Measure cell proliferation --- 3H-labeled thymidine

Virtualab Protocols (Chang Bioscience)
1. Seed 100 l (1000 - 2000 cells) per well in 96 well plate.

2. You may need to add dilution of drugs or other agents at this time or after cells have attached overnight.

3. Incubate cells for the desired time at 37 C.

4. Add to each well 0.2-1 Ci 3H-Thymidine (dilute in media). Incubate cells for 24-48 hours at 37 C.

5. Wash cells twice with ice-cold PBS.

6. Wash cells twice with ice-cold 5% TCA. For the last wash, leave in 5% TCA for 30 minutes at 4 C.

7. Solubilize cells by adding 0.1 ml 0.5 N NaOH. Pipet up and down to completely solubilize cells.

8. Transfer the solubilized cell solution to scintillation vials, add appropriate amount of scintillation fluid and count.

9. Calculate inhibition of proliferation.