Flow cytometric analysis of surface receptor expression and phagocytic function in adipose tissue macrophages. (A) ATM populations were examined for expression of TLR2, TLR4, CD11c, CD274, ABCA1, CD163, SR-B1, LOX-1, MSR1, CD36, RAGE, and CX3CR1. CD14+HLADRII-CD206- (green histograms), CD14+HLADRII+CD206- (blue histograms), and CD14+HLADRII+CD206+ (red histograms) cells are presented compared to FMO control staining for each marker (orange histograms). (B) Mean fluorescence intensity values for each surface marker depicted in (A) were compared in CD14+HLADRII-CD206-, CD14+HLADRII+CD206-, and CD14+HLADRII+CD206+ ATM. Kruskall-Wallis testing with post-hoc Dunn’s multiple comparison testing was performed to determine whether surface macrophage marker expression was statistically different among the different subsets. p values < 0.05 were considered to be statistically significant. (C) The pHrodo system was utilized to demonstrate phagocytosis of opsonized bioparticles by enriched CD14+ monocytes (Monocytes, left panel, positive control), positively selected CD14+ ATM (Enriched ATM, center panel), and whole adipose tissue, gated on CD14+ ATM (Whole ATM, right panel) by flow cytometry. Green dots represent bioparticles incubated with antibody staining cocktail and no cells (acellular negative control). Red dots represent monocytes or ATM incubated with bioparticles at 4°C (cellular negative control). Blue dots represent monocytes or ATM incubated with bioparticles at 37°C. Enriched ATM and Whole ATM were further gated into 3 subpopulations based on HLADRII and CD206 expression. Phagocytosis of opsonized bioparticles is depicted for each subpopulation of Enriched ATM and Whole ATM and is reported as percentages of viable cells containing bioparticles.