Uv live dead fixable stain

Rested, unstimulated PBMCs were cell surface stained to identify the frequency of CD16⁺ expression among CD56^{total/dim/bright} and iNKR^+/- NK cell populations. Following staining with UV Live/Dead Fixable Stain (Invitrogen, Burlington, ON, Canada), surface staining was performed in the dark at room temperature on 1x10⁶ PBMCs per individual with Abs of the following specificities: CD3-BV785 (OKT3), CD56-BV711 (NCAM16.2), 3DL1-BV421 (DX9), CD57-FITC (HCD57), (all from BioLegend, San Diego, CA), NKG2A-PeCy7 (Z199, Beckman Coulter, Mississauga, ON, Canada), CD16-APC-Cy7 (3GB, BD Biosciences, Mississauga, ON, Canada), 2DL1-AlexaFluor700 (143211) and 2DL3-PE (180701; both from R&D Systems, Minneapolis, MN). Stained cells were then washed and fixed with 1% paraformaldehyde solution (PFA, Santa Cruz, Biotechnology, Inc, Dallas, TX). Between 300,000 and 400,000 events per sample were acquired using an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo software v10 (Treestar, Ashland OR). Unstained, single color, and fluorescence minus one controls were used for each subject for multi-color compensation and gating purposes.

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).