Flow Cytometry Protocol

Flow Cytometry (FACS) Protocol

1. Collect and count cells. Pellet by centrifugation and aspirate supernatant.
2. Resuspend cells briefly in 0.5-1 ml PBS. Pellet and re-suspend in 10% Formalin or add Formaldehyde to 2-4%.
3. Fix for 30 minutes at room temperature.
4. Chill tubes on ice for 1 minute.
5. Pellet cells by centrifugation and re-suspend in 90% methanol.
6. Incubate 30 minutes on ice.
7. Proceed with staining or store cells at -20°C in 90% methanol in 0.5x10^6 cell aliquots.
 
NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.
 
8. Aliquot 0.5-1x10^6 cells into each assay tube (by volume).
9. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
10. Resuspend cells in 100 µl Incubation Buffer per assay tube.
11. Block in Incubation Buffer for 10 minutes at room temperature.
12. Add primary antibody at the appropriate dilution to the assay.
13. Incubate for 1 hour at room temperature.
14. Rinse as before in Incubation Buffer by centrifugation.
15. If using a conjugated primary antibody, resuspend cells in 0.5 ml PBS-Tw and analyze on flow cytometer; if using an unlabeled primary antibody, proceed to step 16.
16. Re-suspend cells in fluorochrome-conjugated secondary antibody* or fluorochrome-conjugated avidin, diluted in Incubation Buffer at the appropriate dilution.
17. Incubate for 30 minutes at room temperature.
18. Rinse as before in Incubation Buffer by centrifugation, but be sure to incubate at least as long as secondary antibody incubation.
19. Resuspend cells in 0.5 ml PBS-TW and analyze on flow cytometer; alternatively, for DNA staining, proceed to step E1.
 
Solutions and Reagents:
 
1. 1X Phosphate Buffered Saline (PBS-Tw): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Add Tween-20 to 0.1% (1ml/L). Store at room temperature.  
2. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS-TW. Store at 4°C.

More Resources:

Flow Cytometry (FACS) Illustrated Assay