Cd64 percpcy5

Single cell suspensions of blood (by cardiac puncture) were collected from NRL-CV, NRL and wild-type adult male and female mice and stained for flow cytometry. Antibodies used: CD3-BV785 (17A2, BD Biosciences, cat 564010), CD45-BV711 (30-F11, Biolegend, cat 103147), CD4-PE-Cy7 (RM4–4, Biolegend, cat 116016), CD8-BUV395 (53–6.7, BD Bioscience, cat 563786), CD19-BV605 (6D5, Biolegend, cat 115540), NKp46-BUV737 (29A1.4, BD Bioscience, cat 612805), LY6G-APC-Cy7 (1A8, Biolegend, cat 127623), CD11b-BV650 (M1/70, Biolegend, cat 101259), SiglecF-AF647 (E50–2440, BD Bioscience, cat 562680), CD64-PerCPCy5.5 (X54–5/7.1, Biolegend, cat 139308), TCRγδ-FITC (GL3, Biolegend, cat 118105), CD11c-BV510 (N418, Biolegend, cat 117353) and counterstained with DAPI (ThermoFisher, cat D1306). Samples were acquired on a BD Fortessa instrument. Data were analysed using FlowJo v10.8.1. For cell gating, RFP+ cells were subgated out of CD45+DAPI− single and the immune subsets were subgated out of the RFP+ gate (see Table 1 and Supplementary Figure 4a).

17 week-old mice (n = 7–8 per group from ≥ 4 litters) were perfused with 15 ml of PBS, and one epididymal fat pad was placed in Hank’s Balanced Salt Solution (HBSS; Invitrogen) supplemented with 1% BSA. Adipose tissue was minced and incubated at 37°C in 1mg/ml collagenase (type IV; Worthington Biochemical, Lakewood, NJ). The cell suspension was filtered through a 100 μm filter and centrifuged. The pellet of stromal vascular cells (SVC) was re-suspended in 500 μl of RBC lysis buffer (BioLegend), followed by dilution with PBS containing 1 mM EDTA, 25 mM HEPES and 1% heat-inactivated fetal bovine serum. A minimum of 1x10⁵ cells were aliquoted into single-stain controls, fluorescence minus one controls, and sample tubes, then incubated in Fc Block (rat anti-mouse CD16/CD32) followed by conjugated antibodies. DAPI was used to discriminate live/dead cells. Flow data were acquired on a FACSAria-I (BD Bioscience) and analyzed using Cytobank [32 ]. FMO controls were used to establish polygonal gates. The following commercially available fluorochrome conjugated antibodies were used: CD45 Apc-Cy7 for leukocytes, CD64 PerCp-Cy5.5 for macrophages, CD11c PE-Cy7 for M1 macrophages (BioLegend, San Diego, CA), and CD301 Alexa 647 for M2 macrophages (AbD Serotec, Raleigh, NC).