Re: PI and GFP

Wayne Green (Wayne.Green@genetics.utah.edu)
Tue, 25 Feb 1997 09:03:42 -0700

On Feb 24, 10:34am, Wendy D. Schober wrote:
> Subject: PI and GFP
>
> We are attempting to optimize the procedure to label transfection along
> with cell cycle in HeLa cells. So far we have co-transfected with CD20
> and then labeled the surface with CD20-FITC. We found 5-13% CD20
> positive with good PI patterns after the usual 70% ethanol fixation.
> We used the same cells and fixation procedure with GFP as the reporter and
> hoped to find similar results. Unfortunately, there were few cells GFP
> positive and what may be there are of very low intensity, barely over
> background. We are looking for suggestions for improving our GFP and keeping
> good PI patterns. The GFP is from Invitrogen and they have been somewhat
> helpful, but have not done this with PI.
> Is fixation causing a problem?? If so, is there a better procedure which
> will permeabilize and fix to get both GFP and PI to work?? Ultimately, we
> want this to work in some fibroblast lines, but HeLa is our control.

Wendy:
Yes, fixation is a likely problem. GFP is small enough that appears to
"leak out" of cells permeablized by alcohol fixation. We have used a brief (no
more than 10 min) 1% paraformaldehyde pre-fix at 4 C to cross-link proteins (and
keep GFP inside) followed by a wash in PBS and then addition of EtOH (or
actually MtOH in our case) per your usual procedure.
All the caveats apply for possible detrimental effects of
paraformaldehyde on the quality of DNA histograms that have been published for
dual staining of intracellular antigens and DNA. For a good review of these see
the chapter by Clevenger and Shankey (pp157-176) in "Clinical Flow Cytometry:
Principles and Applications" published by Williams and Wilkins, 1993.
Good luck!

-- 


Wayne Green
wgreen@genetics.utah.edu

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