Hi Simon! I have been using several fixation methods with GFP expressing cells. The best we have found is to use a low concentration of paraformaldehyde - between 0.25% and 1% for as little as 15 minutes seems to do the trick. And theres no need to wash this off before running the samples. In general we are using the red-shifted EGFP from Clontech. The fluorescence is maintained after mild denaturation or with aldehyde fixation but fully denatured GFP is not fluorescent, presumably because the chromophore part of the molecule is obscured. So one does need to avoid other things that may denature the molecule eg extremes of temperature and pH. Aldehyde fixation should also prevent leakage from the cells although a lot of my users are also transfecting in a fusion protein to localise the GFP subcellularly. Pre-fixation in paraformaldehyde is also required if one also wants to assess cell cycle status with PI. Without wishing to advertise, but credit where it is due, Clontech have a good web page about GFP at: http://www.clontech.com/clontech/Manuals/GFP/TOC.html. Hope this helps! Derek On Mon, 6 Oct 1997, Simon Monard wrote: > > Hi All > > 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? > > Thanks > > Simon Monard > ADARC > **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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