Enumeration of thioguanine-resistant lymphocytes using autoradiography

Strauss and Albertini (1979) proposed a method whereby the frequency of thioguanine-resistant lymphocytes could be measured by an autoradiographic technique and suggested that it would provide a measure of in vivo mutation frequency. A number of variables influencing the method were studied and concentration of thioguanine and duration of assay found to be particularly important. The modified method finally arrived at involved culture of PHA-stimulated lymphocytes for 64 h in the presence of 40 microgram/ml of thioguanine, lysis of cells with Nonidet and collection of nuclei on Nucleopore membranes. The frequency of TG-resistant cells was 1.5 x 10(-6) - 1.7 x 10(-5) in young adults and it showed an increase with age. The technique was also adapted for use with continuously cultured lymphocytes, by using a concentration of 60 microgram/ml of thioguanine and an incubation time of 72 h. The frequency of TG-resistant continuously cultured cells was less than 10(-6) - 2 x 10(-5). Consideration of the effect of different variables on the assay suggested that the above estimates for TG-resistant lymphocytes were more valid than the previously published substantially higher estimates.