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My response to Denis Snider's request on obtaining RNA from sorted G2 phase
cells may have been partially misunderstood.
I did NOT mean to say that you should not AT ALL attempt to synchronize your
cells. We had good results by keeping cells (fibroblasts) in low serum for
some time. After replating (and addition of normal serum levels) we
obtained a fairly synchonous wave of cells going through the S-phase.
Synchronous behavior was gradually lost during transit through the cell
cycle.
We had bad experience, however, when we tried to synchronize cells in the G2
phase directly by drug treatment. It turned out that some cells underwent
chromatin endore-duplication. That means, we found cells in the G1 phase
with tetraploid DNA content (looking like G2) and "true" G2 phase cells.
This seems to occur with all G2 blockage protocols we have used thus far.
In 1994 or 1995 Zbigniew Darzynkiewicz published a paper on how one could
distinguish endoreduplicated G1 cells from true G2 cells on the basis of
their differences in cyclin expression. He could also fill you in on his
experience with various techniques of cell synchronization.
SYTOX Green resolves cells in the G1, S, and G2 phase of the cell cycle. It
does so better than PI. We have not yet found a way of distinguishing late
G2 phase cells from early prophase. Their DNA content is similar; only in
metaphase the nucleus breaks up and individual chromosomes will be obtained
(as sub-G1 particles). SYTOX Green works best with the methanol based
fixation procedure I have outlined.
I hope that this may help you.
Martin Poot
martin@probes.mhs.compuserve.com
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