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Protocol for Flow Cytometry Staining
Cell Surface Staining for Whole Blood Samples
  1. Collect whole blood into tubes with anti-coagulant like heparin or EDTA.
  2. Aliquot 100 μL unlysed whole blood for each antibody staining experiment or controls.
  3. Add 5 μL (0.5 μg) antibody conjugate to 100 μL of anti-coagulated whole blood.
  4. Incubate at room temperature for 15-20 minutes in the dark.
  5. Add 2 mL Red Blood Cell (RBC) Lysis Buffer (Catalog 37014) or ReadiUse™ RBC Lysis/Fixation Solution (Catalog 37012) to each sample. Incubate at room temperature for 10 minutes.
  6. Centrifuge at 350g for 2-5 minutes, discard the red supernatant.
  7. Wash 2 times with 2 mL of Cell Staining Buffer followed by centrifugation at 350g for 5 minutes.
  8. Resuspend cells in 0.25-0.5 mL Cell Staining Buffer.
  9. Perform fluorescence activated cell sorting (FACS), or flowcytometric analysis.
Cell Surface Staining for PBMCs or Cells
  1. Resuspend PBMCs or cell lines in Staining Buffer.
  2. Wash the cells twice in cold staining buffer and pellet the cells by centrifugation at 300g at 4 °C.
  3. Resuspend the cell pellet in cold staining buffer to a final concentration of 1 x 107 cells/mL.
  4. Distribute 100 μL aliquots of the cell suspension (106 cells) to each centrifuge tube.
  5. (Optional) To block non-specific Fc-mediated interaction, add Fc Block (Catalog 37000) and incubate for 10 minutes at room temperature.
  6. Add 0.5 μg (5 μL) antibody conjugate to cells and incubate for 20 minutes on ice, protected from light.
  7. Wash it 2 times with 2 mL of Cell Staining Buffer by centrifugation at 350g for 5 minutes.
  8. Resuspend cells in 0.25-0.5 mL Cell Staining Buffer.
  9. Perform fluorescence activated cell sorting (FACS) or flow cytometric analysis.
Document: 02.0247.260102r1
Last updated Fri Jan 02 2026
Protocol for Flow Cytometry Staining
Cell Surface Staining for Whole Blood Samples
Cell Surface Staining for PBMCs or Cells