From: Derek Davies (daviesd2@icrf.icnet.uk)
Date: Tue Feb 10 1998 - 03:10:00 EST
Hi Alice and Anne, On 5 Feb 1998, Alice L. Givan wrote: > We have GFP-fusion-protein-transfected cells and, in the first instance, need > to fix in formaldehyde (not ethanol) to retain the GFP but want to get good > DNA profiles. Is there a stain that works better than PI with formaldehyde- > fixed cells or is there a method for opening up the fixed histones/DNA > so as to improve the PI profile without losing the GFP? I think that this is an inceasing problem with more people using GFP as a marker of transfection and then wanting to assess cell cycle status. As you point out, there is a need for an aldehyde fixation as ethanol tends to abolish GFP staining, but aldehyde fixation gives sub-optimal DNA histograms. The best way I have found round this is to fix briefly (10 mins) in 0.5% paraformaldehyde, and then post-fix as usual (for DNA staining) in 70% ethanol. This gives a compromise between retaining the GFP fluorescence and giving a reasonable DNA profile. You may need to alter the fixation times / strength of fixative to suit your particular cells though. I would still recommend PI as the DNA stain though. An alternative would be to sort GFP+ and GFP- then fix and do a routine PI staining procedure. 50,000 sorted cells should be sufficient to get a statistically fulfilling DNA histogram. Very dull if you have lots of samples though. > In the second instance, we would like to keep the cells alive during this > procedure. Is there a staining method with one of the Hoechst dyes that will > give us histograms sharp enough to allow cell cycle analysis or is there any > better vital alternative? As far as I am aware (and I stand to be corrected!) only the Hoechst dyes can be used to stain the DNA of live cells for cell cycle studies - other dyes such as LDS751 stain DNA but not stoichiometrically. Most workers would use Hoechst 33342. At the right dye concentration, this does give histograms good enough for cell cycle analysis. The dye concentration and the length of staining need to be determined empirically for each cell type as some cell types work hard to pump it out. In my experience the range of dye concentration needed is in the order of 5-20ug/ml for at least 20 minutes (at 37C). And of course you will need a UV light source for excitation. Very nice when it works though, and you can combine with PE/3rd colour for further phenotypic definition. Hope this is of some help! Derek **************************************************************************** * 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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