Na le human fc block

For Fc-receptor blockade, cells were added to the wells in a volume of 100 µl medium. Then, Fc-receptor-blocking reagents (depending on experiment: LEAF anti-human CD16 antibody, clone 3G8, Biolegend; LEAF anti-human CD64 antibody, clone 10.1, Biolegend; functional grade anti-human CD32 antibody, clone 6C4, eBiosciences/Invitrogen; InVivoMAb anti-human CD32a antibody, clone IV.3, BioXCell; NA/LE human BD Fc Block, BD Biosciences; pure anti-human CD16 antibody, clone REA423, Miltenyi Biotec) or respective isotype controls were added to a concentration of 25 µg/ml in 150 µl volume per well, and plates were incubated on wet ice for 30 min. Then, anti-VISTA antibodies were added and cells cultured as described above. Blocking reagents remained in the culture for the whole culture duration.

Prior to cell surface staining, PBMCs were incubated with purified NA/LE Human BD Fc Block (BD, dilution 1:300) and Live/Dead Fixable Aqua or Live/Dead Fixable Blue (both Thermo Fisher, dilution 1:1000) in FACS buffer (2% FCS and 1 mM EDTA in PBS) for 20 min at 4 °C. Cells were then washed and incubated with cell surface antibody cocktails (dilution 1:200) for 30 min at 4 °C. Cells were washed with FACS buffer and fixed using BD Cytofix Fixation Buffer (for intracellular cytokine staining) or eBioscience Fixation/Permeabilization kit (for Foxp3 staining) for 20 min at room temperature. For intracellular staining assays, cells were washed twice with 1× BD Perm/Wash buffer or 1× eBioscience Perm/Wash buffer prior to incubation with intracellular antibody cocktails (dilution 1:200, except for IL-33 antibodies−1:500) for 45 min at room temperature. Cells were washed twice with 1× BD Perm/Wash buffer or 1× eBioscience Perm/Wash buffer and resuspended in FACS buffer. Flow cytometry data were acquired on a BD LSRFortessa or BD FACSymphony flow cytometer.