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1. In theory, PI does not get in to living cells so should not be toxic to
them. In practice, it does get in to living cells, but fairly slowly. It
also binds, I believe, quite reversibly with DNA and therefore can be washed
out of cells quite easily. So living cells will have taken up little PI if
incubation is relatively short and what is inside these cells should be easily
washed out. We have sorted leukocytes in the presence of PI (to gate out the
dead cells). The sorted cells, after washing in fresh medium, seem to
survive and grow. Whether they have been mutagenized by the PI is another
(important) question.....
2. For live/dead discrimination, we prefer to keep the PI concentration low
and the time in PI fairly short (say between 1 minute and 5 minutes) -- in
order to minimize the uptake of PI by living cells. This isn't possible when
sorting -- but you still can get respectable live/dead discrimination after
an hour or more. Just watch the the PI gate -- as the line between live and
dead cells (between PI dim and PI bright cells) will shift with time.
Hope you've won your bet!
Alice Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, NH USA
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