Support Protocols

Cell Surface Staining of Human PBMCs and Suspension Cell Lines

  1. PBMCs1 or cell lines can be resuspended with Pharmingen Stain Buffer containing either BSA (cat. no. 554657) or FBS (cat. no. 554656)2.
  2. Wash the cells twice in cold Stain Buffer and pellet the cells by centrifugation (e.g., 300 x g at 4°C). Resuspend the cell pellet with cold Stain Buffer to a final concentration of 2 x 107 cells/ml.
  3. Distribute 100 µl aliquots of the cell suspension (106 cells) to either tubes (12 x 75 mm Polypropylene Round-Bottom Test Tube) or the round-bottomed wells of microwell plates.
  4. Add antibodies to cells and incubate for 20 min on ice, protected from light3.
  5. Wash the cells two times with either 200 µl (for microwell plates) or 1 ml (for tubes) volumes of Stain Buffer. Centrifuge cells at 300 x g for 5 min. Carefully aspirate (for microwell plates or tubes) or invert and blot away (for tubes) supernatants from cell pellets.
  6. Tap tubes/plates to loosen cell pellet.
  7. For indirect immunofluorescent staining of cells, repeat steps 4 and 5 with either a labeled appropriate secondary antibody or a labeled streptavidin conjugate in 100 µl of Stain Buffer.
  8. Resuspend the cell pellet in either 200 µl (for microwell plates) or 0.5 ml (for tubes) volumes of Stain Buffer.
  9. Analyze stained cell samples by flow cytometry4.

Additional materials required

Microwell plates (round bottom wells) or Tubes (12 x 75 mm Polypropylene Round-Bottom Test Tube)

Footnotes

  1. For PBMC preparation, use manufacturer's directions for Ficoll products.
  2. For most applications, BSA as a blocking agent is sufficient, but investigators may use FBS if more stringent blocking is required.
  3. Determination of optimal antibody concentration may be necessary. For test size antibody products, add the recommended test size volume. Staining time may be increased (> 45 min) depending on the avidity of the fluorescent antibody.
  4. If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde (e.g., Cytofix Buffer; Cat. No. 554655, see product TDS for detailed protocol) for 30 min at 4°C, washed, resuspended in Stain Buffer and then stored at 4°C (protected from light). The fixed cells should be analyzed as soon as possible. We have not tested all fluorescently conjugated antibodies for this fixation; therefore, researchers may need to verify if this fixation will affect antibody binding and fluorescence intensity.