> >To: derek.davies@icrf.icnet.uk >From: Thomas Delohery <t-delohery@ski.mskcc.org> >Subject: Re: DNA: Double wides but single brights >Cc: >Bcc: >X-Attachments: > >We routinely look at FSC vs FL2A (integral fluor) as well as FL2A vs >FL2W. The "double wide" G1 cells are better resolved with FSC than >width. This occurs in primary epithelial cells in response to agents >that induce differentiation; eg, TGF-b. Curiously, the smaller cycling >cells can be fully resolved from the huge G1s with nuclei prepared via >the Nusse protocol but when intact cells fixed with EtOH are used, we >see one broad distribution on both FSC and FL2A. I speculate that it >has to do with cytokeratin production during differentiation. >td > > >>Hi Ray, >> >>You dont say what type of cells you are looking at. If they are large >>epithelial-like cells this could be a bit of non-specific cytplasmic >>uptake but that is unlikely to be confined to the G1 population. Or do you >>see a general width increase as well? >> >>Alternatively it could be FACSRubbish. Are you triggering on FSC or FL2? >>If you trigger on FSC and look at the AvW plot does that wide 'G1" >>population look as if it is heading off towards the origin? I have seen >>this sort of thing when there is a lot of debris or alternatively when the >>culture from which the cells are taken has been contaminated. >> >>In either case, it would probably be productive to have a look down the >>microscope and see if you can determine what could be causing it. >> >>Hope this helps etc >> >>Derek >> >> >>Ray Hetser wrote: >>> Several times over the last few months when analyzing cells that have >>> been stained with propidium iodide we have seen, using pulse processing, >>> cells that have an FL2W signal that would seem to qualify them as >>> doublets and yet their FL2A is similar to that of a singlet. E.g., in >>> the example below, if xxx represent G0/G1 single cells, yyy are G2/M >>> single cells, and zzz doublets, what are the cells represented by ???? ? > > -- ============================================================================== Thomas M. Delohery |Internet: t-delohery@ski.mskcc.org Supervisor, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (917) 432-2333 1275 York Ave. Box 98 | New York, NY 10021 | ==============================================================================
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