Laura,
> When looking at propidium iodide uptake for cell viability, do you typically
> see a large shift in fluorescence in the FL-2 channel? It has been my
> experience that non-viable cells are very bright (3 log shifts over auto),
> with a characteristic change in scatter properties. Someone recently showed
> me data where they claim that a slight shift in fluorescence (< 1 log) of all
> the cells indicates 100% cell death. I find this hard to believe, since the
> experiment consisted only of cells + antibody incubated @ 4oC. If this is not
> cell death, then what can account for the slight uptake of PI?
I agree; 100% dead cells should mostly be very bright provided the PI
concentration is right and the PI has not deteriorated.
Alternative explanation: If the cells were incubated in standard
culture medium they may have taken up something fluorescent other than
PI. Cultured cells often do have a higher fluorescence background
(which we often erroneously refer to as "autofluorescence"). To test
this, you may get some clue from the colour of the fluorescence. If
you are detecting the fluorescence in FL2, look at FL1 vs FL2 or FL3
vs FL2 for these cells and compare with that of *genuinely* PI +ve
cells.
Frank.
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