Re: Mutant GFP

Rochelle A. Diamond (diamond@cco.caltech.edu)
Tue, 4 Jun 1996 16:57:47 -0700 (PDT)

We have been trying to do GFP (green lantern) in conjuction with PI for
cell cycle staining. It seems that the GFP is killed with the ETOH
fixation. We have tried many other types of fix which are compatible
with DNA analysis and the best so far is the Orthopermafix, although the
CV's are horrible compared to ETOH fix. I haven't tried the BD reagent
for intracellular staining yet though.

Our preliminary results suggest a perturbation of the cell cycle in
transfected cells with GFP, has anyone else seen this?

Any suggestions?

Rochelle Diamond
Caltech Flow Cytometry/Cell Sorting Facility

On Tue, 4 Jun 1996, Ken Hook (313) 996-5746 wrote:

>
> A colleague of mine is trying to transfect cells with a gene that should change
> the cell cycle distribution. She used CD20 cotransfection as a reporter gene
> (with modest separation from non-expressors) but hoped to improve upon that
> with (mutant) GFP. Its expression was disappointing on the FACScan even though
> she could see it under the microscope in unfixed cells. My question is: will
> GFP leak out of cells after we EtOH-fix them for PI staining? If, so, I guess
> I'll have to get the plasma tube on my (too long inactive) HeCd replaced.
>
>