On Tue, 8 Aug 2000, William Ross wrote: > what's a good dye to use for simultaneous DNA and GFP detection when your > UV laser has taken an extended vacation? cardiac smooth muscle cells. Hi Bill, Do you need to keep the cells live? If not, you may be able to use a sequential paraformaldehyde and ethanol fixation to retain sufficient GFP expression and still get a reasonable DNA profile. Trial and error experiments with the strength and time of the PF fix will be needed. It will also depend on where the GFP localises and whether you are using co-transfection or a fusion protein. Clontech markets a farnesylated GFP (EGFP-F) which localises in the membrane and is resistant to ethanol fixation so you can use a regular ethanol fix and PI stain. If your GFP fluorescence is destroyed by fixation, you could try permeabilisation (saponin probably best) and then PI, 7AAD or TO-PRO-3. The only 488nm excitable vital DNA quantitation dye that I am aware of, as others have said, is DRAQ5 developed by Paul Smiths group (there are a couple of papers in recent issues of Cytometry). Good luck! Derek ************************************************************************ 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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