Development and validation of a novel flow cytometric macrophage phenotyping method.

Abstract

Abstract Macrophages differentiate into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. M2 macrophages suppress anti-tumor responses, and phenotyping tumor-associated macrophages can be of prognostic value. Expression of mRNA of inducible nitric oxide synthase (iNOS) or arginase 1 is a well-accepted marker of murine M1 or M2 macrophages, respectively. However, macrophages need to be isolated and purified in order to evaluate the mRNA expression. There are no reliable and simple methods for detection of macrophage markers which would not require cell purification. Here we report the development and validation of a novel flow cytometric macrophage phenotyping method. Murine macrophage RAW 264.7 cells were treated with endotoxin and IFN-γ to stimulate iNOS expression. Alternatively, RAW 264.7 cells were stimulated with 8-bromo-cAMP to induce arginase 1 expression. Our new technique allowed successful detection of iNOS expression in RAW 264.7 cells by flow cytometry, which correlated well with NO production measured by Griess assay. We also developed a method which allowed us to successfully detect the Arginase I expression by flow cytometry. Arginase I expression also correlated well with arginase activity as measured by an Abnova arginase assay kit. Our results suggest that murine macrophage phenotype can be successfully determined by flowcytometric measurement of iNOS and arginase 1 expression.