GFP and cell cycle
Ellen Cahir McFarland
ecahir at rics.bwh.harvard.edu
Tue Oct 3 16:54:35 EST 2000
I frequently do cell cycle analysis on GFP transfected cells. GFP will leak
out of the cells with ethanol perm. but if you treat the cells with
paraformaldehyde first and then perm with ethanol or .1% tx-100 and then add
the PI. I have never had a problem with the PI bleeding into the GFP but the
GFP does bleed into the Red channel alot so you do have to compensate, but I
do it like I would for any red green combinantion.
A couple of tricks.
Do not gate on FSC vs SSC but gate on FL2-A vs. FL2-W. If they are using a
cell line it can have a lot of cells with more than 4N DNA or if they are
sticky you get a lot with 2N DNA stuck together. Then compensate with FL2A
Even with paraformaldehyde fix, GFP still leaks so you should run the
samples ASAP after preparation. No matter what I have done, apoptotic cells
are dim for GFP.
Clontech sells a plasmid that encodes a GFP that is farnysylated (added the
signals from Ras)Someone on the list mentioned earlier. It is resistant to
ethanol fixing and would be greatly advantages for this application bc then
you can just ethanol fix as usual. I am going to try it soon in my expt.
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