Saponin based buffer

Multiparametric flow cytometry was used for phenotypic and functional analysis of PBMC, IHL and TILs. Cells were stained with a fixable Live/Dead dye (Invitrogen) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant Stain buffer (BD Biosciences) and 50% PBS (Invitrogen) for 15 min at 4 °C. Cells were fixed and permeabilized for further functional assessment with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s instructions. Cells were incubated with saturated concentrations of mAbs diluted in a 0.1% saponin-based buffer (Sigma-Aldrich) for 20 min at 4 °C for the detection of intracellular proteins. All samples were acquired on Fortessa X20 flow cytometer using FACSDiva software v8.0 (BD biosciences) and analysed using FlowJo v.9 (Tree Star; BD Biosciences). Full details on monoclonal antibodies used can be found in Supplementary Table 5.

Multi-parametric flow cytometry was used for the phenotypic and functional analysis of PBMCs/IHLs/lymph node leukocytes. Cells were stained with saturating concentrations of surface monoclonal antibodies diluted in 50%-Brilliant Violet Buffer (BD Bioscience):50%-1× PBS (Thermo Fisher Scientific). Dead cells were excluded from analysis using a fixable viability dye (Thermo Fisher Scientific). Following surface staining, cells were fixed (and permeabilized) with Cytofix/Cytoperm (BD Bioscience).
Where necessary, intracellular proteins were detected using saturating concentrations of monoclonal antibodies in a 0.1% saponin-based buffer (Sigma-Aldrich). All samples were acquired on a BD Bioscience Fortessa-X20 and analyzed using FlowJo v.9 (TreeStar/BD Bioscience). Full details of all monoclonal antibodies used for flow-cytometric analysis are given in Table S2.

For flow cytometry, cells were stained with saturating concentrations of surface antibodies and a fixable viability dye diluted in 1:1 PBS (Invitrogen): Brilliant Violet Buffer (BD Biosciences). Following surface staining, cells were fixed and permeabilized with cytofix/cytoperm (BD Biosciences) followed by an intracellular staining with antibodies in saturating concentrations diluted in a 0.1% saponin-based buffer (Sigma-Aldrich). Full details on fluorescent monoclonal antibodies can be found in S1 Table. All samples were acquired on a BD Biosciences Fortessa-X20 or Fortessa and analysed using FlowJo v.10 (BD Biosciences).