Multiparameter flow cytometric analysis of lymphocyte subpopulation activation in lectin-stimulated cultures.

Lymphocyte activation is initially associated with changes in membrane permeability followed by DNA synthesis and ceil division. The use of 1 µM Hoechst 33342 (HO342) to detect membrane permeability changes or 10 µM HO342 to estimate DNA content in combination with immunofluorescent staining (W3/25 monoclonal for the rat inducer subset; OX8 monoclonal for the rat suppressor/cytotoxic subset) allowed flow cytometric analysis of lymphocyte subset activation and proliferation kinetics after lectin stimulation. Con A (10 µg/ml) was found to increase membrane permeability to 1 µM HO342 in both inducer and cytotoxic/suppressor subsets at 24 hr, with a greater net increase in membrane permeability in the inducer population. Con A stimulation resulted in proliferation that was most evident in the inducer population at 24 hr, followed by a burst of proliferation by the suppressor/cytotoxic subset such that equal percentages of the inducer and cytotoxic/suppressor subsets were found in the S/G2/M phases of the cell cycle at 48, 72, and 96 hr. In general, Con A stimulation resulted in an increase in suppressor/cytotoxic cell numbers, which led to a decrease in the inducer cellxytotoxic/suppressor cell ratio. The majority of PHA-induced proliferation was found to occur in the inducer population at 48 and 72 hr, and was accompanied by an increase in the inducer:cytotoxic/suppressor cell ratio. This technology provides a rapid and effective means for dissecting the temporal sequence of the activation of individual cells within lymphocyte subsets in response to lectins, and should prove equally valuable in elucidating the sequence of lymphocyte subset activation by alloantigen.