I am looking at activation induced apoptosis in peripheral T
lymphocytes following various pre- or co- treatments of freshly
isolated human PBMC (Ficoll-Hypaque preparation). When I evaluate
apoptosis by gel electrophoresis of fragmented DNA, I can see a
positive effect 95% of the time in a dose dependent manner. However,
I need a method with which I can positively ID which cells from the
PBMC culture are undergoing apoptosis. Recently I switched to a flow
cytometric method using ethidium bromide and CD2-FITC to evaluate
apoptosis within the T cell population. Of the 7 times I have
repeated this experiment, 5 have showed no apoptosis in any of the
treatments at any concentration or level of T cell activation. In
contrast, the 2 other times have shown apoptosis of 5% in controls and
up to 95% in experimentals, with a dose dependent curve for both level
of activation and concentration of the cotreatment variable.
Is anyone aware of minor variations in either the method or culture
conditions which could lead to this apparent discrepancy? It bothers
me that the flow method is giving such an "all or nothing" evaluation
of apoptosis.
One possibility concerns the fact that one method measures DNA
fragmentation, and the other measures loss of membrane integrity. Are
there culture conditions or biochemical processes required for
inducing membrane damage which are independent of apoptotic mechanisms
for DNA fragmentation?
Any other possibilities?
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Dean Lee * We dance 'round in a ring *
Dept of Micro and Mol Genetics * and suppose, *
Loma Linda University * But the secret sits in the middle *
dLee at ccmail.llu.edu * and knows. R. Frost *
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