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Single-cell suspension is required for flow cytometry assays. Thus the adherent cell lines and tissue samples require processing into single-cell suspension before flow cytometry analyzed. A number of protocols are available and involved in mechanical dissociation or enzymatic digestion of the sample.
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Flow Cytometry Protocol: Cell Surface Marker Staining Note: This method is suitable for cell surface staining in flow cytometry. Cell surface markers are expressed on the cell surface and can be used to define cell subtypes as well as function when they are labeled with fluorescent-labeled antibodies and analyzed by flow cytometry. As those surface proteins are accessible to the antibodies, they can be easily stained without permeabilization steps which are critical to intracellular staining. Reagents: PBS: Dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 800 mL distilled water. Adjust the pH to 7.4 with HCl and final volume to 1 liter with additional distilled H2O. Cell staining buffer: Add 0.5% BSA and 0.05% Sodium Azide (NaN3) to PBS. Fc receptor binding reagents Conjugated primary antibodies Conjugated secondary antibodies, if needed Materials Centrifuge Pipettes and pipettors Vortex Procedures: Sample preparation: 1. Harvest the desired tissues and cells, prepare a single cell suspension and adjust the suspension to a concentration of 1 x 106 cells/mL in cell straining buffer. https://www.creative-diagnostics.com/flow-cytometry-protocol-cell-surface-marker- staining.htm
