M1 Macrophage Activation State Markers Overview
Macrophages are activated by exogenous stimuli in their environments such as cytokines, growth factors, or microbial agonists, which can alter their transcriptional programs and functional phenotypes. The historical model of macrophage activation describes two different macrophage activation states, classical and alternative macrophage activation. Classical M1 macrophage activation is stimulated by pro-inflammatory cytokines and microbial products including IFN-gamma and/or lipopolysaccharide or TNF-alpha. The resulting M1 macrophages secrete pro-inflammatory mediators such as TNF-alpha, IL-1 beta, IL-12, IL-18, IL-23, nitric oxide, and reactive oxygen species, which have anti-microbial and tumoricidal properties and drive Th1- and Th17-mediated immune responses. Common cell surface markers used to identify M1 macrophages include high levels of MHC class II, CD68, B7-1/CD80, B7-2/CD86, and iNOS.
The Complex Biology of Macrophages Poster
Detection of Cell Surface Markers on Resting and M1 or M2a Activated Human Macrophages by Flow Cytometry. Enriched human CD14+ monocytes were cultured in the presence of 50 ng/mL Recombinant Human GM-CSF (R&D Systems, Catalog # 215-GM) or 50 ng/mL Recombinant Human M-CSF (R&D Systems, Catalog # 216-MC) for 6 days in a RPMI-based media containing 10% FBS. Following the 6-day maturation period, macrophages were activated for an additional 24 hours to either the classical M1 macrophage phenotype with 50 ng/mL lipopolysaccharide and 50 ng/mL Recombinant Human IFN-gamma (R&D Systems, Catalog # 285-IF; solid blue histograms) or alternatively activated M2a macrophage phenotype with 20 ng/mL Recombinant Human IL-4 and 20 ng/mL Recombinant Human IL-13 (R&D Systems, Catalog # 204-IL and Catalog # 213-ILB, respectively; solid purple histograms). Macrophages that were not activated were cultured for an additional 24 hours in media containing either GM-CSF (light blue histograms) or M-CSF (light purple histograms) so that all macrophages were cultured for a total of 7 days before being compared by flow cytometry. Cells were then stained with either a fluorochrome-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody (R&D Systems, Catalog # FAB140), a fluorochrome-conjugated Mouse Anti-Human B7-2/CD86 Monoclonal Antibody (R&D Systems, Catalog # FAB141), or a fluorochrome-conjugated Mouse Anti-Human CD163 Monoclonal Antibody (R&D Systems, Catalog # FAB1607). Isotype control staining is indicated by the solid gray histograms. Zombie Violet™ cell viability dye labeling and doublet exclusion were used to remove dead cells from the surface marker expression analysis.