Detailed procedures have been described previously [26 (link)]. In brief, freshly isolated human RBCs were biotinylated, conjugated with streptavidin, and incubated with biotinylated pMHC, TCR, or anti-TCR mAb (clone B20.1, eBioscience, San Diego, CA) for adhesion frequency assay with a micropipette. For BFP experiments, biotinylated RBCs were loaded with nystatin (Sigma-Aldrich, Saint Louis, MO, USA) in N2 buffer. To link biotinylated pMHC, TCR, anti-TCR, or 2GSP6 onto glass beads, bead surfaces were silanized first and then covalently linked to streptavidin–maleimide (Sigma-Aldrich). Subsaturating biotinylated pMHC, TCR, anti-TCR, or 2GSP6 was then conjugated onto streptavidinated beads. Wild-type (WT) tetravalent streptavidin was used unless specified otherwise. HPC4-tagged L-selectin was captured by divalent HPC4 mAb (clone HPC4, Roche Life Science, Indianapolis, IN, USA) covalently coupled on beads.