2.9. Flow Cytometry

Following euthanasia, epididymal fat pads were excised and washed in ice cold PBS. Fat pads were shredded, digested in collagenase (Liberase TL, Roche), and passed through a 100 μm filter to remove debris. The stromal vascular fraction was separated by centrifugation and washed in MACS buffer (PBS, 0.5 mM EDTA, 30% BSA). Before applying antibody panels, samples were incubated with an anti-CD16/32 antibody to reduce nonspecific binding. After antibody incubation, samples were washed in MACS buffer, fixed, and analyzed using a FACS Aria flow cytometer (BD Biosciences). Total number of macrophages (F4/80⁺ CD11b⁺ cells) in each sample was calculated by adding Bang’s labs Flow Cytometry Absolute Count Standard, prior to data acquisition. Compensation and data analysis were completed using FlowJo software (Treestar). FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is outlined in Supplemental Figure 1.