96 well microplate AnnexinV-Pe staining vs DNA (7-AAD) for FACS analysis of GFP+ cells

Perform all steps at +4°C, up to the AnnexinV staining.
 

1- Resuspend the cells in complete Phenol red-deficient medium at a convenient concentration (each microtiter well contains 300 µl and you may not stain less than 500.000 cells per well).
2- Place 0.5 to 3 millions cell per well of a U bottom 96 wells plate.
3- Centrifuge at  1000 rpm for 3 min
4- Discard the supernatant  in a basin with a quick wrist-forearm  movement.
5- Vortex the whole plate in order to resuspend the cells in the few µl left.
6- add 250 µl of AnnexinV buffer to each well
7- repeat steps 3 to 5
8- add to each well 50µl AnnexinV buffer containing 2.5 µl AnnV-Pe and 25 µg/ml 7-AAD.
Stock solution of 7-AAD is 1 mg/ml in ETOH. ( equal: 1.25µl stock for 50µl medium.)
9- incubate at RT for 15 min in the dark.
10- add 250 µl AnnexinV buffer.
11- read within 1 hr.  

10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2.

Positive control.

* Exponentially growing Ramos cells are incubated for 4hrs. with 18 µM Camptothecin (CPT; Fisher/ Acros Cat. 27672-1000, FW 348.35. Dissolve in DMSO)  or vector alone. Viability should be in both >90%. You should expect 3x to 10x times apoptotic cells (AnnexinV+, 7AAD-) in the CPT-treated sample. Aliquots of induced and uninduced Ramos can be kept at +4°C in 70% EtOH

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