Mitogenic activation and proliferation of mouse thymocytes: Comparison between isotope incorporation and flow-microfluorometry

Staining with acridine orange and flow microfluorometry was used to measure the distribution over the cell cycle of Concanavalin A (ConA)-stimulated mouse thymocytes. These data were compared with data on isotope incorporation. Mitogen-induced [ 14C]thymidine incorporation into DNA increased 10–20-fold upon addition of 2-mercaptoethanol or spleen-derived glass adherent cells. However, the proportion of proliferating cells as measured by flow microfluorometry increased only by a factor of two. Removal of nylonwool adherent cells nearly abolished ConA-induced thymidine incorporation; this was restored by the addition of glass-adherent cells. The fraction of mitogen-activated cells as measured by flow microfluorometry was hardly affected by nylonwool filtration and addition of mercaptoethanol or glass adherent cells enhanced the proportion of activated cells that can enter DNA synthesis. From these and similar experiments it is concluded that DNA synthesis measured as thymidine incorporation is only proportional to the number of mitogen-activated cells under restricted conditions. Differences in thymidine incorporation in different lymphocyte populations or measured under different conditions are difficult to interpret in terms of number of proliferating cells. The cytofluorographic method is a simple and rapid way to solve these problems quantitatively and rapidly.