Wash C-18 stage tips twice with 100 μL of 0.1% FA and centrifuge for 2 min at maximum speed.
Place the tips into a 1.5 mL low binding tube and elute peptides with 70% ACN 0.1% FA.
Centrifuge the C-18 stage tips at 2,000× g for 5 min to elute the peptides from the C-18 material.
Finally, dry peptides in a speed vac and store at -20 ° C before resuspending in 0.1% FA for further LC–MS/MS analysis.
LC–MS/MS
Peptide samples are dissolved in 20 μL of 0.1% (v/v) FA. Approximately 1 μg of peptide solution is used for each LC–MS/MS analysis. All LC–MS/MS experiments are performed using a Dionex Ultimate 3000 RSLC nanoUPLC system and a Q Exactive Orbitrap mass spectrometer.
Perform separation of peptides by reverse-phase chromatography at a flow rate of 300 nL/min using a reverse-phase nano Easy-spray column (PepMap C18, 2 μm particle size, 100 Å pore size, 75 μm i.d. × 50 cm length). Load peptides onto a pre-column (PepMap 100 C18, 5 μm particle size, 100 Å pore size, 300 μm i.d. × 5 mm length) via the Ultimate 3000 nanoUPLC autosampler with 0.1% FA for 3 min at a flow rate of 15 μL/min.
After loading, switch the column valve to allow elution of peptides from the pre-column onto the analytical column. Solvent A is 0.1% FA in water and solvent B is 80% ACN, 20% water, and 0.1% FA. The linear gradient employed is 2%–40% B in 90 min (the total run time including column washing and re-equilibration is 120 min). In between runs, wash the pre-column and analytical column at least four times to avoid carryover.
Ionize the LC eluant by means of an Easy-spray source. An electrospray voltage of 2.1 kV is applied in order to ionize the eluant. All m/z values of eluting ions are measured in an Orbitrap mass spectrometer, set at a resolution of 35,000, and scanned between m/z 380 and 1,500.
Data-dependent scans (Top 20) are employed to automatically isolate and generate fragment ions by higher energy collisional dissociation [HCD; normalized collision energy (NCE): 25%] in the HCD collision cell, and measurement of the resulting fragment ions are performed in the Orbitrap analyzer, set at a resolution of 17,500. Singly charged ions and ions with unassigned charge states are excluded from being selected for MS/MS and a dynamic exclusion of 20 s is employed.
Data analysis
Protein identification is performed using Sequest HT or Mascot search engine software operating in Proteome Discoverer 2.3 or above (Eng et al., 1994; Koenig et al., 2008), with the following parameters:
Trypsin is set as the enzyme of choice.
Precursor ion mass tolerance 20 ppm.
Fragment ion mass tolerance 0.1 Da.
Maximum of two missed cleavage sites.
A minimum peptide length of six amino acids.
Fixed cysteine static modification by carbamidomethylation.
Variable modification by methionine oxidation & deamidation of asparagine and glutamine.
Data analysis is performed using open-source Bioconductor packages using R and RStudio (Ihaka and Gentleman, 1996; Rstudio Team, 2020; Gatto et al., 2021). Alternatively, the MaxQuant can be used for the identification of proteins using the Andromeda search engine (Tyanova et al., 2016a). Data can then be assessed and visualized in Perseus (Tyanova et al., 2016b).
Expected result
This protocol describes an approach to study the interaction of native membrane proteins with different ligands, like drug molecules. Membrane proteins are known to be difficult to extract in their native conformation, and therefore the development of a method that allows the successful enrichment of native membrane proteins for downstream analysis is of great importance. One of these downstream analyses could be, for example, a protein’s interaction with different ligands and the better characterization of where these molecules bind to the membrane protein. Our protocol describes the enrichment by affinity beads of native nAChRs in SMALP preparations, and as expected, resulting subunits of these receptors can be identified by mass spectrometry. This helps to better characterize receptor–ligand interactions, and our protocol can be applied to various research questions, in a variety of different organisms.
Recipes
Isotonic lysis buffer
0.25 M sucrose, 50 mM Tris-HCl pH 7.4, 10 mM HEPES pH 7.4, 2 mM EDTA, protease inhibitor
SMALP solution
5% styrene maleic acid copolymer, 3:1 average ratio of styrene to maleic acid repeat units, 5 mM Tris-Base, 0.15 mM NaCl, pH 8.0
Coupling buffer
0.25 M NaHCO3, 0.5 M NaCl, pH 8.3
Tris-buffer or TBS
50 mM Tris, 150 mM NaCl, pH 8.0
Tris-buffer or TBS-T
50 mM Tris, 150 mM NaCl, pH 8.0 and add to 500 mL, 1,000 μL Tween 20
Blocking solution
Marvel dried skimmed milk (5% solution) in TBS-T
Laemmli buffer
1 M Tris pH 6.8, 10% SDS, 5% glycerol, 2% bromophenol blue
Sample buffer
98% HPLC water, 2% acetonitrile, 0.1% formic acid
Acknowledgments
We thank Prof. Tim Dafforn for kindly providing us SMA copolymer, David-Paul Minde, Rayner ML Queiroz and Renata Feret for supportive discussions. We are very grateful to Milner Therapeutics Institute excellent infrastructure support. Funding was provided by BBSRC (UKRI-BBSRC BB/P021107/1).
This protocol was derived from the original work of Korona et al. (2022).
Competing interests
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
References
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