Receptor mediated activation of many cell types is followed by motility related events. In T lymphocytes, lateral redistribution of surface receptors is accompanied by aggregation of actin and myosin in cytoplasmic subcaps and both are impaired in T cells from older individuals. Age- related changes both in basal levels of filamentous actin and in further polymerization of actin after activation of resting T cells from C57BL/6 mice with Concanavalin A were previously documented. Because of differences between the CD4 and CD8 positive subpopulations of T cells and technical problems using flow cytometry to document receptor mediated actin polymerization in mixed populations of the cells, a new procedure was developed to separate the subpopulations. Resting lymphocytes were isolated using discontinuous Percoll gradients. Superparamagnetic monodisperse polystyrene coated particles (Dynabeads) were coated with specific monoclonal antibodies to mouse T lymphocyte surface antigens. Coated beads were less efficient than T-cell recovery columns in removing B lymphocytes. Resting T cells were then incubated with CD4 or CD8 antibodies and coated beads using direct or indirect techniques. Negative selection of CD4 and CD8 positive cells in a magnetic field produced cells that were 98-100% viable, and contamination by the eliminated cell types was < 1.0%. These purified subpopulations from aged mice can now be used to characterize actin function, actin gene expression and kinetics of polymerization.
