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.
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