> >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
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