Streptavidin pe dazzle

PBMCs from all experiments were stained extracellularly with Aquavivid live-dead viability dye (Thermo Fisher Scientific, USA), CD3-PeCy5 and PD-1-PE-Dazzle (Biolegend, USA), LAG-3-PE (BD Biosciences, USA) and iNKT CD1d-tetramer (NIH Tetramer Core Facility, USA) for 30 min. In conditions with the biotinylated anti-PD-1 blockade present, cells were first stained with Streptavidin-PE-Dazzle (Biolegend, USA) for 30 min, washed, then stained with the antibodies listed above. Samples from the multi-day (section "Multi-day assay".) and 10-day proliferation assays (section "10-day proliferation assay".) were fixed with Cytofix fixation buffer (BD Biosciences, USA). Samples from the multi-hour kinetics (section "Multi-hour assay".) and 10-h stimulation (section "10-hour stimulation post-iNKT cell expansion model".) assays were permeabilized with Cytofix/Cytoperm solution (BD Biosciences, USA), washed and stained intracellularly with IFN-γ-BUV396 antibody (BD Biosciences, USA) following a protocol from ThermoFisher Scientific³⁶ .
Samples were analyzed on the LSRFortessa flow cytometer (BD Biosciences, USA). Data was acquired and compensated using BD FACS Diva software version 8.0.3. (BD Biosciences, USA) and analyzed using FlowJo v10.8.1 (TreeStar, USA).

CEM cells were electroporated with 2 μg of the following plasmids: GFP-Tax [48 (link)], HBZ-IRES- GFP (pCMV.IRES.GFP.Myc(x2)_HBZ.SP1; [49 (link)]), and/or GFP alone (pCMV.IRES.GFP.Myc(x2); Clontech), or 1 μg HBZ-TTG (pME18Sneo-HBZ TTG; [42 (link)]) plus 1 μg GFP or–GFP-Tax where indicated. Electroporation was carried out using a nucleofector 1 device (program A030) which routinely gave 20–40% transfection efficacy with ~95% viability. Post electroporation cells were placed in warm RPMI 10% FCS at a density of 3x10⁵/ml in the presence or absence of 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.5 μg/ml calcimycin (CAI, Sigma). After 16h culture, cells were harvested, stained with a viability stain and biotinylated anti-CADM1, followed by Streptavidin-PEDazzle (Biolegend). Cells were analysed by flow cytometry within 1 h.