Immobilization of pMHCs on RBCs and Beads

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).