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