Jun 26, 2026
Single-cell suspension for flow cytometry, cell sorting (FACS), and single-cell RNA sequencing
- Karen Lindquist1,
- Jennifer Mecklenburg1,
- Armen Akopian1,
- Alexei Tumanov1
- 1UT Health San Antonio
- RE-JOIN
Protocol Citation: Karen Lindquist, Jennifer Mecklenburg, Armen Akopian, Alexei Tumanov 2026. Single-cell suspension for flow cytometry, cell sorting (FACS), and single-cell RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok8x9l4o/v1
Manuscript citation:
Lindquist, K. A., Shein S. A., Hovhannisyan, A. H., Mecklenburg, J., Zou Y., Lai Z., Tumanov A.V., and Akopian A.N. Associations of tissue damage induced inflammatory plasticity in masseter muscle with the resolution of chronic myalgia. Sci. Reports 2023 Dec 12;13(1):22057. doi: 10.1038/s41598-023-49280-1.
Mecklenburg, J., Shein S. A., Malmir, M., Hovhannisyan, A. H., Weldon, K., Zou Y., Lai Z., Jin, Y.-F., Ruparel, S., Tumanov A.V., and Akopian A.N. Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia. Front Pain Res (Lausanne) 2023 Oct 31:4:1274811. doi: 10.3389/fpain.2023.1274811.
Mecklenburg J, You, Y., Wangzhou A., Garcia D., Lai Z., Tumanov A.V., Dussor G, Price TJ and Akopian AN Transcriptomic sex differences in sensory neuronal populations of mice Sci. Reports 1. 2020 Sep 17;10(1):15278. doi: 10.1038/s41598-020-72285-z.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working for primary cell culturing, single-cell RNA sequencing, flow cytometry and FACS sorting.
Created: July 29, 2024
Last Modified: June 26, 2026
Protocol Integer ID: 104264
Keywords: TMJ, Masseter Muscle, Single-cell suspension, facial skin, flow cytometry, single-cell RNA sequencing, cell sorting, FACS, cell suspension for flow cytometry, sequencing flow cytometry, cell suspension, flow cytometry, gene plasticity in tissue, cell rna, cell, tissue type, humans in pain condition, tissue, use of tissue, gene plasticity
Funders Acknowledgements:
NIH/NIDCR
Grant ID: R01 DE029187
NIH/NIAMS
Grant ID: UC2 AR082195
Disclaimer
All authors declare that they have no competing interests.
Abstract
Flow cytometry, cell sorting and single-cell RNA sequencing are widely used approaches to identify cell and gene plasticity in tissues from animals and humans in pain conditions. These experiments start from preparations of single-cell suspensions.
This protocol details steps for preparing singled-cell suspension from a variety of tissues isolated from mice, primates and humans. The core of the approach is the same. Only difference is step 4, which depends on tissue type and source (mouse, primate or human). The protocol was adapted from the study published by Lindquist et al Sci Rep. (2023), Mecklenburg et al Front Pain Res (Lausanne) (2023) and Mecklenburg et al Sci Rep. (2020). Specifically, unlike the original studies, this protocol includes a portion describing the use of tissues from primates and humans.
Guidelines
All personnel handling specimens of primate and human fluids and tissues, or other potentially infectious materials (e.g., unfixed tissues) should be trained and vaccinated as required by their Institutional guidelines. It is crucial to take precautionary measures and use at least biosafety level 2 (BSL2) protocols for preparation primate and human single-cell suspensions, which includes the use of proper personal protective equipment (PPE) and biosafety cabinets. Additionally, the safe transport and disposal of biological materials is essential to mitigate the risk of infection or contamination.
Materials
50 mL Centrifuge Tube (Falcon) Catalog # 352098
15 mL Centrifuge Tube (Falcon) Catalog # 352097
Phosphate Buffered Saline 1X (Gibco) Catalog # 19344-10
Penicillin G benzathine tetrahydrate (Millipore Sigma) Catalog 1502009
Streptomycin sulfate salt (Millipore Sigma) Catalog S9137
Fetal bovine serum (Millipore Sigma) Catalog # F4135
L-Glutamine Solution (Millipore Sigma) Catalog # G7513
Cell strainer-100 µm (50/package) (Corning) Catalog # 352360
Cell strainer-70 µm (50/package) (Corning) Catalog # 352350
DMEM, 1x (Corning) Catalog # 15-017-CV
HBSS, 1x (Corning) Catalog # 21-021-CV
DPBS (Corning) Catalog # 21-031-CV
Liberase‱ TL Roche (Millipore Sigma) Catalog #05401020001
Dispase II (neutral protease, grade II) Roche (Millipore Sigma) Catalog #4942078001
Safety warnings
None
Ethics statement
The reporting in the protocol follows the recommendations in the ARRIVE guidelines V2 (PLoS Bio July 14, 2020; https://doi.org/10.1371/journal.pbio.3000410
We also followed guidelines issued by the National Institutes of Health (NIH) and the Society for Neuroscience (SfN) to minimize the number of animals used and their suffering. All animal experiments conformed to protocols approved by the University Texas Health Science Center at San Antonio (UTHSCSA) Institutional Animal Care and Use Committee (IACUC). Protocol numbers are 20190114AR, 20200021AR, and 20220069AR.
Before start
Some experiments can use reporter mice to highlight specific type of cells. In this case FACS sorting should be able to detect these fluorophores.
Before tissue retrieval, every mouse should be perfused with PBS or saline pH7.2 to avoid presence of blood cells in the tissue. For human and primate samples, it is important to mince tissue and wash it with Hank's solution.
Before tissue retrieval by surgery, prepare pre-labeled sterile containers for maintaining tissue ready for different downstream processes.
Tissue preparation for single-cell suspension
Immediately after a tissue isolation from mice, primate or human participants, place a tissue in 3 or 6cm-Petri dish placed on wet ice, mince a tissue into small pieces and submerge a tissue in 10 ml cold Hank's solution (calcium-magnesium free) in 15ml-tube. Put tube on wet ice.
Wash a tissue twice with 10ml Hank's solution pre-warmed to 37°C.
Submerge a tissue in 1ml (for mice) or 3ml (for primates; humans) of pre-warmed Hank's solution.
Add 50-100ul/ml Liberase (2.5mg/ml) and 15ul/ml dispase II (50mg/ml). Incubate 60-90 min at 37°C (water bath). Mix tubes gently every 10-15 min.
Note
Amount of Liberase and incubation time are defined by types of tissues. Thus:
- muscle tissue 100ul/ml Liberase and 90 min incubation;
- TMJ tissues 100ul/ml Liberase and 70 min incubation;
- facial skin tissue 100ul/ml Liberase and 90 min incubation;
- dura mater from mice 50ul/ml Liberase and 60 min incubation;
- dura mater from primates 100ul/ml Liberase and 70 min incubation;
- trigeminal ganglia 50ul/ml Liberase and 60 min incubation.
Note
- Concentration of Dispase II is not defined by types of tissues. It is constant independent on tissue type; only Liberase is adjusted.
- Concentrations of Liberase and Dispase II depend on batch and company producing these enzymes. Therefore, pilot experiments (i.e. digestions) should be run to determine optimal Liberase and Dispase II concentrations when using a new batch.
Single-cell suspension preparation
Add up to 5ml total volume of pre-warmed filtered media (DMEM with 2% fetal calf serum (FCS) with 2 mM L-glutamate, 100U/ml penicillin and 100ug/ml streptomycin).
Spin 1000rpm for 0.5 min. Discard supernatant. Add 6 ml pre-warm filtered media (see Step 5)
Mix gently. Spin 1000rpm for 0.5 min. Discard supernatant. Add 1-2 ml pre-warm filtered media (see Step 5).
Prepare single-cell suspension by pipetting with 1 ml tip. Pass this single-cell suspension through 100 μm strainer.
Note
For flow cytometry experiments, 70 μm strainer should be used.
Done. Prepared single-cell suspension is kept on ice before starting the next step.
Protocol references
Lindquist, K. A., Shein S. A., Hovhannisyan, A. H., Mecklenburg, J., Zou Y., Lai Z., Tumanov A.V., and Akopian A.N. Associations of tissue damage induced inflammatory plasticity in masseter muscle with the resolution of chronic myalgia. Sci. Reports 2023 Dec 12;13(1):22057. doi: 10.1038/s41598-023-49280-1.
Mecklenburg, J., Shein S. A., Malmir, M., Hovhannisyan, A. H., Weldon, K., Zou Y., Lai Z., Jin, Y.-F., Ruparel, S., Tumanov A.V., and Akopian A.N. Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia. Front Pain Res (Lausanne) 2023 Oct 31:4:1274811. doi: 10.3389/fpain.2023.1274811.
Mecklenburg J, You, Y., Wangzhou A., Garcia D., Lai Z., Tumanov A.V., Dussor G, Price TJ and Akopian AN Transcriptomic sex differences in sensory neuronal populations of mice Sci. Reports 1. 2020 Sep 17;10(1):15278. doi: 10.1038/s41598-020-72285-z.