I have been trying to measure rates of apoptosis in epithelial cells by marking apoptotic cells with annexin V conjugated with phycoerythrin (PE) and sorting using flow cytometry. Unfortunately, the cells are adherent, and have been cultured in a collagen coated plate. Thus, to suspend them for flow cytometry, I have been using a trypsin treatment. Unfortunately, my results have been quite poor. I believe that there are several problems. First, the trypsin treatment is likely to induce the disruption of integral membrane proteins, potentially causing the externalization of phosphatidylserine (PS) (to which the annexin binds) on the outer plasma membrane of cells. Since the cells were incubated with the annexin V following suspension with trypsin, this could have led to a substantial increase in apparent rates of apoptosis, and masked real differences between rates of apoptosis in WT and KO cells. Furthermore, I was informed that epithelial cells, in order to maintain their structural integrity, have a relatively large ratio of cytoplasm to nucleus, and a prominent cytoskeleton resulting in increased background noise (greenish in color). In order to solve these problems I was considering several alternatives, such as changing the fluorescent marker that annexin V is conjugated with to reduce autofluorescent interference, using a different marker of apoptosis (in the place of annexin V), or, if I were to continue using annexin V, I think that incubating with annexin V followed by washing off of excess annexin, followed only then by trypsin treatment could potentially solve one of my various problems (or then again, it might not). Please help me find a suitable protocol for flow cytometric analysis of apoptotic markers in adherent epithelial cells. Additionally, if you know of any studies which utilize such methods, please direct me to them. Thanks in advance.

  • Consider trypsin is a serine protease and your annexin is a protein. – CKM Jul 12 at 15:15

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