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What is the general protocol for analysis of surface expression of a CD marker using flow cytometry?
Posted May 18, 2020
Answer
- 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.
- Blocking Fc receptors is optional but useful to reduce non-specific immunofluorescent staining. Add 1 ug of Fc receptors binding reagents per 1 x 106 cells and incubate for 10 minutes at room temperature.
- Add appropriate CD antibody with previously determined optimum concentration and vortex. Incubate on ice for 15 to 30 minutes.
- 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.
- Repeat Step 4 twice.
- Add an appropriate fluorescent-conjugated secondary antibody with recommended concentration. Incubate on ice for 15-20 min in the dark.
- Repeat Step 4 three times.
- Resuspend cell in 200-500 uL cell staining buffer for final flow cytometric analysis.
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