Activation of OT1 T Cells with pMHC Monomers

To activate OT1 T cells with pMHC monomers, 8-well coverglass chamber slides (Nunc LabTek II Chambered #1.5, Thermo Scientific, Waltham, MA) were incubated sequentially for one hour each with 1 mg/ml BSA-biotin, 1 mg/ml streptavidin (SA), and 10 μg/ml pMHCs plus 5 μg/ml mouse recombinant CD80 (Sino Biological, Beijing, China). Three wash steps were performed between each addition. Activation of OT1 T cells on glass-supported planar lipid-bilayer was performed as described previously [45 (link),50 ]. In brief, biotin-CAP-PE and DGS-Ni were passed through a mini-extruder kit (Avanti Polar Lipids, Alabaster, AL) to generate liposomes, which were then mixed and added to a flow chamber formed between a clean glass slide and a cover slip. Following a one hour incubation at RT, the chamber was blocked using 5% w/v casein in 1X PBS. Finally, the flow chamber was incubated with 1 mg/ml SA, 10 μg/ml pMHCs, and 10 μg/ml recombinant mouse His-ICAM-1.