Hi Candace, Ethanol fixation generally reduces GFP fluorescence and the way round this (other than using Hoechst for DNA which you cant do!) is to fix initially in paraformaldehye which will cross-link the protein and preserve a large amount of the GFP fluorescence. DNA profiles following aldehyde fixation alone arent generally that good - CVs are increased, accessibility is reduced and cell cycle analysis isnt as accurate. You can _to an extent_ circumvent that by having a sequential fixation in paraformaldehyde and then ethanol or even permeabilistaion with saponin. The times concentrations etc need to be empirically determined. Low GFP signals can be a problem as any fixation may reduce them. We never really see a problem with PI overwhelming a GFP signal, but if you have a two laser Calibur you can always try TO-PRO-3 as your DNA stain. I think there are several protocosl out there for 7AAD DNA analysis, I think Ingrid Schmid has done quite a bit of this so a literature serach should pull something out. Good luck! Derek On Mon, 2 Oct 2000, Candace Enockson wrote: > I have a client who wants to do cell cylce on GFP transfected cells. Since > I do not have a sorter, I need to be able to do this on a Calibur. She has > found some comments that claim you can do DNA with a formaldehyde fix > rather than ethanol. Is that true? Also, I read on this list a while back > that PI may be so bright that it may overwhelm low GFP signal so 7AAD would > be the better choice. I have not been able to find a protocol for cell > cycle with 7AAD. Any help would be greatly appreciated, as always. ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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