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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