Facslyrictm

Stromovascular cells (SVC) and adipocytes from gonadal white adipose tissue (gWAT) were fractionated, as previously described (Lee et al., 2012 (link); Lee et al., 2017 (link); Cho et al., 2019 (link)). Live cells were processed for cell surface marker staining. Antibodies used for flow cytometry analysis were the following: anti-F4/80-APC and CD11c-BV421 (Biolegends). Anti-F4/80-FITC (Biolegends), anti-Cx43 (rabbit, 1:100, Cell Signaling), and goat anti-Rabbit IgG (H+L) secondary antibody Alexa Fluor^TM 594 (rabbit, 1:100, Invitrogen). Analytic cytometry was performed using BD FACSLyric^TM (BD Biosciences) flow cytometers. Raw data were processed using FlowJo software (Tree Star). For the identification of cell types in flow cytometry data, at least 10,000 cells were analyzed per sample.
For macrophage isolation, dissociated adipose tissue was fractionated by magnetic cell sorting (MACS) with Anti-F4/80-FITC/anti-FITC-microbeads (Miltenyi Biotech).

Cells were incubated with 7AAD or fixable viability dye 700 and with FcR Blocking Reagent (all BD biosciences, Franklin Laker, NJ, USA) followed by the staining with various fluorescence-labeled antibodies (Table S1). For intracellular staining, cells were fixed and permeabilized with the eBioscience^TM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. To measure NO and ROS production, we used hROS Detection Kit (Cell Technology Inc., Minneapolis, MN, USA) and diaminofluorescein-FM diacetate (DAF-FM DA, Cayman Chemical, Ann Arbor, MI, USA) respectively. Acquisition was performed by ten-color flow cytometry by BD FACSLyric^TM with FACSuite^TM software (BD Biosciences, Franklin Laker, NJ, USA). The results were analyzed by FlowJo^TM V10 software (BD Biosciences, Franklin Laker, NJ, USA). Up to fluorescence minus four controls were used to define the gating strategy. For CD73 and PD-L1 staining, isotype controls were used (Table S2).