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 vs FL1. 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. Ellen
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:38 EST