Hi flow-experts, Fc-blocking (or not) on human PBMC prior direct or indirect staining is a never ending discussion or story in our lab. And the literature is confusing. Some claim to use an IgG solution of the same origin as the used cells are. Others an IgG solution of the same origin as the monAb (direct coupled with fluorochromes) or the secondary polyclonal Ab are. What is the flow community thinking about that issue and is there really a need? Some more questions: What is the best wash and staining buffer for human PBMC staining protocols? Some people add BSA or HSA and some FCS in different concentrations (0.1 - 5%) to a PBS-buffer. And what is the cell biological need to add serum components? I suppose not blocking Fc-receptors. The explanation I've red that cells feel at home and comfortable is unsufficient (for me). And finally the question of speed in centrifugation of the cells for washing after staining. Mostly published protocols differ extremely. The range in speed starts with 200g up to 1800g or more for 1 to 10 minutes. It depends on the source of cells that's clear but even for human PBMC there are such differences outlined. PBMC's are hard taking cells but where is the balance between cell loss by damage (high g) or cells loss by aspirating due to less pelleting of the cells (low g)? All responses are highly welcome. Best regards VolkerVolker Eckstein PhD Dept. of Internal Medicine V Medical School of the University University of Heidelberg GERMANY volker_eckstein@med.uni-heidelberg.de
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