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)

1X PBS

0.2% Triton-X100

2 mM EDTA