Ryan: Most, if not all, of your problem is likely to stem from your model. Camptothecin is a topoisomerase I inhibitor. As such, it binds to the enzyme-DNA complex, stabilizing it and preventing religation of the DNA single strand break. This, by itself is necessary but probably not sufficient to induce an apoptotic response. The conventional wisdom is that collision of the DNA polymerase molecule with the stabilized complex during replication causes a double strand break which triggers apoptosis when the number of strand breaks exceed some threshold. Simply stated, you need the cells to be going through DNA synthesis to get camptothecin to cause apoptosis and even then, it will only occur in S phase cells. In fact, if you inhibit DNA synthesis in an actively growing cell line prior to addition of camptothecin you will not see any apoptosis even at the appropriate concentration which, by the way, is in the range of 150 nM. The small and variable amount of apoptosis you seem to be observing is either spontaneous apoptosis which will occur in mononuclear cell cultures over time. If any granulocyte elements remain in your system they will also contribute to this population. You will probably need to trigger apoptosis through cell surface Fas receptors or by using agents which do not require cell proliferation for activity. Hope this helps. Frank Traganos, Ph.D. Cancer Research Institute at NY Medical College >>> Ryan Hung <rhung@vcn.bc.ca> 6/9/97, 02:58pm >>> I've just started using FACS analysis for analyzing apoptosis in PBMC a couple of months ago. I figured that I would first try the inexpensive technique of PI staining. However, to date, having tried both direct staining with PI (with sodium citrate, Triton X-100, RNAase A) and ethanol-fixation prior to PI staining, I have not been able to get either to work reliably. My model system itself consists of PBMC extracted from healthy human donors at a concentration of 3x10^6 cells/ml in 1ml of RPMI growth medium in 24 well plates. I'm using camptothecin in a concentration of 5 ug/ml as my positive control of apoptosis. I incubate for between 14-18h at 37oC in 5% CO2. With direct staining with PI, I've typically obtained two closely-spaced high fluorescent intensity peaks when reading FL3 on a histogram plot. I see virtually no lower fluorescent intensity peaks. With ethanol fixation, I've had problems with gating when using a linear scale on FSC, so I've tried using a log scale. I understand from previous discussion that this may be incorporating significant amounts of cellular debris. What I observe in terms of histograms are a single high-fluorescence peak, with a lower fluorescent peak at about 1/5 the intensity of the high peak, rather than the expected 1/2. Since my results from both techniques don't seem to correspond well with what I've found in literature, I've been finding it difficult to analyze. Any ideas or suggestions would be much appreciated. Ryan. _/ \__/ \__/ \__/ \__/ \__/ \__/ \__/rhung@vcn.bc.ca__/ \__/ \__/ \_Apoptosis=programmed cell death/ \__/ \rwhung@netinfo.ubc.ca \__/ \__ _/ --you can't live without it!/ \__/ \http://www.vcn.bc.ca/people/rhung \__/ \__/ \__/ \__/ \__/ \__/ \__/ \My words Copyright (C) 1997 \__
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