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 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? THanks in advance for your advice Heather Heather Medbury PhD Department of Surgery Westmead Hospital Westmead, NSW, 2145 Australia ph (612) 9845 7680 fx (612) 9893 7440
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