FixNCut: A Practical Guide to Sample Preservation by Reversible Fixation for Single Cell Assays

Materials and reagents

Biological materials

1. Tissue samples and cell suspensions can both be used. For cells, the protocol is set up for up to 2 × 10⁶ cells. For more cells, scale up the fixation accordingly. Tissues larger than 3 mm in diameter or edge length need to be partitioned into smaller pieces to facilitate fixative penetration

   
   

1. DSP (Dithio-bis-succinimidyl propionate), also known as Lomant's reagent (Thermo Fisher, catalog number: 22586)

2. DMSO, anhydrous (Thermo Fisher, Molecular Probes, catalog number: D12345)

3. Liberase^TM research grade, 10 mg (Roche, catalog number: 5401119001)

4. Protector RNase inhibitor (40 U/μL) (Roche, catalog number: RNAINH-RO)

5. UltraPure^TM 1 M Tris-HCI buffer, pH 7.5 (Thermo Fisher, Invitrogen, catalog number: 15567027)

6. 10× PBS buffer, pH 7.4 (Invitrogen, catalog number: AM9624 or AM9625)

7. Ambion nuclease-free water (Invitrogen, catalog number: AM9932)

8. Bovine serum albumin (BSA) solution, sterile filtered and cell-culture tested (Sigma Aldrich, catalog number: A1595)

9. Tris base, UltraPure Tris buffer (powder format) (Thermo Fisher, Invitrogen, catalog number: 15504020)

   
   

1. Fixation concentrate stock solution (1 mL, for 100 standard assays) (see Recipes)

2. PBS with 1% BSA (see Recipes)

3. 1 M Tris pH 7.5 (optional, if not using ready-made buffer) (see Recipes)

   
   

1. Fixation concentrate stock solution (1 mL, for 100 standard assays)

   Reagent	Final concentration	Amount
   DSP (powder)	n/a	50 mg
   DMSO	n/a	1 mL
   Total	50 mg/mL (w/v)	1 mL

   
   

2. PBS with 1% BSA

   Reagent	Final concentration	Amount
   10× PBS	n/a	50 mg
   BSA 10%	n/a	1 mL
   Total	n/a	1 mL

   
   

3. 1 M Tris pH 7.5 (optional, if not using ready-made buffer)

   Start with 30 mL of water and adjust pH to 7.5 by adding 5 M HCl to a final volume of 50 mL.

   Reagent	Final concentration	Amount
   Tris base	n/a	6.05 g
   Nuclease-free water	n/a	50 mL
   Total	1 M	50 mL

   
   

Laboratory supplies

1. DNA LoBind tubes 1.5 mL (Eppendorf, catalog number: 022431021)

2. DNA LoBind tubes 2.0 mL (Eppendorf, catalog number: 022431048)

3. Flowmi^® cell strainers, porosity 40 μm, for 1000 μL pipette tips (Sigma, Scienceware, catalog number: BAH136800040)

4. pluriStrainer mini 70 μm (pluriSelect, catalog number: 43-10070-40)

5. Falcon conical centrifuge tubes (Corning, catalog number: 352070 for 50 mL, 352095 for 15 mL)

6. Sterile serological pipettes with pipettor

7. Rainin Pipet-Lite LTS pipette tips (Rainin, catalog number: 30389240, 30389213, 30389226)

Equipment

1. Centrifuge 5810/5810R (Eppendorf, catalog number: EP022628188) with rotor S-4-104

2. Rotor F-35-6-30 (Eppendorf, catalog number: EP5427716009)

3. S-4-104 rotor adapters for 50 × 1.5/2 mL tubes (Eppendorf, catalog number: 58-257-40009)

4. Standard heavy-duty vortex mixer (VWR or Fisherbrand, catalog number: 97043-562)

5. ThermoMixer C (Eppendorf, catalog number: 05-412-503) or thermoblock with minimal temperature fluctuation

6. Ice bucket with cool blocks or Cool Rack CFT30 (Corning, catalog number: CLS432052)

7. Automated cell counter, e.g., Luna-FX7 (Logos Biosystems) or hemocytometer

8. CoolCell freezing container for 12 × 1 mL or 2 mL cryogenic vials (Corning, catalog number: 432000) or Mr. Frosty freezing container (Thermo Scientific, catalog number: 5100-0001)

9. Tissue-Tek cold plate (VWR Scientific, catalog number: 25608-942)

Procedure

1. 50× fixation concentrate stock preparation (50 mg/mL)

   1. DSP equilibration

      Thoroughly equilibrate the DSP package to room temperature for 30 min prior to first opening. Critical: Only open the package after equilibration.

      Note: The NHS-ester of DSP is susceptible to hydrolysis upon contact with the humid atmosphere and is therefore moisture sensitive. Thorough equilibration is crucial in preventing condensation and premature inactivation of DSP.

   2. 50× working solution stock preparation

      Dissolve 50 mg of DSP in 1 mL of high-quality anhydrous DMSO for best performance. Final concentration: 50 mg/mL.

   3. Working stock storage

      1. For short-term storage, dispense 10 μL from the stock into 1.5 mL or 2 mL Eppendorf safe-lock microtubes and store at -80 °C in a sealed container or bag with desiccants.

      2. Alternatively, 10 μL aliquots can be stored in cryogenic vials with compression O-rings to prevent moisture absorption. Avoid freeze-thaw cycles by limiting the size of aliquots (up to 100 μL). Any opened and unused stock must be discarded.

         
         

2. Working solution preparation (1 mg/mL)

   Note: Time-sensitive step.Prepare fresh working solution immediately before the fixation process.

   1. Using a P200 pipette, slowly and drop-by-drop add 490 μL of PBS to an Eppendorf tube containing 10 μL of 50× stock while vortexing (Video 1).

      Caution: Choose “touch mode” and maximal speed (3,500 rpm or no less than 2,500 rpm) on the vortex instead of the “on mode”. Using a P1000 pipette is acceptable too, but adding the PBS too fast will cause DSP to precipitate, which perturbs the effectiveness of fixation. It is not concerning to see tiny precipitates on the wall of the tube initially; however, in case of substantial precipitates, repeat by slowing down the PBS addition or start with a new DSP aliquot.

      
      
      

      Video 1. Working solution preparation

      
      

   2. Filter the working solution once using a 40 μm Flowmi strainer and transfer it to a new 1.5 or 2 mL tube to remove larger precipitates.

   3. Critical: Working solution standing for more than 10 min should not be used and must be discarded. If sample preparation takes longer than 10 min, prepare the working solution during the process to avoid leaving it standing.

      
      

3. Fixation

   Note: For this section, follow instructions based on your sample type.

   1. Fixation of tissue samples

      1. For larger tissue samples or organoids (3 mm or larger in diameter/edge length), finely cut or mince the tissue with a sterile razor blade on a cold plate to facilitate working solution penetration. For effective fixation, tissue samples should be no thicker than 3 mm, with 1–2 mm being preferable for uniformity. This is because fixatives penetrate through passive diffusion, and the higher molecular weight of DSP (14.5-fold paraformaldehyde and 4-fold glutaraldehyde) significantly reduces its penetration speed and depth in tissue.

         Note: Fine mincing without separating the tissue minimizes sample loss during washing and transferring into a new tube.

      2. Add the minced tissue to a new Eppendorf tube containing 500 μL of working solution and incubate at ambient temperature for 30–45 min.

      3. Gently invert the tube every 15 min. For certain tissue types, if the tube becomes overly bloody or cloudy, consider replenishing once with fresh working solution.

   2. Fixation of cells

      1. Wash the cells in cold, sterile RNase-free PBS.

         1. Centrifuge the cells using an appropriate speed depending on type and fragility: usually between 150× g for 3 min (large cells) and 600× g for 10 min (small cells). Keep the cell pellet and remove the supernatant. Resuspend in cold, sterile RNase-free PBS.

         2. Repeat this step for a minimum of two washes.

            Note: Remnant media containing FBS (see General notes, Tip 4), storage additives, or sheath fluid components may interfere with the crosslinking.

      2. Pellet the cells, resuspend in 500 μL of working solution, and incubate for 30 min at ambient temperature.

      3. Gently invert the tube once at the 15-min mark.

         
         

4. Quenching of reactive DSP

   Note: For this section, follow instructions based on your sample type.

   1. For cells

      1. Add 10 μL of 1 M Tris-HCl pH 7.5. Then, mix thoroughly on a vortex for 2–3 s followed by a 15 min incubation at room temperature.

         Note: As an amine-reactive crosslinker, excessive reactive DSP must be quenched with Tris-HCl buffer before proceeding. The quantity is stoichiometrically optimized and must be adjusted accordingly if more fixative was used for larger tissues or a higher number of cells.

      2. Centrifuge at 500× g for 5 min at ambient temperature. Remove supernatant.

      3. Resuspend the cell pellet in 1,000 μL of PBS. Mix by vortexing for 2–3 s.

      4. Centrifuge at 500× g for 5 min at ambient temperature. Remove the supernatant.

      5. Repeat steps D1c–D1d for a total of two washes.

      6. Resuspend the cells in 0.5–1 mL of cold PBS with 1% BSA.

         Note: For maximal RNA integrity, add 0.2–1 unit/μL RNase inhibitor to the final resuspension buffer.

      7. Filter the final resuspension through an appropriately sized filter for the cell type. Commonly used mesh sizes for single-cell suspensions are between 40 and 70 μm.

      8. Count cells, bring concentration to 1,000–1,500 cells per microliter, and proceed to encapsulation.

   2. For tissues

      1. Add 10 µL of 1 M Tris-HCl pH 7.5. Then, mix thoroughly on a vortex for 2–3 s followed by a 15 min incubation at room temperature.

      2. Centrifuge at 500× g for 20 s or for 5–10 s in a mini spinner and remove supernatant.

      3. Add 1 mL of 200 μg/mL Liberase in PBS.

      4. Incubate the tissue with digestion buffer at 37 °C for 30 min with agitation at 800 rpm on a ThermoMixer C with heated lid.

         Note: The length of digestion may need to be optimized according to tissue type.

      5. Pipette to mix the sample 5–10 times every 15 min to facilitate digestion.

      6. After digestion is completed, filter the sample through a 70 μm filter and into a 15 mL falcon tube to remove coarse debris.

      7. Add 10 mL of ice-cold PBS and mix well.

      8. Centrifuge the sample at 500× g for 5 min in a pre-cooled centrifuge with swinging bucket rotor.

      9. Discard the supernatant and resuspend the pellet again in 10 mL of ice-cold PBS with 1% BSA. Mix well.

      10. Repeat steps D2g–D2h for three washes.

      11. Filter cells with an appropriate filter depending on cell size and resuspend the cells in 0.5–1 mL cold PBS with 1% BSA. Count cells, bring the concentration to 1,000–1,500 cells per microliter, and proceed to encapsulation.

          Note: For maximal RNA integrity, add 0.2–1 unit/µL RNase inhibitor.

          
          

5. Cryopreservation of samples (Optional)

   1. Resuspend the cell pellet in 500 μL of fresh PBS or media.

   2. Count and record the cell number.

   3. Add an appropriate volume of chilled cryopreservation medium to obtain a cell concentration of 1–2 × 10⁶ cells per milliliter.

   4. Dispense cell suspension aliquots of 1–2 mL into pre-cooled cryovials and place the cryovials inside a pre-cooled cell freezing container, e.g., CoolCell FTS30, to ensure gradual freezing.

   5. Place the cell freezing container in a -80 °C freezer for ≥4 h. After 4 h, transfer the cryovials to liquid nitrogen for long-term storage.

   6. Thaw in a 37 °C water bath and wash cells twice with PBS + 0.5%–1% BSA before proceeding to cell encapsulation.

Validation of protocol

The current protocol has been routinely performed as a standard protocol on human, mouse, and rat tissues and primary cells at the Spatial Technologies Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, being considered robust and reproducible. The whole procedure was repeated once after the completion of this manuscript by a person without previous knowledge using the current version to ensure details are correct, comprehensible, and executable.

Additional verification has been conducted in various studies, as documented, and systematically tested and optimized in Jiménez-Gracia et al. [23] (Figures 1–7) and in Aney et al. [24] (Figure 1).

Authors have validated the following sample types with successful single-cell RNA sequencing: human PBMC, mouse pancreas, mouse and rat liver, mouse lung, mouse colon, human colon biopsies, and human prostate.

General notes and troubleshooting

General notes

Tip 1: Only use freshly prepared DSP

Make single-use aliquots (20–50 μL) for 2–5 fixations. Do not re-freeze leftovers. Keeping the stock fixative away from water is key because it neutralizes the NHS-esters quickly. Currently, this can be a limitation in clinical settings, since clinical staff requires bandwidth, proper instrument setup, and training for the fixation process.

Tip 2: Avoid using Tris, glycine, or any other amine-active buffer to prepare the fixative

Avoid buffer components with primary amines such as Tris and glycine buffers, as they compete with proteins in the sample. DSP reacts with non-protonated aliphatic amine groups, including the N (amine) terminus of polypeptides and the ϵ-amino group of lysine (K) side chain.

Tip 3: Never bypass the stock preparation step by directly dissolving DSP solid powder in PBS

DSP is hydrophobic and needs to be dissolved in DMSO before being added to the aqueous reaction mixture. Besides the 50 mg (22586) size, DSP is also available in 10 × 1 mg (A35393) or 1 g (22585) packaging. Although a smaller size unit is more expensive per sample, consider using a smaller size matching the experimental scale to avoid exposure to moisture absorption. Lot-to-lot differences in DSP may exist; testing one aliquot on the optimization sample from the batch can be a QC option.

Tip 4: DSP crystal formation is normal but needs to be controlled; not all crystals are from DSP

DSP does not possess a charged group; it is lipophilic and membrane-permeable, making it suitable for intracellular and intramembrane crosslinking. However, it is water-insoluble and can form crystal precipitates when preparing the working solution. These crystals are removed by filtering in step B3. However, usage of BSA and even a high percentage of FBS are common to promote cell survival and boost viability. Still, calcium oxalate crystals can often be found in commercial fetal bovine serum (FBS) [25], being mistakenly perceived as DSP crystals. Using BSA is not concerning but a high percentage of BSA will deplete reactive DSP and lead to cloudy samples.

Tip 5: Calculate viability before fixation and re-count before storage

Viability is no longer a good measure for single-cell sample quality because DSP permeabilizes the cell membrane. Therefore, it is advisable to measure viability immediately before the fixation. Some cells will inevitably be lost during wash steps, so recounting cells before freezing and storage is necessary. Because fixed cells are not alive, no additive beyond DMSO or glycerol is required, such as FBS, ascorbic acid, or cell culture media components.

Tip 6: Pay attention to other potentially interfering components

The list here is not exhaustive and only includes common examples. With the incorporation of new components into the FixNCut workflow, caution is advised. The central disulfide bridge in DSP provides a reducible link that can be cleaved by reducing agents such as DTT (dithiothreitol) or β-mercaptoethanol. In single-cell assays, DTT is commonly used to inhibit RNase activity, inactivate reverse transcription inhibitors, and dissolve gel beads by breaking their disulfide bonds, thus releasing oligonucleotides essential for mRNA capture. However, DTT also de-crosslinks DSP-induced fixation. DTT, often used with sodium dodecyl sulfate (SDS), can rapidly break down cells. Therefore, even fixed cells should not be left in the reaction mix for more than a few min before chip loading. In contrast, EDTA in cell sorting buffer does not interfere with the crosslinking process. Once cells are fixed, EDTA cannot alter intracellular protein structure through chelating divalent cations or prevent cell clumping by inhibiting calcium-dependent adhesion, making its use in FACS buffer non-essential.

Final remark: As a reactive compound, DSP esters can cause eye and skin irritation and may be harmful if swallowed or inhaled. Therefore, it should be handled with care in a controlled laboratory environment. Always follow safety guidelines when handling chemicals, including wearing appropriate personal protective equipment and working in a well-ventilated area. The waste generated in this protocol is inactive but contains trace contents of DMSO and should be disposed of according to local regulations.

Acknowledgments

This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (IMI 2 JU) under grant agreement No 831434 (3TR; Taxonomy, Targets, Treatment, and Remission). The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA. Also, this project has received funding from the European Union’s H2020 research and innovation program under grant agreement No. 848028 (DoCTIS; Decision On Optimal Combinatorial Therapies In Imids Using Systems Approaches), and the Commonwealth Standard Grant Agreement 4-F26M8TZ. L.J.-G. has held an FPU PhD fellowship (FPU19/04886) from the Spanish Ministry of Universities. Australian Prostate Cancer BioResource (APCB) collection in Adelaide is supported by funding from the South Australian Immunogenomics Cancer Institute and the South Australian Health and Medical Research Institute. S.W., A.A.A. and I.S.V. acknowledge support from Spatial Technologies Unit of Precision RNA Medicine Core (RRID:SCR_024905), as well as the National Institutes of Health under award number P01AI179405 and U54HL165440. The authors further thank Dr. Tatsuyuki Sato and Dr. Joji Fujisaki for their valuable feedback and discussion. The graphics were created with BioRender.com.

Competing interests

H.H. is a co-founder and shareholder of Omniscope, a scientific advisory board member of MiRXES and Nanostring, and a consultant to Moderna and Singularity. L.G.M is an advisor and shareholder of Omniscope, and advisor for ArgenTAG and BioScryb. Omniscope has filed a patent related to the application of the FixNCut protocol. All other authors declare no competing interests.

References

1. Ziegenhain, C., Vieth, B., Parekh, S., Hellmann, I. and Enard, W. (2018). Quantitative single-cell transcriptomics. Briefings Funct Genomics. 17(4): 220–232. https://doi.org/10.1093/bfgp/ely009
2. Lafzi, A., Moutinho, C., Picelli, S. and Heyn, H. (2018). Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies. Nat Protoc. 13(12): 2742–2757. https://doi.org/10.1038/s41596-018-0073-y
3. Kazer, S. W., Aicher, T. P., Muema, D. M., Carroll, S. L., Ordovas-Montanes, J., Miao, V. N., Tu, A. A., Ziegler, C. G. K., Nyquist, S. K., Wong, E. B., et al. (2020). Integrated single-cell analysis of multicellular immune dynamics during hyperacute HIV-1 infection. Nat Med. 26(4): 511–518. https://doi.org/10.1038/s41591-020-0799-2
4. Litviňuková, M., Talavera-López, C., Maatz, H., Reichart, D., Worth, C. L., Lindberg, E. L., Kanda, M., Polanski, K., Heinig, M., Lee, M., et al. (2020). Cells of the adult human heart. Nature. 588(7838): 466–472. https://doi.org/10.1038/s41586-020-2797-4
5. Slyper, M., Porter, C. B. M., Ashenberg, O., Waldman, J., Drokhlyansky, E., Wakiro, I., Smillie, C., Smith-Rosario, G., Wu, J., Dionne, D., et al. (2020). A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med. 26(5): 792–802. https://doi.org/10.1038/s41591-020-0844-1
6. Denisenko, E., Guo, B. B., Jones, M., Hou, R., de Kock, L., Lassmann, T., Poppe, D., Clément, O., Simmons, R. K., Lister, R., et al. (2020). Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biol. 21(1): 1–25. https://doi.org/10.1186/s13059-020-02048-6
7. de Ruiter, K., van Staveren, S., Hilvering, B., Knol, E., Vrisekoop, N., Koenderman, L. and Yazdanbakhsh, M. (2018). A field‐applicable method for flow cytometric analysis of granulocyte activation: Cryopreservation of fixed granulocytes. Cytometry Part A 93(5): 540–547. https://doi.org/10.1002/cyto.a.23354
8. Kotsakis, A., Harasymczuk, M., Schilling, B., Georgoulias, V., Argiris, A. and Whiteside, T. L. (2012). Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples. J Immunol Methods. 381: 14–22. https://doi.org/10.1016/j.jim.2012.04.004
9. Horie, M., Castaldi, A., Sunohara, M., Wang, H., Ji, Y., Liu, Y., Li, F., Wilkinson, T. A., Hung, L., Shen, H., et al. (2020). Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker. Cells. 9(11): 2460. https://doi.org/10.3390/cells9112460
10. Alles, J., Karaiskos, N., Praktiknjo, S. D., Grosswendt, S., Wahle, P., Ruffault, P. L., Ayoub, S., Schreyer, L., Boltengagen, A., Birchmeier, C., et al. (2017). Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol. 15(1): 1–4. https://doi.org/10.1186/s12915-017-0383-5
11. Cao, J., Packer, J. S., Ramani, V., Cusanovich, D. A., Huynh, C., Daza, R., Qiu, X., Lee, C., Furlan, S. N., Steemers, F. J., et al. (2017). Comprehensive single-cell transcriptional profiling of a multicellular organism. Science (1979). 357(6352): 661–667. https://doi.org/10.1126/science.aam8940
12. Chen, J., Cheung, F., Shi, R., Zhou, H., Wenrui, W., Consortium, C., Candia, J., Kotliarov, Y., Stagliano, K. R., Tsang, J. S., et al. (2018). PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing. J Transl Med. 16(1): 1–11. https://doi.org/10.1101/315267
13. García-Castro, H., Kenny, N. J., Iglesias, M., Álvarez-Campos, P., Mason, V., Elek, A., Schönauer, A., Sleight, V. A., Neiro, J., Aboobaker, A., et al. (2021). ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. Genome Biol. 22(1): 1–34. https://doi.org/10.1186/s13059-021-02302-5
14. Sánchez-Carbonell, M., Jiménez Peinado, P., Bayer-Kaufmann, C., Hennings, J. C., Hofmann, Y., Schmidt, S., Witte, O. W. and Urbach, A. (2023). Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus. Front Mol Neurosci. 16: e1223798. https://doi.org/10.3389/fnmol.2023.1223798
15. Wang, X., Yu, L. and Wu, A. R. (2021). The effect of methanol fixation on single-cell RNA sequencing data. BMC Genomics. 22(1): 420. https://doi.org/10.1186/s12864-021-07744-6
16. Lomant, A. and Fairbanks, G. (1976). Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol. 104(1): 243–261. https://doi.org/10.1016/0022-2836(76)90011-5
17. Akaki, K., Mino, T. and Takeuchi, O. (2022). DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex. Bio Protoc. 12(15): e4478. https://doi.org/10.21769/bioprotoc.4478
18. Attar, M., Sharma, E., Li, S., Bryer, C., Cubitt, L., Broxholme, J., Lockstone, H., Kinchen, J., Simmons, A., Piazza, P., et al. (2018). A practical solution for preserving single cells for RNA sequencing. Sci Rep. 8(1): 2151. https://doi.org/10.1038/s41598-018-20372-7
19. Subramanian Parimalam, S., Oguchi, Y., Abdelmoez, M. N., Tsuchida, A., Ozaki, Y., Yokokawa, R., Kotera, H. and Shintaku, H. (2018). Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate). Anal Chem. 90(21): 12512–12518. https://doi.org/10.1021/acs.analchem.8b02338
20. Reyes, M., Billman, K., Hacohen, N. and Blainey, P. C. (2019). Simultaneous Profiling of Gene Expression and Chromatin Accessibility in Single Cells. Adv Biosyst. 3(11): e201900065. https://doi.org/10.1002/adbi.201900065
21. Nesterenko, P. A., McLaughlin, J., Cheng, D., Bangayan, N. J., Burton Sojo, G., Seet, C. S., Qin, Y., Mao, Z., Obusan, M. B., Phillips, J. W., et al. (2021). Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning. Proc Natl Acad Sci USA. 118(3): e2021190118. https://doi.org/10.1073/pnas.2021190118
22. Mutisheva, I., Robatel, S., Bäriswyl, L. and Schenk, M. (2022). An Innovative Approach to Tissue Processing and Cell Sorting of Fixed Cells for Subsequent Single-Cell RNA Sequencing. Int J Mol Sci. 23(18): 10233. https://doi.org/10.3390/ijms231810233
23. Jiménez-Gracia, L., Marchese, D., Nieto, J. C., Caratù, G., Melón-Ardanaz, E., Gudiño, V., Roth, S., Wise, K., Ryan, N. K., Jensen, K. B., et al. (2024). FixNCut: single-cell genomics through reversible tissue fixation and dissociation. Genome Biol. 25(1): 81. https://doi.org/10.1186/s13059-024-03219-5
24. Aney, K. J., Jeong, W. J., Vallejo, A. F., Burdziak, C., Chen, E., Wang, A., Koak, P., Wise, K., Jensen, K., Pe’er, D., et al. (2024). Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis. Gastroenterology. 166(6): 1100–1113. https://doi.org/10.1053/j.gastro.2024.01.043
25. Pedraza, C. E., Chien, Y. and McKee, M. D. (2007). Calcium oxalate crystals in fetal bovine serum: Implications for cell culture, phagocytosis and biomineralization studies in vitro. J Cell Biochem. 103(5): 1379–1393. https://doi.org/10.1002/jcb.21515