Anti f4 80 fitc anti fitc microbeads

Stromovascular cells (SVC) and adipocytes from eWAT were fractionated, as previously described [21 (link), 26 (link)]. For gene expression analysis, dissociated adipose tissue was fractionated by magnetic cell sorting (MACS) with anti-PDGFRα-PE/anti-PE-microbeads, anti-F4/80-FITC/anti-FITC-microbeads, and anti-CD31-APC/anti-APC-microbeads (Miltenyi Biotech). For adipogenic differentiation, MACS-isolated PDGFRα was expanded in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, and then differentiated by DMEM supplemented with a standard adipogenic cocktail for 7 days, as described previously [25 (link)]. To determine levels of adipogenic differentiation, differentiated cells were labeled with 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY 558/568 C12) (Invitrogen Molecular Probes) or Oil Red O (Sigma-Aldrich). Mitochondrion-labeling in live cells was performed using red-?uorescent mitochondrion-selective probe MitoTracker Red CMXRos (Thermo Fisher, Waltham, MA, USA).

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).