Apoptosis: Miniassay

1) Aliquot 5 X 106 total cells in 5-8 ml 2% media. 2) Add 30 µl 3H-thymidine. 3) Incubate for approximately 16 hours under normal growth conditions. 4) Spin down cells and wash 2-3 times with PBS (or serum free media) to remove any unincorporated label. 5) Resuspend cells at 5 X 105/ml in serum free media. 6) Aliquot approximately 400 µl of cell suspension into each well of a 24 well plate. 7) Treat cells (ALWAYS HAVE TIME-MATCHED CONTROLS!!). 8) Pellet cells gently and count supernatant. --> supernatant = A 9) Resuspend pellet in lysis buffer. 10) Microfuge cell suspension for 15 minutes at maximum speed. 11) Collect pellet and supernatant and count both. --> supernatant = B pellet = C 12) To determine the percent fragmentation use the following equation: --> (A + B)/(A + B + C)

Lysis buffer 1X PBS 0.2% Triton-X100 2 mM EDTA