Anti ifn γ 4s b3

Cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Invitrogen) and surface antibodies for 30 min at 4 °C. For intracellular cytokine staining, cells were treated with 50 nM phorbol-12-myristate-13-acetate (MilliporeSigma) and 250 nM ionomycin (MilliporeSigma) for 4 hours in the presence of Brefeldin A (BD Biosciences) before harvesting. Cells were washed and fixed with BD Cytofix™ Fixation Buffer (BD Biosciences) for 10 min at RT, then washed with PBS. Intracellular cytokines were stained in permeabilization buffer (eBioscience) for 30 min at 4 °C. The following antibodies were used: anti-LAG-3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-TIGIT (MBSA43, eBioscience), anti-Tim-3 (F38-2E2, Biolegend), anti-IFN-γ (4S.B3, eBioscience), and IL-10 (JES3-9D7, Biolegend). Cells were acquired on a BD Fortessa flow cytometer and data was analyzed with FlowJo software v10 (Threestar).

PBMC were stained for extracellular markers (clones in parenthesis) with anti-CD2 (RPA-2.10, BioLegend, San Diego, CA, USA), anti-CD3 (BW264/56), anti-CD16 (3G8), anti-CD56 (REA196), anti-CD57 (TB03), anti-NKG2A (REA110), all from Miltenyi Biotec, Auburn, CA, USA and anti-NKG2C (134591, R&D Systems, Minneapolis, MN, USA). LIVE/DEAD Fixable Far Red (Invitrogen) was used to exclude dead cells. Fixation and permeabilization were performed using MACS Inside Stain Kit (Miltenyi Biotec) with polyclonal goat anti-human FcεRIγ (EMD Millipore, Etobicoke, ON, Canada), anti-IFN-γ (4S.B3), and anti-TNF-α (MAb11) from eBioscience, San Diego, CA, USA added for intracellular staining. The fluorescence minus one strategy was used to adjust multicolor compensation. Phenotypic analysis was performed using Kaluza Flow Cytometry Software 1.2 after data acquisition on a MoFlo Astrios EQ flow cytometer (both Beckman Coulter, Brea, CA, USA).