Proliferation Assay: [3H] Thymidine incorporation

Labelling:

1) Seed cells in 6 well plates ( in normal growth media) such that there are 5 X 104 cells per well.

2) Incubate cells 2 nights.

3) Remove media, wash cells one time with 1X PBS, and add serum free media.

4) Rest cells for 2-4 hours then treat.

5) Six-twenty four hours before cells are to be harvested, add 1 µCi/ml [3H]-TdR to each well.



Harvesting:

6) Wash cells 2 times with ice cold PBS.

7) Wash cells 2 times with 5% TCA.

8) Solubilize cells by adding 0.5 ml 10.25 N NaOH (pipet up and down to completely solubilize cells).

9) Collect 400 µl of the solubilized cell solution into 4 ml scintillation vials and count.