Anti cd16 antibody clone 3g8

The following reagents and antibodies were used: anti‐4‐1BB antibody (clone 4B4‐1, BioLegend, 309811; clone BBK‐2, LS‐C88297, LifeSpan BioSciences Inc; urelumab, produced internally), anti‐CD28 antibody (clone CD28.2, 16‐0289‐85; eBiosciences), anti‐NKp30 (clone P30‐15, 325204; BioLegend), anti‐NKp46 (clone 9E2, 331904; BioLegend), anti‐CD16 antibody (clone 3G8, 302050; BioLegend), anti‐CD20 (rituximab, hcd20‐mab1; Invivogen), anti‐CD3 (clone OKT3, 317326; BioLegend), anti‐TCR γδ (clone B1, 331236; BioLegend), MART‐1 (26‐35) peptide (EAAGIGILTV) (H‐4102.0005; Bachem), and NY‐ESO‐1 (157‐164‐V) peptide (SLLMWITQV; custom peptide synthesis, 96% pure; Bachem).

Anti-2B4 (clone C1.7, 3 μg/ml; BioLegend) and anti-CD16 antibody (clone 3G8, 15 μg/ml; BioLegend), as well as NKG2D ligand MICA (R&D system, 2.5 μg/ml) and ULBP1 (R&D system, 2.5 μg/ml), and LFA-1 ligand ICAM-1 (R&D system, 2.5 μg/ml) were used to stimulate NK cells. Antibodies and ligands were diluted with PBS and coated on 6-well and 24-well plates at 4°C overnight. After incubation, plates were washed once with PBS. NK cells were added to the coated plate and incubated at 37°C (5% CO2) for 4 or 6 h as indicated. Cells were harvested for subsequent metabolic and flow cytometry analyses.

Anti-CD16 antibody (clone 3G8) was purchased from BioLegend. BUV395-CD3 (clone UCHT1), PE-Cy™7-CD56 (clone B159), BV421-CD210a (clone 3F9), BV421-IgG2a (clone R35-95), FITC-CD107a (clone H4A3), PE-FasL (clone NOK-1) were purchase from BD Biosciences. Antibodies used for western blot were as follows: mouse anti-β-actin was purchased from Santa Cruz Biotechnology. Rabbit anti-phospho-S6 ribosomal protein (Ser235-Ser236) antibody, rabbit anti-phospho-p70 S6 kinase (Thr389) antibody, rabbit anti-p70 S6 kinase antibody, and mouse anti-phospho-STAT3 (Tyr705) antibody were purchased from Cell Signaling Technology.
Human IL-10 was purchased from Miltenyi Biotec, and human recombinant IL-2 was from R&D System. Cell-Tak™ Cell and Tissue Adhesive was purchased from Corning. Rapamycin and CellTrace Violet were from Invitrogen. Ficoll® Paque Plus, 2-deoxyglucose (2-DG), etomoxir, UK5099, BPTES, oligomycin, FCCP, rotenone, antimycin and concanamycin A were purchased from Sigma-Aldrich. Human IFN-γ ELISA MAX™ Standard Set was from BioLegend. Human Granzyme B ELISA development kit was from Mabtech. Lactate Assay Kit was from Sigma-Aldrich. Fixable viability dye eFluor 780 (FVD) was purchased from eBioscience. 2-NBDG was purchased from Life Technologies. Stattic was from Santa Cruz Biotechnology. SuperSignal West Pico/Dura chemiluminescent substrate was from Pierce.