Mini extruder kit

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.

Triton X-100, Calcein, cholesterol (CH), eosin Y, ergosterol, hematoxylin, hexamethyldisilazane (HMDS), and XTT were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). PE (from the brain), PC (from eggs), sphingomyelin (SM, from egg), PI (from bovine liver), and a mini-extruder kit were from Avanti Polar Lipids (Alabaster, AL, USA). Ethyl cyanohydroxyiminoacetate (oxyma) and 9‑fluorenylmethoxycarbonyl (Fmoc) amino acids were obtained from CEM Co. (Matthews, NC, USA) N,N′-diisopropylcarbodiimide (DIC) was acquired from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). SYTOX Green, 5/6-carboxy-tetramethyl-rhodamine succinimidyl ester (NHS-rhodamine), and MitoSOX Red were purchased from Molecular Probes (Eugene, OR, USA). All other reagents were of analytical grade.