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 monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested 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 used prior to staining) and processed (permeabilized prior to staining) cells. Light-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 microscopic examination of Trypan blue-stained cells and flow cytometric detection 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 DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V 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 nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.
Koester
S K
SK
Department of Scientific Research, Coulter Technology Center, Miami, Florida 33196, USA.
Roth
P
P
Mikulka
W R
WR
Schlossman
S F
SF
Zhang
C
C
Bolton
W E
WE
D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000276:immunology*; D008928:Mitochondria / Q000276:immunology*; D019014:fas Receptor / Q000494:pharmacology
D017304:Annexin A5; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D004273:DNA, Neoplasm; D008565:Membrane Proteins; D019014:fas Receptor