Analysis of early lymphocyte activation events by fluorescence polarization flow cytometry

After short-term (up to 4 h) stimulation with mitogen or antigen, lymphocytes were incubated with fluorescein diacetate and the polarization of fluorescence from intracellular fluorescein was measured on a specially adapted FACS II. This flow cytofluorimetric method to assay early changes in activated lymphocytes gave a reproducible response to the mitogens phytohaemagglutinin (PHA), concanavalin-A and the monoclonal antibody OKT3, recognized at 1 h by decreased polarization. A response by immune spleen cells to the antigen dinitrophenyl-ovalbumin was revealed at 4 h. The calcium ionophore A23187 induced an increase in polarization after only 10 min. The PHA polarization response was shown to be dependent on PHA binding, PHA dose, T cells, calcium ions and an intact cytoskeleton. The cellular events monitored by the polarization change are presumably altered fluidity of the probe's microenvironment due to conformational change in macromolecules to which the probe has bound or to dissociation of the probe into the aqueous phase. The fluorescein fluorescence polarization assay is a reliable and sensitive monitor of early lymphocyte activation events and, coupled with the use of a flow cytometer, permits study of particular subpopulations of responding cells.