Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:50)

Protocol for immunostaining of cell surface markers for flow cytometric analysis. Applicable to antibodies with recommended working dilution of 1:50, in PBS/EDTA/BSA buffer. 

Protocol

  • The recommended antibody dilution for labeling of cells and subsequent analysis by flow cytometry is 1:50 for up to 106 cells in final volume of 100 µL.
  • Volumes given below are for up to 106 nucleated cells. When working with fewer than 106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
  1. Determine cell number.
  2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
  3. Resuspend up to 106 nucleated cells per 98 µL of buffer.
  4. Add 2 µL of the antibody.
  5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C). 
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. 
  6. Wash cells by adding 1−2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. If biotinylated primary antibody was used, repeat washing step.
  7. (Optional) If biotinylated primary antibody was used, resuspend the cell pellet in buffer and stain with fluorochrome-conjugated Biotin Antibody according to the manufacturer’s recommendations. 
  8. Resuspend cell pellet in a suitable amount of buffer for analysis.

Materials