Mark Kukuruga wrote-
"The easiest -- assuming one has multi-lasers and a UV source -- is to
combine Hoechst
33342 DNA staining in live cells with GFP expression measurements. This is
a fairly
straightforward two laser application, well documented in methods literature.
I've also seen discussions where aldehyde fixed cells are permeabilized
with ethanol
for labeling with PI (or 7-AAD). We've done some of this, and it seems to
work.
I would suspect that the trick will be in the nature of the GFP expression, and
differential sensitivity to permeabilization."
-this was in response to Tim Kute's query-
"A fellow investigator has asked if the expression of FGP
(fluorescent green protein) is cell cycle related? Are there any flow
people that have tried to answer this question. If so, what was the
results and how did you do it by flow?"
The Hoechst/GFP methodology Mark suggests is less susceptible to artifact
than a PI/GFP technique would be, but, as Mark noted, "the trick will be in
the nature of the GFP expression". When GFP is used as a reporter gene,
its pattern of expression should be the same as the pattern of expression
of the protein with which it is associated. If the expression of this
protein varies with cell cycle, so will GFP expression; if not, one would
not expect to see cell cycle variation. Or is Tim's fellow investigator
asking whether GFP cloned into cells by itself (perhaps as a control) will
vary in expression with the cell cycle?
-Howard
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