Discrepancies between flow cytometric analysis and [3H]thymidine incorporation in stimulated lymphocytes

The DNA synthesis system of freshly isolated tonsillar lymphocytes and those stimulated by phytohaemagglutinin were compared by different methods. Both cell populations had high DNA polymerase alpha and thymidine kinase activities, as well as a high rate of incorporation of [3H]thymidine into DNA. However, the two cell populations differed when their DNA distributions were compared by flow cytometry. Freshly isolated cells contained many less (6%) cells in S phase than were found in phytohaemagglutinin-stimulated lymphocytes (18%) as detected by flow cytometry. The labelling of different subpopulations of lymphocytes was studied by sorting them electrically with a fluorescence-activated cell sorter. Analysis of the radioactivity of [3H]thymidine pulse-labelled cells, sorted according to their DNA content, showed that cells in the G1 peak of DNA distribution had a significant amount of incorporated [3H]thymidine. Sorting of cells according to their size (i.e., by light scattering) revealed that only large cells were labelled with [3H]thymidine.