Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PM phi) by flow cytometry are not ideal for elicited or activated PM phi. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac-1 antigens on resident PM phi. In contrast, high concentrations (33-50% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PM phi elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2). However, even low concentrations of goat serum masked the expression of the Mac-2 antigen on TG and MVE-2 PM phi. Thus, within a given elicited or activated PM phi population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE-2 PM phi.