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Protocol: Cell Surface Antibody Staining for Flow Cytometry

Extracellular Staining for Flow Cytometry

There are many protocols for staining cells for flow cytometry. Protocols may to need be optimized for different cell types, targets, or applications. This is our basic protocol for extracellular staining of cell surface epitopes in suspension cells for flow cytometry. For intracellular staining, see our Protocol: Intracellular Antibody Staining for Flow Cytometry.

Materials required:

Workflow overview:

  1. Aliquot cells to flow tubes
  2. Primary antibody incubation (30 min.)
  3. Wash and centrifuge (5 min.) 2x
  4. Fixation followed by wash (optional)
  5. Secondary antibody incubation (not required for labeled primary antibody) (30 min.)
  6. Wash and centrifuge (5 min.) 2x (optional stopping point if fixation is performed)
  7. Analyze by flow cytometry


  1. Detach adherent cells from substrate by trypsinization or with a commercial non-enzymatic cell lift solution.
  2. Optional: To exclude dead cells from analysis, resuspend cells in PBS or other protein-free buffer and stain cells with a fixable dead cell dye,  such as our Live-or-Dye™ Fixable Viability Stains, according to the product protocol.
    Note: If cell fixation will not be performed, a non-fixable dead cell stain, such as PI or 7-AAD, can be added together with primary or secondary antibody.
  3. Adjust cell density to 107 cells per mL in flow buffer.
  4. Aliquot 100 uL of cell suspension per flow cytometry tube for a total of 106 cells per tube. Place tubes on ice.
  5. Add primary antibodies to tubes and vortex gently to mix. Incubate tubes on ice (or at 4°C) for 30 min. If using directly conjugated fluorescent primary antibodies, tubes should be protected from light.
    Note: Primary antibody concentration must be optimized for different applications, but 0.5-1 ug antibody per tube is a common starting concentration.
  6. Wash by adding 1 mL flow buffer to each tube and pellet cells by centrifugation for 5 min. at 350 x g.
  7. Pour off the wash buffer from the tubes into a waste container.
  8. Repeat wash (steps 6-7).
  9. Optional: Cells can be fixed at this step with your preferred fixative. After fixation, wash as in steps 6-7.
  10. If using directly labeled primary antibodies, proceed to step 14. If using labeled secondary antibodies, continue with step 11.
  11. After pouring off wash buffer, resuspend cells in residual buffer (~100 uL) by gentle vortexing.
  12. Add 1 ug of each secondary antibody to each tube and vortex gently to mix. Incubate at room temperature, protected from light, for 30 min.
    Note: For biotinylated primary antibodies, Streptavidin conjugates can be used for detection, typically at 0.25 ug/tube.
  13. Wash cells twice in flow buffer (repeat steps 6-7).
  14. After pouring off wash buffer, add 500 uL flow buffer per tube.
  15. Analyze by flow cytometry in the correct channel for your conjugate. Mix by gentle vortexing before loading each sample on cytometer.
    Note: If fixation is performed in step 7, cells can be stored at 4°C, protected from light, for several days before analysis.