Proliferative assays for the assessment of radiosensitivity of tumor cell lines using 96‐well microcultures

We have compared the effectiveness of three short‐term proliferative assays [3(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction, methylene blue staining, and [ 3 H]‐thymidine incorporation] in predicting the results of clonogenic assay of radiosensitivity in five mammalian cell lines (SCCVII, AA8, V3, FME, and MM‐96). Cells were cultured in 96‐well microculture plates and a lead wedge was employed to provide a range of radiation doses ( 60 Co source) up to 10 Gy. Radiation dosimetry was determined by a novel modification of the Fricke/thiocyanate dosimetry technique to allow direct determination in 96‐well plates. Cells were cultured following irradiation to determine clonogenic survival curves and to calculate the survival at 2 Gy (SF 2 ), the initial slope of the fitted linear quadratic function (α), and the mean inactivation dose (D). A broad range of radiosensitivity was obtained (SF 2 = 0.14–0.92) with the clonogenic assay. All three proliferation assays provided measures of radiosensitivity which correlated with clonogenicity at low radiation dose. The thymidine incorporation assay, which provided excellent linear correlation (r = 0.98) with clonogenicity for SF 2 and D, used semiautomated methods for harvesting and scintillation counting to give sample processing times comparable to the colorimetric methods. It offered the advantages of superior assay linearity and dynamic range, and reduced interference by non‐proliferating cells at high radiation dose.