Anti cd16 32 monoclonal antibody

LV tissues were harvested, minced with a fine scissors, placed in 4 mL RPMI-1640 (Thermo Fisher Scientific) with 5% fetal bovine serum, 450 U/mL collagenase type I, 125 U/mL collagenase type XI, 60 U/mL DNase I, and 60 U/mL hyaluronidase (all from Sigma-Aldrich), and shaken at 37 ºC for 40 minutes. Tissues were then triturated through 70-μm nylon mesh and centrifuged (1,000 rpm, 5 minutes, 4 ºC). Cells were incubated with anti-CD16/32 monoclonal antibody (BD Pharmingen) to block nonspecific binding of primary monoclonal antibody and then stained with a combination of allophycocyanin conjugated anti-mouse CD45 (BD Pharmingen), fluorescein isothiocyanate conjugated anti-mouse TCRβ (BD Pharmingen), and phycoerythrin conjugated αGC-loaded murine CD1d tetramer (MBL International) for 1 hour at 4°C. Cells were washed, and 7-Amino-Actinomycin D (Immunostep) was added to distinguish dead cells. αGC-loaded CD1d tetramer⁺ TCRβ⁺ cells were identified as iNKT cells. Stained cells were acquired with FACSVerse flow cytometer (BD Biosciences) and analyzed with FlowJo (Tommy Digital Biology).

Single cells were generated from mouse tumors or spleens, and then pre–incubated with the anti–CD16/32 monoclonal antibody (BD Biosciences) to block nonspecific binding. Appropriate dilutions of various combinations of fluorescently‐labeled antibodies were used and are listed in Table S1. BODIPY 493/503 staining was performed as previously described.²² For sorting cells, the gate was set to discriminate TAM with normal lipid content (TAM‐NL) versus TAM with higher lipid content (TAM‐HL) in the tumor‐bearing host using the fluorescence of naïve macrophages as the background defining normal lipid levels. For the cell cycle assay, the cells were stained with propidium iodide (PI, 20 μg/mL). Apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit I (BD) following the manufacturer’s protocol. All flow experiments were performed using FACS Aria II machines (BD).