Zombie aqua fixable live dead exclusion dye

To sort iNKT cells, the following antibodies were used for staining: anti-Vα24-Jα18 (clone 6B11; BioLegend #342912), anti-CD3 (clone OKT3; BioLegend #317318). For staining of purified lymphocyte populations, cells were first stained with Zombie Aqua fixable live/dead exclusion dye per manufacturer’s instructions (BioLegend cat #423101), followed by surface staining in FACS buffer containing 2.5% FBS. For intracellular staining, cells were fixed and permeabilized using the Becton Dickinson Cytofix/Cytoperm kit, according to manufacturer’s instructions (BD Biosciences cat #554714) and stained with antibodies against granzyme B (clone QA16A02; BioLegend #372208) or mouse IgG1k isotype control (clone MOPC-21; BD Biosciences cat #556650), or Cpt1a (clone8F6AE9; Abcam cat# 171449) and rabbit IgG monoclonal isotype control (clone EPR25A; Abcam cat# 199091). For all flow cytometry studies, rested and stimulated iNKT cells and T_CONV were run on the same in-house instrument [FACSVerse cytometer (BD Biosciences)] on different days, and normalized to isotype controls within each sample, in order to minimize day-to-day variations. Mean fluorescent intensity (MFI) values of intracellular proteins (granzyme B and Cpt1a) were calculated by subtracting the isotype control MFI in each sample. Flow cytometry analysis was conducted using FlowJo software (Tree Star Inc.).