In the past, I used 2.5 ug of Human BD Fc Block (564220) in each flow tube containg 0.5-1 x 10^6 monocytes from peripheral blood. The experiment results were very good. However, I used M-CSF to polarize the monocytes to
become monocyte-derived macrophages recently. I add 2.5 ug of Human BD Fc Block in each flow tube containg 0.5 x10^6 monocyte-derived macrophages. Unfortunately, the fluorescence intensity of
isotype tube is very high (> 10^3-4). I guess that there might be more Fc receptor in macrophages than monocytes. Should I add more
human BD Fc Block (564220) in each flow tube (e.g. 5 or 10 ug of Human BD Fc Block (564220) in each flow tube) or change to other methods (e.g. human serum or other company's Fc receptor block)? I read a article and the authors use beriglobin to block Fc receptor. However the beriglobin is clinically used for hypoglobinemia and expensive. Does anyone have good recommendations of better protocols about Fc receptor block? Thanks.
Is your isotype in FITC by any chance?
Were it Fc-mediated binding, you would see high background for all the other antibodies in the panel as well. Is this the case? Did you try other isotypes in different colors?
As the other commentators have noted, macrophages naturally have high autofluoresence, but it is usually manageable even in M-CSF derived macrophages. Are you adding the Fc block in with your antibody staining step, or as a separate step before staining? Sometimes it can help to do both, and to up the blocking concentration and/or use some human serum. Have you noticed any effect of the Fc blocking at all? What happens if you run an Fc-blocked sample and compare it to an un-blocked sample? Do you see any improvement?
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