In Vitro [3H Thymidine Incorporation Assay

[3H] Thymidine incorporation assays are conducted to demonstrate that the agents are incorporated into the DNA. Tumor cells are plated at 50,000 cells/well in

200 mL RPMI, 10% FCS. Concentration of the EC-agents (0.1-1 mg/well) and saline (control) are added to this 96-well culture plate and incubated in 5% CO2/air at 37°C. After 24 hours, each well is pulsed with 0.5 mCi/10 mL [3H] thymidine and incubated for 24 hours. Cells are then harvested, typsinized with 100 mL of typsin, and incubated for 10 minutes in the incubator. The cells are counted with a liquid scintillation counter. Cellular uptake of [3H] thymidine in the control group is normalized to be 100 (baseline).

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