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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
Antibodies and ProteomicsCluster of Differentiation (CD Antibodies)ProtocolSpectral Flow Cytometryflow cytometry
Answer
  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.
  2. 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.
  3. Add appropriate CD antibody with previously determined optimum concentration and vortex. Incubate on ice for 15 to 30 minutes.
  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.
Additional resources
Fundamentals of Flow CytometryAvailable CD AntibodiesLeif, R. C. (1986). Practical flow cytometry, by Howard M. Shapiro. Alan R. Liss, New York, 1985, 295 pages. Cytometry, 7(1), 111–112. doi:10.1002/cyto.990070119 Cluster of Differentiation (CD Antibodies)

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