Comprehensive Flow Cytometry Analysis of T Cell Subsets

Antibodies were purchased from eBioscience (San Diego, California, USA), Biolegend (San Diego, California, USA) and BD Biosciences (Mississauga, Ontario, Canada). Vγ6-specific staining was accomplished in some experiments using undiluted supernatants from the 17D1 hybridoma (Sunnybrook Core Antibody Facility)^{66 (link)}. A negative gating strategy was used in most experiments to identify Vγ6⁺ T cells, as previously described^{67 (link)} and shown in Fig. 7b. Cells were washed and incubated with Fc blocking antibody (clone 2.4G2, Sunnybrook Core Antibody Facility, 1 mg/ml), followed by extracellular staining for surface CD4 (clone GK1.5), CD8α (clone 53–6.7), CD3 (clone 145-2C11), TCRγδ (clone GL3), TCRβ (clone Η57–597), Vγ4 (Vγ2; clone UC3-10A6), Vγ5 (Vγ3; clone 536), Vγ1 (Vγ1.1; clone 2.11), Vγ7 (clone F2.67), CD27 (clone LG3-1A10), CD24 (clone M1/69) and/or CD73 (clone TY11.8). All flow cytometric analyzes were performed with a Becton-Dickenson (BD) LSRII, Diva software, and FlowJo software. Sorting was performed on a FACSARIA (BD). Representative gating strategies are shown in Supplementary Fig. 11.