Adipocyte Isolation and Purification

scABD and scFEM tissues were treated with a HEPES/type 1 collagenase solution for 1 h at 37°C to separate adipocytes and the stromal-vascular fraction [40 (link)]. As previously described [41 (link)], the adipocytes were purified by incubation with a cocktail of biotinylated antibodies against markers of endothelial cells (anti-human CD31; eBioscience, Thermo Fisher Scientific Life Sciences, Waltham, MA, USA; 1:200), hematopoietic cells (anti-human CD45; BioLegend, San Diego, CA, USA; 1:400), and mesenchymal stem cells (anti-human CD34; eBioscience, Thermo Fisher Scientific Life Sciences, Waltham, MA, USA 1:00) for 15 min at room temperature using an EasySep Cell Separation kit (EasySep, Stem Cell Technologies, USA). Cells attached to these antibodies were bound by magnetic nanoparticles (EasySep) and removed, while immuno-purified adipocytes were retained. Adipocytes were flash frozen in liquid N₂ and stored at −80°C until DNA extraction.