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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.


  • 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


  • Centrifuge
  • Pipettes and pipettors
  • Vortex


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.

Learn more about sample preparation for flow cytometry.

Blocking Fc-receptors (optional):

2. Blocking Fc receptors is useful to reduce non-specific immunofluorescent staining. Add 1 μg of Fc receptors binding reagents per 1 x 106 cells and incubate for 10 minutes at room temperature.

  • For mouse cells, purified anti-mouse CD16/CD32 antibody specific for Fcγ R III/II (cat. no. CABT-45660RM) can be used to block the Fc receptors.
  • For human cells, purified human Fc receptor binding inhibitor solution can be used to eliminate non-specific staining.
  • In the absence of an effective/available blocking antibody for Fc receptors, an alternative approach is to block cells with excess irrelevant purified IgG from the same species and same isotype as the antibodies used for immunofluorescent staining.

Note: Do not wash excess blocking reagents from this reaction.


3. Add appropriately primary antibody with previously determined optimum concentration and vortex. Incubate on ice for 15 to 30 minutes.

Note: If using primary antibodies directly conjugated with fluorochrome, the incubation should be carried out in the dark, and then skip to Step 7.

4. Wash cells with cell staining buffer to remove any unbound antibodies. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard supernatant.

5. Repeat Step 4 twice.

6. Add an appropriate fluorescent-conjugated secondary antibody with recommended concentration. Incubate on ice for 15-20 min in the dark.

7. Repeat Step 4 three times.

8. Resuspend cell in 200-500 uL cell staining buffer for final flow cytometric analysis.

Browse all the antibodies for flow cytometry.

Learn the principle of flow cytometry.

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