fix and permeabilize Cells

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Tue Jul 10 07:36:51 EST 2007


On Mon, 9 Jul 2007, aakash wrote:

> I would be glad if any of you can share your experience about how to fix
> and permeabilize GFP expressing cells without quenching/killing the GFP
> for cell cycle analysis using PI. GFP is not fusion protein. I used
> paraformaldehyde and ethonol, but i loose complete gfp expression.

You don't say what analysis method this is for; i'm guessing flow
cytometry.

I don't do that myself, but i do do fluorescence microscopy with GFP. We
fix in 4% methanol-free formaldehyde in a fancy buffer; it should be
around pH 6.8, but a quick measurement puts my tube at about pH 5.0. It
still seems to work okay! PBS at pH 7.4 is basically also fine, so i guess
pH is not absolutely crucial at this step. We fix for 10-30 min. We
permeabilise with 0.5% Triton X-100 in PBS, for 10 min.

Anyway, GFP fluorescence seems fine in those conditions.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk


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