Trustain fcx anti cd16 32 clone 93

Seven and 14 days after implant, epididymal fat pads were harvested following euthanasia and washed in ice cold PBS (Sigma). Fat pads were minced, digested in collagenase (Liberase TL, Roche), and passed through a 100 μm filter. The stromal vascular fraction was harvested by centrifugation, washed in MACS buffer (PBS, 0.5 mM EDTA, 30% BSA), and incubated with anti-CD16/32 prior to adding an antibody cocktail against extracellular antigens. The following antibodies were purchased from Biolegend: anti-CD45 clone 30-F11, anti-Ly6G clone 1A8, anti-F4/80 clone BM8, anti-NK1.1 clone PK136, anti-CD19 clone 6D5, anti-CD11b clone M1/70, anti-CD3 clone 17A2, and Trustain fcX (anti-CD16/32) clone 93. The following isotype controls were also purchased from Biolegend: mouse IgG2a clone MOPC-173; rat IgG2a clone RTK2758; rat IgG2b clone RTK4530. After antibody incubation, cells were washed, fixed, and analyzed using a FACS Aria flow cytometer (BD Biosciences). The number of CD45 cells in each flow cytometry sample was calculated using Bang’s labs Flow Cytometry Absolute Count Standard, which was added prior to data acquisition. FlowJo software (Treestar) was utilized to compensate and analyze data. FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is depicted in Figure S3.