Jun 29, 2026
Cell sorting (FACS) for viable cells, non-neuronal and neuronal cells from mice, primates and humans
- Jennifer Mecklenburg1,
- Sergey Shein1,
- Alexei Tumanov2,
- Armen Akopian2
- 1UT Health at San Antonio;
- 2UT Health San Antonio
- RE-JOIN
Protocol Citation: Jennifer Mecklenburg, Sergey Shein, Alexei Tumanov, Armen Akopian 2026. Cell sorting (FACS) for viable cells, non-neuronal and neuronal cells from mice, primates and humans. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rd5dg1b/v1
Manuscript citation:
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.
Hovhannisyan, A., Son, H., Mecklenburg, J., Barba-Escobedo, P.A., Tram, M., Gomex, R., Shannonhouse, J., Zou, Y., Weldon, K., Ruparel, S., Lai, Z., Tumanov, A. V., Kim, Y-S., and Akopian, A.N. Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system. Sci. Reports 2021 Sep 8;11(1):17813. doi: 10.1038/s41598-021-97084-y.
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 Scientific Reports 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 routinely use this protocol and it's working
Created: August 04, 2024
Last Modified: June 29, 2026
Protocol Integer ID: 104677
Keywords: FACS, immune cells, non-neuronal cells, sensory neurons, cell sorting, mice, primates, human, sorting live cell, humans cell sorting, sorted cell, purity of sorted cell, cell rna sequencing, helix np nir dye, live cell, viable cell, using calcein violet, specific type of cell, neuronal cell, cell, steps for cell, helix np nir, cell suspension, human cell, dead cell, defining cell, neuronal cells from mice, calcein violet, cell culturing, specific types of sensory neuron, bulk rna, sensory neuron
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
Cell sorting is a key step for many multi-step procedures defining cell and gene plasticity, such as single-cell RNA sequencing, bulk RNA sequencing from specific type of cells, RT-PCR from specific population of cells, cell culturing and electrophysiology.
This protocol outlines the steps for cell sorting (FACS) for live cells, for specific non-neuronal cell types and for specific types of sensory neurons. Accordingly, this protocol outlines the detailed steps for preparing and sorting live cells using Calcein Violet-AM and Helix NP NIR dyes, followed by cell sorting. The procedure includes the preparation of working solutions, cell suspension, labeling, and sorting of viable cells. Calcein Violet-AM is used to label live cells, while Helix NP NIR is utilized to exclude dead cells, ensuring high viability and purity of sorted cells.
The protocol was adapted from established methodologies and our multiple studies, including Mecklenburg et al., Front Pain Res (Lausanne) (2023); Hovhannisyan et al., Scientific Reports (2021); and Mecklenburg et al., Scientific Reports (2020). Specifically, unlike the original studies, this protocol is validated for primate and human cells.
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 preparing primate and human tissues for slicing, 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
FACSAria Fusion Cell Sorter (BD Biosciences) RRID:SCR_025715
Corning‱ LSE‱ low speed orbital shakers (Millipore Sigma) Catalog #CLS6781FP-1EA
Eppendorf‱ Centrifuge 5430/5430R (Millipore Sigma) Catalog #EP022620596-1EA
C100/C100-SE Automated Cell Counter (RWD Life Science)
Phosphate Buffered Saline 1X (Gibco) Catalog # 19344-10
50 mL Centrifuge Tube (Falcon) Catalog # 352098
15 mL Centrifuge Tube (Falcon) Catalog # 352097
Fisherbrand‱ Premium Microcentrifuge Tubes: 1.5mL (Fisher scientific) Catalog # 05-408-129
RPMI-1640 (Corning) Catalog # 10-040-CV
FBS (Gibco) Catalog # 10437-028
DPBS (Corning) Catalog #21-031-CV
EDTA (Fisher Bioreagents) Catalog #BP2482
Penicillin Streptomycin 100X (Corning) Catalog # 30-002-CI
Acridine Orange and Propidium Iodide staining kit (Logos Bio) Catalog # F23001
Calcein Violet-AM (Biolegend) Catalog #425203
Helix NP NIR (Biolegend) Catalog #425301
UltraComp eBeads (Invitrogen) Catalog # 01-2222-42
Corning‱ syringe filter. Diam. 25 mm, pore size 0.2 μm (Millipore Sigma) Catalog # CLS431224-50EA
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.
Human experiments are approved by IRB protocol - HSC20220807H Title: "Retrodiscal, Capsular and Discal TMJ Tissue Analysis and Repository".
Before start
Before tissue retrieval, every mouse should be perfused with cold PBS to avoid presence of blood cells in the tissue. For human and primate samples, it is important to mince tissue and thoroughly wash with PBS. Cell types can be identified by labeling with marker antibodies, using reporter mice (e.g., tdTomato, GFP), or retrograde tracers (e.g., WGA, Fluoro-Gold), or viral particles containing plasmid expressing tdTomato or GFP injected into peripheral tissues innervated by the target neurons. Conversion of dissected tissue into single-cell suspension is described in separate protocol (https://dx.doi.org/10.17504/protocols.io.5qpvok8x9l4o/v1).
Preparation of Working Solutions
Prior to reconstitution, spin down the vial of lyophilized Calcein Violet-AM in a microcentrifuge to ensure the reagent is at the bottom of the vial. Prepare a 1 mM stock solution by reconstituting 1 vial of lyophilized Calcein Violet-AM dye with 40 µL of anhydrous DMSO. Store it at -20°C. Prepare a 1 µM working solution by diluting 1 µL of the 1 mM Calcein Violet-AM stock solution in 1 mL DPBS.
Prepare a 1 µM working solution of Helix NP NIR by diluting 1 µL of the 1 mM stock solution in 1 mL DPBS. Keep the working solutions at +4°C in the dark and use them on the same day.
Prepare RPMI-1640 with 3% and 10% FBS along with 1% of Penicillin Streptomycin 100X solution. Warm RPMI-1640 with 3% FBS to 37°C.
Prepare FACS buffer by supplementing 1X PBS with 2% FBS and 0.5 mM EDTA (use a 0.5 M
EDTA stock solution, diluting it 1:1000).
Preparation of Cells
Filter the single-cell suspension through a 100 μm mesh to remove aggregates.
Note
Preparation of the single-cell suspension is in a separate protocol (https://dx.doi.org/10.17504/protocols.io.5qpvok8x9l4o/v1).
Take an aliquot for unstained control.
Perform a quality/cell viability check by adding 2 µL of AO/PI reagent to 18 µL of cell suspension, then load onto the C100 automated cell counter (RWD).
Spin down cells from single-cell suspension at 800g for 5-10 min.
Resuspend cells in warm RPMI-1640 containing 3% FBS and adjust the cell suspension to 5x105 cells/mL.
Cell viability reaction: Calcein Violet-AM Labeling
Add 10 µL of the 1 µM Calcein Violet-AM working solution to each 0.1 mL of cell suspension to achieve a final concentration of 100 nM.
Incubate cells for 40 minutes at 37°C, protected from light.
Note
Incubation temperature could be room temperature for 1-1.5 hours. We did not note substantial difference.
Dilute the 1 µM Helix NP NIR working solution 1:50 in FACS buffer to achieve a final concentration of 20 nM.
Spin down cells at 800g for 5-10 minutes and resuspend in FACS buffer containing 20 nM Helix NP NIR.
After incubation at for 40 minutes at 37°C, Calcein Violet-AM labeled cells are ready for downstream applications or analysis.
Note
Again, incubation could be room temperature 1-1.5 hours.
Non-neuronal cell sorting
Note: this section describes sorting all viable non-neuronal cells, surface antibody marker-labeled non-neuronal cell type, and non-neuronal cells highlighted in reporter mice.
Set up BD FACSAria Fusion (BD Biosciences) equipped with violet and red lasers and a 100 μm nozzle.
Gate Calcein Violet-positive (400 nm/452 nm) and Helix NP NIR-negative (640 nm/660 nm) cells using unstained cells as the negative control.
Note
Laser and gating has unique setting for each case of surface antibody marker-labeled non-neuronal cell type, and non-neuronal cells highlighted in reporter mice
Collect sorted cells into a 1.5 mL DNase/RNase-free tube containing 200 µL of RPMI-1640 with 10% FBS.
Perform post-sort analysis of the collected cells.
Check cell concentration and viability using Acridine Orange and Propidium Iodide on the C100 cell counter (RWD).
Keep the sorted cells on ice until further use.
Sorting of Fluorescence Labeled Sensory Neurons
Set up BD FACSAria Fusion (BD Biosciences) equipped with a 130 μm nozzle and appropriate lasers.
Note
Sorting medium to large neurons with diameter >30
Note
Sensory neurons are labeled with WGA back-traced from peripheral tissues or with tdTom, GFP, etc. reporter.
Gate the neurons using unstained cells as the negative control.
Use single color controls for compensation if two or more overlapping fluorochromes were used.
Collect sorted cells into a 1.5 mL DNase/RNase-free tube containing 200 µL of RPMI-1640 with 10% FBS.
Perform post-sort analysis of the collected cells.
Check cell concentration and viability using Acridine Orange and Propidium Iodide on the C100 cell counter (RWD).
Note
It is better to check morphology of viable neurons using high power (x40 objective) microscopy as well.
Keep the sorted cells on ice until further use.
Protocol references
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.
Hovhannisyan, A., Son, H., Mecklenburg, J., Barba-Escobedo, P.A., Tram, M., Gomez, R., Shannonhouse, J., Zou, Y., Weldon, K., Ruparel, S., Lai, Z., Tumanov, A. V., Kim, Y-S., and Akopian, A.N. Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system. Sci. Reports 2021 Sep 8;11(1):17813. doi: 10.1038/s41598-021-97084-y.
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 Scientific Reports 2020 Sep 17; 10(1): 15278. doi: 10.1038/s41598-020-72285-z.
Yajun Shou, Ekaterina Koroleva, Cody M. Spencer, Sergey A. Shein, Anna A. Korchagina, Kizil A. Yusoof, Raksha Parthasarathy, Elizabeth A. Leadbetter, Armen N. Akopian, Amanda R. Muñoz and Alexei V. Tumanov Redefining the Role of Lymphotoxin Beta Receptor in the Maintenance of Lymphoid Organs and Immune Cell Homeostasis in Adulthood. Frontiers in Immunology 15 July 2021.