Hello, We are using a human stem cell line which has been transfected with an adenovirus carrying a specific membrane protein. This membrane protein has a V5 tag on the C-terminal (intracellular) end, and I have an anti-V5-FITC antibody available to detect it. Our primary goal is to determine transfection efficiancy by flow. My question is what protocol or kit would people recommend using to permeabilize the cell membranes for optimal detection of this membrane protein? I am concerned that permeabilizing too much will disrupt the membrane protein and drastically reduce my signal. Does anyone have experience with this? We may eventually want to sort cells based on expression of this membrane protein. My other question is: is it possible to sort live cells based on intracellular staining? Will the permeabilization required for the staining kill the cells? If anyone has any tips or references they can point me in the direction of I would appriciate it. Thanks, Jennifer Kennedy
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