Tritiated-thymidine uptake in mixed leucocyte cultures: effect of specific activity and exposure time

Abstract

The degree of in vitro transformation of lymphocytes to blast cells can be estimated by measuring the uptake of radioactive precursors into DNA. We have used ³H-thymidine uptake to quantitate blastogenesis in mixed leucocyte cultures. In experiments designed to standardize this procedure, the kinetics of thymidine uptake were studied by adding 4 μCi of ³H-thymidine of varying specific activities to 5-day mixed cultures. With high specific activity (0·194 μg total thymidine/culture), the rate of uptake was constant for only about 6 hr, then declined. There was no further cellular uptake between 8 and 24 hr, even though the total radioactivity of the supernatant medium did not diminish appreciably. This decreasing rate of uptake was at least partly the result of thymidine degradation to thymine and dihydrothymine by cellular enzymes. It is also possible that eventual radiation damage to blast cell nuclei may have retarded DNA synthesis when ³H-thymidine specific activity was high. With decreasing specific activity (up to 19·4 μg thymidine/culture), the rate of uptake became more nearly linear throughout 24 hr exposure to the isotope. The possible effects of thymidine degradation and radiation damage should be considered when measuring radioactive thymidine uptake in vitro, and short labelling times should be used whenever feasible.