Thymidine incorporation is highly predictive of colony formation and can be used for high-throughput screening.

The primary goal of anticancer chemotherapy is to kill cancer cells. Therefore, it is of critical importance that any assay that is used to determine the toxicity of a potential anticancer drug accurately measures viability. While colony formation is widely regarded as the most accurate measure of viability following drug treatment, it is laborious, time consuming, and difficult to carry out with non-adherent cells. For these reasons, it is not suitable for moderate- to high-throughput screening applications. We sought to identify a convenient and reliable assay that would accurately reproduce colony formation results and be amenable to high-throughput applications. Here, we describe a modification of the 3H-thymidine incorporation assay that meets these criteria. The assay can be carried out in 96-well plates with minimal handling of reagents and media. It can be performed with non-adherent and adherent cell lines. Most importantly, LC50 values obtained with this assay show excellent agreement with colony formation results. Taken together, these advantages make the modified 3H-thymidine incorporation assay well suited for high-throughput viability assays in anticancer drug discovery and development.