Ebioscience fixable viability dyes efluor 506 and 780

PBS57-loaded and unloaded human CD1d tetramers were obtained from the National Institutes of Health Tetramer Core Facility (Atlanta, GA, USA). The following antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA) or BioLegend (San Diego, CA, USA): anti-CD3 (HIT3a/OKT3), anti-CD4 (RPA-T4), anti-CD8 (HIT8a), anti-CD25 (BC96), anti-IFN-γ (4S.B3), anti-IL-4 (MP4-25D2), anti-IL-17 (BL168). Fluorescence minus one controls were used for proper gating. To stain dead cells, eBioscience Fixable Viability Dyes eFluor™ 506 and 780 (ThermoFisher Scientific, Waltham, MA, USA) were used. Data were acquired on a LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyses were performed with FlowJo 10.2 (Tree Star, Ashland, OR, USA).

Fresh or thawed PBMC were resuspended in staining buffer consisting of phosphate-buffered saline (PBS; Thermo Fisher Scientific) supplemented with 2% fetal bovine serum (FBS; Biochrom) followed by staining with fluorochrome-conjugated monoclonal antibodies (Ab). The following Ab were purchased from BD Biosciences or BioLegend: CD4 (RPA-T4), CD127 (A019D5), CD3 (OKT3), CD8 (HIT8a), CD19 (SJ25C1), and CD25 (BC96). To exclude dead cells, eBioscience™ Fixable Viability Dyes eFluor™ 506 and 780 (ThermoFisher Scientific) were used. Phycoerythrin (PE)-labeled PBS57-loaded CD1d tetramers were provided by the National Institutes of Health Tetramer Core Facility. Stained cells were measured using a LSR Fortessa flow cytometer (BD Biosciences), and analysis was performed with FlowJo 10.2 (Tree Star).