1. Culture 1-2 x 10⁶ cells/mL in 1 mL CGM (RPMI + 2% each pen.-strep. and AB serum) with  desired stimuli/toxins for 24 to 48 hours.
2. Spin cells down and dump supernatant.
3. Wash* once.
4. Fix 20 minutes, room temperature, with 1 mL 4% paraformaldehyde in PBS.
5. Spin & dump.
6. Permeablize 10 minutes with 0.15% Triton X-100/0.1% BSA in TBS.
• (Seki uses 0.1% Triton X-100/0.1% BSA in TBS for 5 minutes.)
7. Spin & dump.
8. Wash once.
• (Seki washes a total of three times at this point.)
9. Stain in a total volume of 100 mL for 30 minutes (dark/4C).
• (Seki stains for 20 minutes. Other protocols call for including detergent in the staining mixture.)
10. Raise volume to 2 mL and wash.
11. Dump supernatant, raise volume to 500 mL, and read the same day by flow cytometry.
• For FITC conjugated antibodies, excite with 488 nm and read fluorescence at 530 nm.

Controls should include unstained/"cells only", FITC-IgG₁/"conjugate control", and FITC-bcl-2 labeled unstimulated/untreated cells.

For both Dako FITC-bcl-2 (cat #F 7053, 100 mg/mL, F:P=4-5) and BDIS FITC-IgG₁ (cat #349041, 50 mg/mL, F:P=?), use roughly 2 mL of Ab solution per tube. (Dako calls for 10 mL; BDIS calls for 20 mL.)