A sensitive assay for 6-thioguanine-resistant lymphocytes

We have defined variants (V) resistant to 6-thioguanine (TG) by their ability to divide at least once during a 72-h incubation in a medium containing 0.2 mM TG. We blocked cytokinesis by adding cytochalasin B (CB) after 30 h in culture. The cells that had undergone nuclear division were identified by their content of 2 or more nuclei. The long incubation period allowed slow growing V to be counted. As a result we scored an order of magnitude more V than have been reported in assays using the conventional 30-40 h culture times. In the gamma-ray dose range of 0-0.5 Gy we scored 80 V per 1000 surviving lymphocytes per Gy--a result some two orders of magnitude larger than has been reported previously.