MONITORING EARLY CELLULAR-RESPONSES IN APOPTOSIS IS AIDED BY THE MITOCHONDRIAL-MEMBRANE PROTEIN-SPECIFIC MONOCLONAL-ANTIBODY APO2.7

A recently described mitochondrial membrane protein-specific monoclona l antibody, APO2.7, was examined for monitoring early apoptotic respon ses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harves ted at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent us ed prior to staining) and processed (permeabilized prior to staining) cells, right-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light micro scopic examination of Trypan blue-stained cells and flow cytometric de tection of tubulin, showed little change until after 6 h, In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DN A fragmentation was demonstrated by flow cytometry and gel, electropho resis; however processed tells stained with APO2.7 antibody showed sig nificant increase in staining after 1.5 h. Detection, using annexin ti and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but ne arly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis, The data presented here in indicate that the mitochondria. membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells. ( C) 1997 Wiley-Liss, Inc.