Anti cd16 32 clone 2.4g2

Single-cell suspensions were pretreated with anti-CD16/32 (clone 2.4G2; TONBO Biosciences, CA, USA) antibody to block Fc receptors. For additional flow cytometry methods, please see the Supplementary Data.

FACSCantoII (BD Bioscience) was used for the flow cytometry analysis. Cells were treated with an Fc blocker (anti-CD16/32, clone 2.4G2, TONBO Bioscience) prior to cell surface staining. The cells were stained with the indicated antibodies at the final concentration of 1 to 2 μg/ml. To exclude dead cells, 7-aminoactionomycin D (7AAD) was added to the cell suspension at the final concentration of 0.3 μg/ml. For the intracellular staining, a Foxp3 Staining Buffer Set (eBioscience) was used according to their manufacturer’s protocol. The antibodies used for flow cytometry were as follows: CD4 (GK1.5), CD5 (53-7.3), CD8α (53-6.7), TCRβ (H57-597), CD3 (17A2), B220 (RA3-6B2), Ly6C (HK1.4), NK1.1(PK136), CD45RB (C363-16A), CD44 (IM7), CD25 (PC61), CD24 (30-F1), CD27 (LG.3A10), Gr-1 (RB6-8C5), TCRδ (GL3), TCR-Vγ1 (2.11), TCR-Vγ4 (UC3-10A6), TCR-Vγ5 (536), TCR-Vγ7 (GL1), IL-17A (TC11-18H10.1), IFN-γ (XMG1.2), phospho-ERK (p-ERK, 197G2) and phospho-AKT (p-Akt; D9E). The monoclonal antibody 17D1, specific for TCR-Vγ6/Vδ1 and TCR-Vγ5/Vδ1, was provided by Robert E. Tigelaar (Yale University, New Haven, Connecticut, USA) and used as described previously (23 (link)). Anti-Vγ7 monoclonal antibody (GL1) was provided by the late professor Leo Lefrançois (University of Connecticut Health Center, Farmington, Connecticut, USA) (24 (link)).