Live dead stain zombie uv dye

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.

Single cell suspensions were derived from hepatic digestion as described and stained with directly conjugated monoclonal antibodies or isotype controls (all eBioscience) [3 (link), 17 (link)]. To determine IL-17A and IL-17F 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 as described [18 (link)–20 (link)]. Briefly, cells were treated with FACS fix buffer (BD Biosciences) for 15 minutes, washed and incubated in PBS supplemented with 2% FBS and were subsequently stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-AF700 (104), TCRβ-PE (H57-597), CD4-APCef780 (GK1.5), CD8-ef450 (53–6.7), CD11b-PE (M1/70), CD11c-Percp (N418), F4/80-APCef780 (BM8), Ly6C-Percp (HK1.4) Gr1-FITC (RB6-8C5), NK1.1-PECy7 (PK136), B220-ef450 (RA3-6B2), IL-17RA-APC (PAJ-17R), TNF-BV650 (MP6-XT22), IL-17A-Percp (17B7) and IL-17F-PE (18F10) [all antibodies from e-Bioscience] for 30 minutes. Flow cytometry data were then collected using a LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7).