Quantification of Immune Cell Populations

Immune infiltration into adipose and liver tissues and splenic composition was quantified by flow cytometry as previously described^{13 (link),22 (link)–25 (link)}. Briefly, single cell suspensions from indicated tissues were obtained by enzymatic digestion. To determine cytokine production, total single cells were stimulated for 5 h with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 1 μg/ml Ionomycin (Calbiochem), in presence of brefeldin A (10 μg/mL, Sigma-Aldrich). Subsequently, flow cytometry was used to enumerate immune cell populations. Briefly, cells were incubated in PBS supplemented with 2% FBS and were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly conjugated monoclonal antibodies to CD3-AF700(145-2C11), TCRβ-BV711 (H57-597), CD8-PECʏ−7 (53-6.7), CD4-APC (RM4-5) (all antibodies from eBioscience) for 30 min. For intracellular staining, cells were fixed and permeabilized using eBioscience buffer and stained with FoxP3-PB (FJK-16s) and IL-10-PE (JES5-16E3). Flow cytometry data were collected using an LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7) and FACS Diva Software.