Hello All, I have a user who is looking at the cell line U205, which is an osteosarcoma line. Part of her study is to look for apoptosis after UV treatment. By morphology the cells look like they are apoptotic but she wants also to demonstrate this by flow. Initially we looked for a sub-G1 peak but without success. OK, this may be a true result if the DNA is not breaking up into very small fragments. So, I went on to look for strand breaks by TUNEL. My problem is that although the treated and untreated cells do show differences, they are quite small. If I run a thymocyte control in parallel I get a good log and a half shift in the dUTP fluorescence. In contrast in the U205 cells, I only really see positive cells if I run on linear and even then it is maybe a two-fold increase. I have tried upping the concentrations of dUTPs, enzyme and Av-FITC without success (I don't use a kit BTW). Has anybody any tips, or has anyone any experience with these cells? Still, at least, its nearly Christmas, eh? 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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