Proliferative kinetics of human lymphocytes in culture measured by autoradiography and sister chromatid differential staining

A simple combination of autoradiography, to determine when a cell synthesized DNA, and sister chromatid differential staining, to determine how many times a cell has divided, was used to follow up the proliferating fate of human lymphocytes in culture. Cells were incubated continuously with 5-bromodeoxyuridine (BrdU) and pulse-labelled with 0.1 muCi/ml [3H]thymidine at various times after stimulation with phytohemagglutinin (PHA). The cells were then harvested at 4 h intervals up to 72 h, and the percentage of labelled mitoses was determined separately in first, second, or third division cells. The data showed that the cycling cells, whether they began cycling at earlier or later times after stimulation, had about the same generation times of 12--14 h. This confirms that the heterogeneity of cell generations seen in short-term lymphocyte cultures is in large part due to the difference in the times when cells began cell cycling in response to PHA.