Anti γδtcr pe cy7

Immune cells from the colon were isolated according to recent protocol^{63 (link)}. The isolated immune cells were resuspended in FACS buffer (2% FCS in PBS) and were passed through a 70-μm strainer (BD Biosciences). Samples were analysed with following antibodies: anti-CD11b-PE (1:100 dilution, 101208, Biolegend), anti-CD11c-PE-Cy7 (1:100 dilution, 117318, Biolegend), anti-γδTCR-APC (1:100 dilution, 118116, Biolegend), anti-γδTCR-PE-Cy7 (1:100 dilution, 118123, Biolegend), anti-βTCR-FITC (1:100 dilution, 109205, Biolegend), anti-βTCR-PE (1:100 dilution, 553172, BD Biosciences), anti-CD45-V510 (1:100 dilution, 103137, Biolegend), anti-CD45-PE-CF594 (1:200 dilution, 562420, BD Biosciences), anti-CD45-APC (1:200 dilution, 103112, Biolegend), anti-CD90.2-PE-Cy7 (1:200 dilution, 140309, Biolegend), anti-CD3-PE-CF594 (1:200 dilution, 562332, BD Biosciences), anti-F4/80-APC-Cy7 (1:50 dilution, 123119, Biolegend), anti-CD19-FITC (1:100 dilution, 115505, Biolegend), anti-CCR-6-V421 (1:50 dilution, 129817, Biolegend), anti-IL-6R-PE (1:50 dilution, 115805, Biolegend) and LIVE/DEAD Fixable Violet Dead Cell Stain Kit or LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (1:1000 dilution, L34955 and L34957, ThermoFisherScientific). Data were acquired on MACSQuant Analyzer (Miltenyi) and MACSQuant VYB (Miltenyi) and data were analysed with FlowJo software (Treestar).

Single-cell suspensions were stained with PE or APC-labelled IA^b/NP_311-325 or APC-labelled D^b/NP_368-374 tetramers (NIH tetramer core) at 37°C, 5% CO₂ for 2 h in complete RPMI (RPMI with 10% foetal calf serum, 100 μg/ml penicillin-streptomycin, and 2 mM l-glutamine) containing Fc block (24G2). Surface antibodies were added and the cells were incubated for a further 20 min at 4°C. Antibodies used were: anti-CD3 BV785 (BioLegend; clone: 17A2), anti-CD4 BUV805 (BD Bioscience; clone: RM4-5), anti-CD8 BV421 (ThermoFisher; clone: 53-6.7), anti-CD44 BUV395 (BD; clone: IM7), anti-CD45.2 BV605 (BioLegend; clone: 104), anti-CD69 PE-Cy7 (ThermoFisher; clone: H1.2F3), anti-CD127 APC (ThermoFisher; clone: A7R34), anti- γδ TCR PE-Cy7 (BioLegend; clone: GL3), anti-ICOS BV785 (BioLegend; clone: C398.4A), anti-NK1.1 APC-Cy7 (BioLegend; clone: PK136), anti-PD1 BV711 (BioLegend; clone: 29F,1A12), and ‘dump’ antibodies: B220 (RA3-6B2), F4/80 (BM8), and MHC II (M5114) all on eFluor-450 (ThermoFisher) or PerCP-Cy5.5 (ThermoFisher; B220 and F4/80, and BioLegend; MHCII). Cells were stained with a fixable viability dye eFluor 506 (ThermoFisher). Stained cells were acquired on a BD LSR Fortessa and analysed using FlowJo (version 10, BD Bioscience).