On Mon, 2 Oct 2000, Kenichi Fujise wrote: > I would be appreciative if somebody could tell me whether Annexin > experiment using flow cytometry can be performed in adhesive cells. My > concern was that harvesting adhesive cells using Trypsin-EDTA would > reduce the Annexin signals significantly. Hi Ken, I have several users who use a slightly modified trypsin-EDTA (with slightly lower strength enzyme) to harvest adherent cells. You have to be careful with EDTA that you make sure you have sufficient calcium later on in your staining buffer to get the annexin binding. Also you need to be careful that you dont induce additional cell death during harvesting but the usual controls will be there to adjust for that of course. > If anybody has tried to gate cells that are successfully transfected > with a certain gene somehow, I would be appreciative of knowing how it > was done. I wonder if anybody has used GFP to achieve this. Again, we do this. GFP is one of the most common markers of transfection here so we have in a lot of cases moved away from Annexin-FITC. As I have a dual laser FACS Calibur, what I normally do is use an Annexin-Cy3 (MBL certainly do one and I am sure there are others!) and the use TO-PRO-3 as my dead cell discriminatory dye. It works well! Hope that helps, 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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