Clone epr25a

rMAL-CHO cells were grown in 6-well dishes to 90–100% confluence. Cells were treated with or without 50 nM ETX in 2 mL of media for 1 h. Cells were then washed and trypsinized to suspend cells. Trypsinized cells were fixed in 2% PFA for ten min, then blocked in 10% FBS in PBS for 30 min. Control and ETX-treated cells were both stained with individual antibodies in cell staining buffer at 0.2 μg/mL (Biolegend, San Diego, CA, USA). Cells were also stained with a monoclonal isotype control (Abcam, Clone EPR25A, Cambridge, UK) at 0.2 μg/mL. Finally, cells were also stained with AP204 at 1:100 dilution and Normal Rabbit Serum Control (Invitrogen) was used neat as a polyclonal isotype control. Cells were stained for 30 min at RT. Cells were washed in PBS + 0.2%FBS, and antibody binding determined using a phycoerythrin PE conjugated anti-rabbit antibody (Thermo Fisher Scientific). Cells were incubated with PE conjugate Donkey anti-rabbit IgG (minimal x-reactivity) antibody (Biolegend) at 0.4 μg/mL for 30 min at RT. Cells were washed in PBS and analyzed on a FACSVerse (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (FlowJo, LCC, Ashland, Oregon, USA). Alternatively, media containing 50 nM of pETX-647 was pre-incubated with 15 μg/mL of anti-ETX antibodies for 30 min at 37 °C prior to treatment of rMAL-CHO cells.

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.).