Visceral WAT Stromal Vascular Fraction Isolation

Perigonadal visceral WAT was collected, the stromal vascular cell (SVC) fraction was separated, and the mature adipocyte fraction was enriched using a collagenase‐based approach, as described previously (Cho et al., 2014 (link)). Red blood cell (RBC) lysis was performed on SVC by incubating the cells in a 0.8% ammonium chloride solution (StemCell Technologies, Cambridge, MA), and the cells were subjected to staining for flow cytometry analysis. Cells were stained with the UV LIVE/DEAD fixable stain (Invitrogen) and then surface labeled for different combinations of the following markers: CD45, CD11b, CD11c, CD206, CD301, CD19, TCRβ, Ly6G, F4/80, and Ly6C (Biolegend, San Diego, CA) and fixed with 1% paraformaldehyde (Sigma Aldrich, St. Louis, MO). Samples were analyzed on an LSRII cytometer (BD Biosciences) or an Aurora cytometer (Cytek Biosciences). All flow cytometry data analysis was performed using FlowJo Software version 10.6.1 (BD Biosciences).