Identification of mitogen responding lymphocytes by fluorescence polarization

Measurements of the intensity and polarization distributions of fluorescein fluorescence in human lymphocyte populations show changes within minutes after exposure of the cells to the mitogens phytohemagglutinin and Concanavalin A. The distributions of polarization, which before mitogen exposure show essentially a single peak, after exposure develop a second peak at a lower value of polarization. Simultaneously, the intensity distributions show a shift to higher levels of intensity. These shifts can be modeled on the basis of a subpopulation of lymphocytes responding to mitogen. Interpretation of the results is complicated by many factors that may influence the shape of the polarization distributions. In particular we show that the measurements are sensitive to the concentrations of phytohemagglutinin, Concanavalin A, Ca++, K+, fluorescein diacetate, the incubation time and the specific donor. Nevertheless, fluorescein fluorescence measurements provide a rapid and sensitive method for studying the physiology of lymphocytes and for identifying responding lymphocyte subpopulations.