Dear colleagues, I struggle with stable transfected HEK-293 cells. I need to compare the expression level of a certain (transfected) receptor on these cells. I have difficulties with the extended aggregation that take place during the labeling procedure which affects mean fluorescence. Do you have any good protocol to decrease the extent of aggregation, or at least standardize it. Thanks for your help, Best whishes, Andras Boros My protocol is : Suspension of cells with EDTA containing D'PBS, wash and fixation for 20 min in 4% PF. Then labeling with primary antibody O/N, wash and labeling with FITC-conjugated secondary antibody (2.5 h), wash and analysis with FACScan. (LOG amplification of all parametrs)
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