Cell Surface

Cell Surface Staining of Human PBMCs and Suspension Cell Lines

Reagents and Materials Required
 

Optional Reagents
 

Procedural Notes

• For peripheral blood mononuclear cell (PBMC) preparation, use the manufacturer's directions for Ficoll products.
• For most applications, BSA as a blocking agent is sufficient, but investigators may use FBS if more stringent blocking is required.
• No wash is needed prior to antibody staining.
• 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 minutes) depending on the avidity of the fluorescent antibody.
• If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde (for example, BD Cytofix buffer, see the product TDS for a detailed protocol) for 30 minutes 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.

Procedure

1. If necessary, resuspend PBMCs or cell lines with Stain Buffer.
2. Wash the cells twice in cold Stain Buffer and pellet the cells by centrifugation at 300 g at 4°C.
3. Resuspend the cell pellet with cold Stain Buffer to a final concentration of 2 x 10⁷ cells/mL.
4. Distribute 100-µL aliquots of the cell suspension (10⁶ cells) to either tubes or the round-bottomed wells of plates.
5. (Optional) To block non-specific Fc-mediated interaction, add 2.5 μg of Fc Block per 10⁶ PBMCs per tube (or per well) and incubate for 10 minutes at room temperature.
6. Add antibodies to cells and incubate for 20 minutes on ice, protected from light.
7. Wash the cells two times with either 200-µL (for plates) or 1-mL (for tubes) volumes of Stain Buffer. Centrifuge cells at 300 g for 5 minutes.
8. Carefully aspirate (for microwell plates or tubes) or invert and blot away (for tubes) supernatants from cell pellets.
9. Tap tubes/plates to loosen the cell pellet.
10. For indirect immunofluorescent staining of cells, repeat steps 5 and 6 with either a secondary antibody or a streptavidin conjugate in 100 µL of Stain Buffer.
11. Resuspend the cell pellet in either 200-µL (for microwell plates) or 0.5-mL (for tubes) volumes of Stain Buffer.
12. Analyze stained cell samples by flow cytometry.