Effect of mitogenic and high, nonmitogenic concentrations of phytohemagglutinin and concanavalin A on the number of human lymphocytes in culture.

A number of different investigators have observed a significant decrease in the number of lymphocytes in culture within 24 to 48 hr after the addition of mitogenic concentrations of phytohemagglutinin (PHA) and concanavalin A (Con A). In order to investigate the mechanism of this apparent decrease in cell number, we compared the number of cells in culture, determined by the pronase-cetrimide technique, to the amount of cell DNA and cell protein. Although cell number appeared to decrease following the addition of PHA or Con A in a dose-related fashion, cell DNA and cell protein did not change. When lymphocytes that were incubated with Con A were treated with alpha-methyl-D-mannoside prior to counting, a decrease in cell number or viability was not observed and there was close agreement between the number of cells and amount of cell DNA in culture. We conclude that the number of lymphocytes in culture does not decrease during the first 24 hr after the addition of mitogenic concentrations of PHA and Con A, and that previous reports to this effect may be accounted for by problems associated with counting lymphocytes that are agglutinated by mitogens.