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.

Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry.

The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT-4 and MDA-MD-175-VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice-cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4 degrees C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT-4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25-1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed < 5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation.

Expression of proliferation-associated antigens (PCNA, p120, p145) during the reentry of G0 cells into the cell cycle.

Flow cytometric bivariate analysis was used to evaluate the expression of PCNA, p120, and p145 during the G0 reentry of CHO-K1 cells into the cell cycle. CHO-K1 cells were placed in a G0-like state using serum depletion and stimulated to reenter the cell cycle by replating into fresh, serum-containing medium. At discrete intervals after stimulation, replicate samples were stained for either PCNA, p120, or 145; stained for DNA (Coulter DNA-Prep); evaluated on the EPICS Profile I; and analyzed on the EPICS ELITE workstation. PCNA stained less than 10% of the G0 cells; in contrast, however, 30-35% of the G0 cells were positive for p120 and p145. Eight hours after stimulating G0 cells to reenter the cell cycle (during G0/G1), p120 reached 88% positivity, while p145 and PCNA were 63% and 30% positive, respectively. Cells in S phase (12 and 16 h following G0 stimulation) were greater than 90% positive for all three antigens. PCNA had the greatest change throughout the G0 reentry process, both in percentage positive and quantitatively (mean channel fluorescence). This report indicates that all three proliferation-associated antigens studied are differentially expressed during the reentry of G0 cells into the cell cycle. Furthermore, these antigens may be useful in the early detection of G0 recruitment.

Expression of proliferation associated antigens in the cell cycle of synchronized mammalian cells.

Flow cytometric bivariate analysis was used to investigate the expression of PCNA, p120 and p145 during the cell cycle of a mammalian cell line (CHO-K1). Initially, aliquots of cells in exponential and plateau (G0) phase were analyzed for proliferation associated antigen expression. Expression of PCNA and p145 during G0 was markedly depressed (less than 12% positive) while 54% of the G0 cells stained positive for p120. The fluorescent intensity (mean channel fluorescence) of these G0 positive p120 cells, however, was only slightly above the mean channel fluorescence (MCF) of cells stained with a negative isotype control. In asynchronous cultures, all three antigens were expressed in greater than 70% of the cells, with PCNA staining being greater than 95%. Cells were then synchronized using mitotic selection (mitotic index of 97%) and antigen levels were measured as cells progressed synchronously through the cell cycle. From DNA analysis histograms, it appeared that the degree of synchrony was approximately 90% throughout the remainder of the cell cycle. The bivariate DNA/PCNA, DNA/p120, and DNA/p145 histograms for mitotic cells indicated that both p120 and p145 expression were elevated (percent positive and MCF) while PCNA levels were near controls (MCF). In early G1, all three markers were depressed (less than 12% positive); however PCNA levels rose precipitously in mid-G1 (greater than 50% positive). In late G1 to early S, p145 levels increased concomitantly with increases in p120. All three antigens were elevated throughout S phase and began to decline as cells moved from G2/M to G1 of the next cell cycle with p145 expression decreasing first. This report indicates that all three proliferation associated antigens studied are differentially expressed in the cell cycle and therefore may be useful in detecting and assessing the proliferation state.