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.