Hello, everyone: I just ran an experiment (flow cytometry, but would expect the same results by confocal) staining T47D breast Ca cells with DiOC6(3) and PI to detect apoptosis. We had Jurkat cells stimulated with camptothecin for controls. Also, m-chlorophenylhydrazone (CCCP) was used to trigger loss of mitochondrial membrane potential in some of the controls. The Jurkat cell results were exactly as expected. The results of the T47D cells, on the other hand, were very difficult to interpret. For those unfamiliar with this assay, the unperturbed cells fall into the lower right-hand quadrant when DiOC is plotted on the x-axis and PI is plotted on the Y-axis. It should also be noted that the T47D cells grow attached to the flask and were trypsinized ( and washed 2 times) treatment with CCCP or staining with DiOC and PI. The results for the T47D cells were as follows. I gated on the viable cell population based on fwd vs. side scatter. 1. Untreated cells stained with both DiOC and PI were a little more fluorescent for both stains than were Jurkat cells, so I adjusted the lines that define the quadrants accordingly. 93% of the untreated cells were in the lower rh quadrant. 2. Treatment with CCCP, which should have moved the cells as a whole across the line into the lower left hand quadrant, instead resulted in a second DiOC peak forming to the right of the original (-) peak, both still located for the most part in the lower rh quadrant. 3. An attempt to induce apoptosis by depriving the cells of serum for 2 days resulted in the cells (DiOC/PI) being located in the lower rh quadrant with a few cells leaking over into adjacent quadrants. But, for the most part, the cells remained located in the lower rh quadrant, but divided between 2 peaks. 4. Starvation plus CCCP not only resulted in a minor shift half-way across the vertical quadrant line to the left, but also a shift completlely across the horizontal quadrant line into the PI (+) space. Remember that these cells were gated on the live cell population by scatter. Changing the scatter gate to include only the "dead" cells resulted in a DiOC/PI quad plot that was virtually indistinguishable from that generated through the "live cell" gate. This pattern was consistent for a duplicate set of samples. Do any of you have any experience with this assay and/or with this cell line? Any explanation of the results? Thanks in advance. Rick Meister * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Richard K. Meister Email: meister.1@osu.edu * * The Ohio State University Voice: (614) 292-9716 * * Dept. of Veterinary Biosciences FAX: (614) 292-6473 * * Cytometry Instrumentation Lab * * 1925 Coffey Road * * Columbus, OH 43210 U.S.A. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
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