Macrophage phenotype analysis by flow cytometry

Adipose tissue macrophages (ATM) and their respective pro- or anti-inflammatory phenotype markers were characterized by flow cytometry as described earlier (24). SVF cells were stained with the following antibodies: CD11b-AlexaFluor700 (eBioscience, CA, United States), F4/80-eFluor450 (eBioscience, CA, United States), CD45-APC (Biolegend, CA, United States), CD11c-PE, CD301-AlexaFluor647 (AbD Serotec, NC, USA). All samples were measured on a LSR II cytometer (BD Bioscience, CA, USA) and the data was analyzed by FlowJo (OR, United States). After exclusion of singlets by forward and sideward scatter, macrophages were defined as CD45⁺, CD11b⁺, F4/80⁺ cells. CD11c was used as a pro-inflammatory macrophage marker, while CD301 served as an anti-inflammatory marker. To characterize intracellular protein levels of pro-and anti-inflammatory markers, SVF fraction cells were surface-stained with CD11b-AlexaFluor700 (eBioscience, CA, United States), F4/80-eFluor450 (eBioscience, CA, United States), CD45-APC-Cy7 (BD Pharmingen, CA, United States) and then fixed and permeabilized with Fix/Perm Buffer (eBioscience FoxP3/Transcription Factor Buffer Set, Invitrogen/Thermo Fisher Scientific, CA, United States) according to manufacturer’s instructions. After fixation and permeabilization, the cells were stained intracellularly for MCP1-APC (R&D Systems, MN, United States), TNFα-PE (eBioscience, CA, United States), Arginase1-FITC (R&D Systems, MN, United States) and TGFβ-PerCP-Cy5.5 (Biolegend, CA, United States). All samples were measured on an LSR II cytometer (BD Bioscience, CA, USA) and the data was analyzed by FlowJo (OR, United States). Adipose tissue macrophages were defined as CD45⁺, CD11b⁺, F4/80⁺ cells and then further analyzed for intracellular expression of the pro-inflammatory markers MCP1 and TNFα as well as the anti-inflammatory markers TGFβ and Arginase1.