The trace and subgrouping of lymphocyte activation by dynamic fluorescence intensity and polarization measurements

Cell activation involves conformational changes of cytosolic enzymes, and/or their regulatory proteins, as well as intracellular matrix re-organization. In this work, these changes were monitored by dynamic measurements of fluorescence polarization in single cells incubated with or without phytohaemagglutinin (PHA), using the Cellscan mark S (CS-S) cytometer. This instrument and the procedure used proved to be a powerful tool for distinguishing subpopulations of cells. Grouping of cells by their staining rates (the time rate of change of the fluorescence intensity) yielded three major subgroups. For each subgroup, the fluorescence depolarization (FDP) induced by the incubation with PHA was measured. The kinetics of the subgroups indicate that the major FDP is contributed by the cells with the lowest staining rate. This FDP is approximately 1.5 times greater than that of a bulk population. It is believed that the analysis of kinetic probing might yield an important and more sensitive method for functional marking of subgroups of cells by their response characteristics.