Re: GFP, Fixation?

David Chambers (davidc@flosun.salk.edu)
Mon, 06 Oct 1997 19:06:05 -0700

>
>Simple question: Does formaldehyde fixation affect GFP fluorescence? I've
>seen some protocols involving fixing in 4% formaldehyde for an hour or more
>then washing cells in PBS before running on the cytometer, surely
>unnessesary? Can GFP leak out of fixed cells?
>

Complicated answer 8^> : (para)formaldehyde fixation reduces GFP fluorescence
a little, and unless the cells are hazardous or the experiment is a long
one, I
don't see any reason to fix before running them.

I doubt that GFP will leak out of HCHO fixed cells; my understanding is
that there
is a great deal of cross-linking. On the other hand, ethanol fixation causes
immediate loss of GFP. Which brings me to ask: Does anyone have a good
protocol
for doing cell cycle with PI on paraformaldehyde fixed cells? One that
produces
halfway decent CVs?

-- David