Streptavidin maleimide

Human red blood cells (RBCs) were isolated from the whole blood of healthy volunteers obtained by finger-prick according to protocols approved by the Ethical Review Board of Zhejiang University. The procedures to prepare biotinylated RBCs and pMHC-coated glass beads were as described previously (Liu et al., 2014 (link)). Briefly, fresh RBCs were first washed twice with coating buffer (0.1 M NaHCO₃, 0.1 M Na₂CO₃, pH 8.5, ~180 mOsm) and then covalently linked to biotin-PEG3500-SGA (JenKem, China) by incubation for 30 min at room temperature (RT) at a concentration of 1.5 mg/mL. The biotinylated RBCs were then treated with 5 mg/mL nystatin (Aladdin, China) for 1 h at 4°C in N2 buffer (265.2 mM KCl, 38.8 mM NaCl, 0.94 mM KH₂PO₄, 4.74 mM Na₂HPO₄, 27 mM sucrose; pH 7.2, 588 mOsm) and stored in N2 buffer for BFP experiments.
To prepare pMHC-coated glass beads, borosilicate glass beads (2 μm in diameter, ThermoFisher) were first modified with mercapto-propyl-trimethoxysilane (Aladdin) and then coupled to streptavidin-maleimide (Sigma-Aldrich) (Chen et al., 2008 (link)). Finally, streptavin-coated beads were incubated with the desired concentrations of biotinylated pMHCs for 30 min at RT and resuspended in HBS with 0.5% BSA to achieve 10–15% adhesion frequency in BFP experiments for measuring TCR-pMHC bond lifetimes under force.

These procedures have been described^{5 (link), 51} . Briefly, human RBCs were isolated from the whole blood of healthy volunteers according to a protocol approved by the Institutional Review Board of the Georgia Institute of Technology. pMHCs were coupled onto RBCs by first biotinylating cells with Biotin-XNHS (Calbiochem, San Diego, CA) at pH 7.2 for 30 min at room temperature, next incubating with 0.5 mg/ml streptavidin (Pierce, Rockford, IL) for 30 min at 4 °C, finally incubating with pMHCs. To coat proteins on glass beads for BFP experiments, borosilicate glass beads (Duke Scientific, Palo Alto, CA) were first covalently coupled with mercapto-propyltrimethoxy silane (United Chemical Technologies, Bristol, PA), followed by covalently linking to tetravalent streptavidin-maleimide (Sigma-Aldrich, St. Louis, MO) in phosphate buffer saline (PBS) (pH 6.8) by overnight incubation at room temperature. Streptavindylated beads were then incubated with biotinylated pMHCs for 2 h at room temperature and finally resuspended in HEPES buffer saline (HBS) plus 0.5% bovine serum albumin (BSA).