From: medbury@renal.wsahs.nsw.gov.au Organization: Renal Medicine, Westmead Hospital. To: cyto-inbox Date: Fri, 19 Dec 1997 13:01:26 EST+1000 Subject: Apoptosis again..... Priority: normal I set out to answer my own questions from 19/12/97 so here are my questions, with my answers Hi, I have started doing annexin V binding (clontech) to monitor apoptosis in endothelial cells. I have a couple of questions I am getting what looks like non specific binding, a slight shift to the right by the main population (compared to the no ab control), separate to my specific peak which is further to the right. Any ideas why? Does the use of Trypsin EDTA affect the cells and alter there binding pattern? Or is it perculiar to EC's The non specific binding was perculiar not to EC's but to the antibody I was using. Until my antibody came in I was using another guys antibody. When mine came in I compared the two. I only got the non specific binding with his antibody. Both were clontech. Dont know why the difference between the two. I am taking the cells at different time points, ie at 6 hours and at 10 hours. I was fixing the cells because in the annexin binding method you dont wash away excess antibody. The cells would be sitting around till the next day, and I thought that they would be slowly dying and reacting with the antibody. Is fixing the cells okay? will they continue to react with the ab if not fixed? Okay so I compared fixing with non fixing. I took cells at 8hrs, added the antibody. After 15min I fixed tube A but didnt fix tube B It was 1hr before the tubes were analysed on the facs. THe result was that tube A and tube B both gave a specific peak of about the same height, however, the specific peak for the fixed tube A was not as far to the right as the unfixed tube B. I tested two different samples with the fixing and unfixing and the result was the same. If fixation opens the cells and exposes more PS you would expect the shift to be further to the right, but it wasn't. If in the unfixed tube more cells were dying with the extended time and putting PS on their surface and therefore binding the antibody, then you would expect a higher peak for the unfixed cells, but there wasn't. It suggests that in fixation of cells, there is no change in the number of cells binding, but a decrease in the intesity of the fluorescence. Hope this helps others who have the same questions further comments are welcome Merry Christmas Christ was born to save. Heather Heather Medbury PhD Department of Surgery Westmead Hospital Westmead, NSW, 2145 Australia ph (612) 9845 7680 fx (612) 9893 7440 Heather Medbury PhD Department of Surgery Westmead Hospital Westmead, NSW, 2145 Australia ph (612) 9845 7680 fx (612) 9893 7440
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