High protein binding 96 well plates

High protein-binding 96-well plates (Corning) were coated with recombinant human ICAM-1-Fc (Acro Biosystems) or an isotype-matiched negative control mAb (clone MOPC21, Sigma Aldrich), then washed with PBS to remove unbound ICAM-1. iNKT cells (50 ×10⁵ per well) were added in culture medium lacking IL-2. Where indicated, the following compounds were included in the culture medium: 20 U/ml recombinant human IL-12p70 (Peprotech); the indicated concentrations of U0126 (Invivogen) or Cyclosporin A (Sigma Aldrich); 1 μg/ml anti-human CD11a blocking mAb (Invitrogen, Clone HI111); 1 μg/ml anti-human CD18 blocking mAb (Biolegend, Clone TS1/18); 5 μg/ml anti-CD1d blocking mAb (clone CD1d55); or the indicated concentrations of JAK Inhibitor I (Calbiochem). Where indicated, instead of ICAM-Fc simulation the iNKT cells were exposed to plate-bound CD1d-Fc that had been pulsed with 25 ng/ml α-GalCer, as previously described (40 (link)). Each stimulation condition was performed in 3–4 replicate wells. iNKT cells were stimulated for 18–24h at 37 ^oC and 5% C0₂, then culture supernatants were harvested and assayed for IFN-γ using a sandwich ELISA (capture antibody clone MD-1 from Biolegend; biotinylated detection antibody clone 4SB.3 from BD Pharmingen). IFN-γ concentrations were determined by comparison to a standard curve of recombinant human IFN-γ (Peprotech) assayed in parallel.