As a surrogate of antigen tethered on APCs, a PLB was used to tether anti-Ig as a model membrane-associated antigen. PLBs were prepared as described previously (51 (link)). Briefly, PLBs were prepared in 8- or 18-well chambered cover glass made by nanostrip-cleaned #1.5 cover glass adhered on it with either 8-well Lab-Tek chambered cover glass (Nunc, 155411) replaced for the bottom coverglass or sticky Slide (ibidi, 81818). A working solution of 100 μM small, unilamellar vesicles was prepared from the stock solution, consisting of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC, 850375) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (DOPE-cap biotin, 870273C) from Avanti Polar Lipids at a ratio of 100:1. To bind anti-Ig to the PLB, the wells containing PLB were incubated at room temperature with streptavidin (2.5 μg/ml) for 10 min, which was followed by incubation with biotinylated goat F(ab′)2 anti-human κ + λ (1 μg/ml) for 20 min with washing between the incubations with PBS.