IDENTIFICATION AND QUANTITATION OF APOPTOTIC CELLS FOLLOWING ANTI-CD3ACTIVATION OF MURINE G(0) T-CELLS

Multiparameter flow cytometry and cell sorting were used to examine th e process of apoptosis after activation of murine resting T cells with immobilized anti-CD3. Activated T cells treated with Hoechst 33342 (H O-33342) and analyzed by flow cytometry showed two major cell populati ons of high and low fluorescence. These populations were sorted and th e DNA extracted and subjected to electrophoresis. Electrophoresis of D NA extracted from T cells showing a low level of HO-33342 fluorescence (HO-Low) resulted in a typical ladder pattern characteristic of inter nucleosomal DNA degradation associated with apoptosis, whereas the cel lular DNA of the cells showing a high level of fluorescence (HO-High) showed a narrow high molecular weight band. Multiparameter analysis fu rther indicated that cells with HO-High characteristics possessed corr esponding high-FSC/low-SSC properties, whereas HO-Low cells formed a c luster of low-FSC/high-SSC cells. Analysis of the DNA extracted from c ells sorted on the basis of scatter properties alone confirmed that th e low-FSC/high-SSC population contained the apoptotic cells and that t he high-FSC/low-SSC population was comprised of viable cells. This met hodology allowed us to determine the percentage of apoptotic cells fol lowing anti-CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8 + cells in the cell cycle. (C) 1993 Wiley-Liss, Inc.