Cd16 32 monoclonal antibody 2.4g2

We isolated stromal vascular cells using previously described methods [7 (link)], with some modifications. Mice were sacrificed under general anesthesia after systemic heparinization. eVAT was removed and ground into small pieces. Samples were incubated for 40 min in collagenase II/DNase I solution (1 mg/ml collagenase II and 50 μg/ml in HBSS solution) with gentle stirring. Digested tissue was then centrifuged at 1000 g for 10 min. The resulting pellets were washed twice with cold PBS and filtered through a 70-mm mesh. Red blood cells were lysed with erythrocyte-lysing buffer (Biolegend) for 10 min and resuspended in RPMI-1640 supplemented with 10% FBS. Single-cell suspensions of adipose stromal vascular fraction (SVF) were blocked with CD16/32 monoclonal antibody (2.4G2; BD Biosciences) at 4°C for 5 min. Cells were stained with a mixture of antibodies at 4°C for 20 min. Flow cytometric analysis and sorting were performed on a FACSAriaIII instrument (BD Biosciences) and analyzed using the FlowJo software (Tree Star). Antibodies were specific to CD3 (14A-2; Biolegend), CD4 (GK1.5;Biolegend), CD8a (OKT8; Biolegend), CD44 (IM7; Biolegend), CD11b (M1/70; Biolegend), F4/80 (BM8; Biolegend), PD-1 (29F.1A12, Biolegend), CD19 (1D3, eBioscience), and CD153 (RM153, Biolegend)