Cells were stained with Fixable Viability Dye eFluor 780 (eBioscience), then stained for surface markers for 25 minutes at room temperature (RT). Cells were then fixed with 2% PFA (EMS)/PBS for 20 minutes at 4°C, or fixed and permeabilized for intracellular/intranuclear staining. For intracellular cytokine staining, cells were permeabilized using BD cytofix/cytoperm, according to the manufacturer’s instructions, then stained in 1x BD fix/perm buffer for 25 minutes at RT. For intranuclear staining, cells were fixed with 4% PFA for 10 minutes at RT, and permeabilized with ice-cold (−20°C) methanol (Sigma) for 15 minutes on ice. Intranuclear staining was then performed in FoxP3 buffer (eBioscience) for 45 minutes at RT. All flow cytometry data was acquired on a BD LSR II or BD Fortessa, and analyses were performed on FlowJo v10.
For cell sorting, freshly isolated PBMC were lineage-depleted with FITC-conjugated antibodies (anti-CD3, CD14, CD19, CD16, CD94, CD11c, CD123) and anti-FITC beads (Miltenyi Biotec) according to the manufacturer’s instructions, with minor modifications. Twenty microliters of beads per 107 cells were used in most experiments. After staining with viability dye and surface markers, cells were sorted on a BD FACS ARIA II using a 70 μm nozzle, and collected into Eppendorf tubes containing cRPMI/10%HS.
For cell sorting, freshly isolated PBMC were lineage-depleted with FITC-conjugated antibodies (anti-CD3, CD14, CD19, CD16, CD94, CD11c, CD123) and anti-FITC beads (Miltenyi Biotec) according to the manufacturer’s instructions, with minor modifications. Twenty microliters of beads per 107 cells were used in most experiments. After staining with viability dye and surface markers, cells were sorted on a BD FACS ARIA II using a 70 μm nozzle, and collected into Eppendorf tubes containing cRPMI/10%HS.