Glass surfaces were coated with either stimulatory antibody (C305) or streptavidin (SA). Surfaces were then washed, and biotinylated monomeric pMHC complexes were incubated with streptavidin surfaces to immobilize them. Surfaces were then washed and used for antigen presentation. To assess synapse formation, J.OT1 cells were dropped onto cover glass coated with various stimulatory reagents for 30 min at 37°C. Cells were subsequently fixed with 4% paraformaldehyde. After fixation, the cell membrane was stained using either AF-594 anti–human-CD45 (Biolegend) or AF-594 wheat germ agglutinin (Thermo Fisher Scientific). Fixed immunological synapses formed on glass surfaces were imaged with the Nikon Ti Microscope using total internal reflection fluorescence microscopy at the UCSF Nikon Imaging Center. The areas of synapses were processed and quantified using ImageJ software. The pMHC complexes were provided by the Palmer laboratory and the NIH tetramer core.

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