Fluorescence-Activated Cell Sorting (FACS)-Based Cell Sorting
Materials Required
Principle
Fluorescence-Activated Cell Sorting (FACS) separates cells in suspension after flow-cytometric measurement of light scatter and fluorescence; classic droplet-based instruments interrogate cells with a laser, convert optical signals into electronic signals, charge droplets containing target cells, and electrostatically deflect them into collection vessels[1][2][3]. FACS detects phenotypes defined by fluorescent antibodies, fluorescent proteins, viability dyes, or intracellular markers, and the readout is the sorted fraction, purity, recovery, and post-sort viability of the gated population[2][3][4].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use a single-cell suspension in an appropriate isotonic staining/sort buffer, because FACS requires cells to pass individually through the fluidic stream[1][2][3].
• Use viability-discrimination reagents such as 7-AAD for unfixed live-cell sorting or amine-reactive fixable dyes when fixed/permeabilized samples are required[5][6].
• Use fluorophore-conjugated antibodies or endogenous fluorescent reporters to define target populations, and titrate antibodies during panel setup because staining performance depends on reagent selection and staining specificity[2][4].
• Use unstained, single-stained compensation controls, and fluorescence-minus-one controls when multicolor gating boundaries require spillover-aware interpretation[4][7].
• Use a flow cytometer with sorting capability, appropriate excitation lasers and emission filters for the chosen fluorophores, droplet-generation and deflection hardware for droplet sorters, and collection tubes or plates compatible with the downstream assay[1][2][3].
• Use viability-discrimination reagents such as 7-AAD for unfixed live-cell sorting or amine-reactive fixable dyes when fixed/permeabilized samples are required[5][6].
• Use fluorophore-conjugated antibodies or endogenous fluorescent reporters to define target populations, and titrate antibodies during panel setup because staining performance depends on reagent selection and staining specificity[2][4].
• Use unstained, single-stained compensation controls, and fluorescence-minus-one controls when multicolor gating boundaries require spillover-aware interpretation[4][7].
• Use a flow cytometer with sorting capability, appropriate excitation lasers and emission filters for the chosen fluorophores, droplet-generation and deflection hardware for droplet sorters, and collection tubes or plates compatible with the downstream assay[1][2][3].
Experimental Procedure
• Prepare a clean single-cell suspension from cultured cells or tissue-derived cells, remove aggregates before sorting, and keep only parameters that preserve the intended biological state for the specific sample type[2][4].
• Design gates before sorting by acquiring unstained, single-color, viability, and FMO controls, because compensation corrects spectral overlap and FMO controls help define positive/negative boundaries in multicolor panels[4][7].
• Stain cells with the selected antibody or reporter-compatible dye panel, include a viability dye, wash away unbound reagents, and resuspend cells as a single-cell suspension before acquisition[2][5][6].
• Acquire events on the sorter, gate sequentially on cells, singlets, viable cells, and the fluorescence-defined target population, then sort target cells into tubes or plates using the instrument’s validated sort mode for the downstream application[2][3][4].
• For single-cell applications, index sorting can be used to retain the fluorescence phenotype of each deposited cell and link that phenotype to downstream single-cell measurements[8].
• Assess sort performance by reanalyzing a small post-sort fraction for purity, recording event counts and recovery, and comparing sorted populations with the predefined gating controls[2][3][4].
• For downstream RNA or functional assays, consider that published evaluations found cell sorting can cause minor gene-expression or stress-signaling effects under tested conditions, so matched unsorted or mock-sorted controls are appropriate when the downstream readout is stress-sensitive[9][10].
• Design gates before sorting by acquiring unstained, single-color, viability, and FMO controls, because compensation corrects spectral overlap and FMO controls help define positive/negative boundaries in multicolor panels[4][7].
• Stain cells with the selected antibody or reporter-compatible dye panel, include a viability dye, wash away unbound reagents, and resuspend cells as a single-cell suspension before acquisition[2][5][6].
• Acquire events on the sorter, gate sequentially on cells, singlets, viable cells, and the fluorescence-defined target population, then sort target cells into tubes or plates using the instrument’s validated sort mode for the downstream application[2][3][4].
• For single-cell applications, index sorting can be used to retain the fluorescence phenotype of each deposited cell and link that phenotype to downstream single-cell measurements[8].
• Assess sort performance by reanalyzing a small post-sort fraction for purity, recording event counts and recovery, and comparing sorted populations with the predefined gating controls[2][3][4].
• For downstream RNA or functional assays, consider that published evaluations found cell sorting can cause minor gene-expression or stress-signaling effects under tested conditions, so matched unsorted or mock-sorted controls are appropriate when the downstream readout is stress-sensitive[9][10].
Troubleshooting
Problem: Low purity.
• Possible Cause: Gate boundaries were poorly defined in a multicolor panel.• Literature-supported Solution: Rebuild gates using unstained, single-stained compensation controls, and FMO controls before sorting[4][7].
Problem: High dead-cell contamination.
• Possible Cause: Dead cells were not excluded reliably by scatter alone.• Literature-supported Solution: Include 7-AAD for unfixed samples or amine-reactive fixable viability dyes when fixation/permeabilization is required[5][6].
Problem: Downstream transcriptomic or functional perturbation is suspected.
• Possible Cause: Sorting conditions or sorter-induced cellular stress may affect stress signaling or gene-expression measurements.• Literature-supported Solution: Include unsorted and mock-sorted controls and interpret post-sort RNA or function data against those controls[9][10].
References:
- [1]. Bonner WA, et al. Fluorescence activated cell sorting. Rev Sci Instrum. 1972;43(3):404-409. [Content Brief]
- [2]. Basu S, et al. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. 2010;(41):1546. [Content Brief]
- [3]. Telford WG. Flow cytometry and cell sorting. Front Med (Lausanne). 2023;10:1287884. [Content Brief]
- [4]. Cossarizza A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol. 2019;49(10):1457-1973. [Content Brief]
- [5]. Schmid I, et al. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry. 1992;13(2):204-208. [Content Brief]
- [6]. Perfetto SP, et al. Amine-reactive dyes for dead cell discrimination in fixed samples. Curr Protoc Cytom. 2010;Chapter 9:Unit 9.34. [Content Brief]
- [7]. Feher K, et al. Cell population identification using fluorescence-minus-one controls with a one-class classifying algorithm. Bioinformatics. 2014;30(23):3372-3378.
- [8]. Penter L, et al. FACS single cell index sorting is highly reliable and determines immune phenotypes of clonally expanded T cells. Eur J Immunol. 2018;48(7):1248-1250. [Content Brief]
- [9]. Box A, DeLay M, Tighe S, et al. Evaluating the effects of cell sorting on gene expression. J Biomol Tech. 2020;31:100-111. [Content Brief]
- [10]. Pfister G, et al. An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions—Are your sorted cells getting SICS? J Immunol Methods. 2020;487:112902. [Content Brief]
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