An in vitro thymidine incorporation assay for human cancers: technical details revisited

Among the choices of laboratory methods for in vitro testing of human tumor chemosensitivity, a thymidine incorporation assay (TIA) has been described. Advantages of a TIA over a colony-forming assay include greater probability of tumor growth, requirement for fewer tumor cells, less dependence on an absolute single-cell suspension, shorter time to completion, and the ability to measure up to three logs cell-kill. Specific technical details which are reported in the literature for performing a TIA are incomplete and/or conflicting between one laboratory and another. This paper reports details of methods designed to remove unbound 3[H]-thymidine from the agarose, standardize background counts, and determine optimal cell-plating densities, labeling time, optimal concentration of 3[H]-thymidine, and the best day after seeding cultures to add the 3[H]-thymidine. Care has been taken to compare the end point of drug sensitivity determinations following these methodologic changes, both among serial TIAs as well as with colony-forming assays.