Polystyrene beads coated with streptavidin

DNA oligomers were purchased from Integrated DNA Technologies (www.idtdna.com) and further purified by 10% denaturing PAGE gel and stored at −20 °C. All the enzymes required for the preparation of DNA constructs were purchased from NEB (www.neb.com). The polystyrene beads coated with streptavidin or anti-digoxigenin for the single-molecule experiments were purchased from Spherotech (Lake Forest, IL). All chemicals were purchased from VWR (Radnor, Pennsylvania) with purity >98%.

Polystyrene beads coated with streptavidin of size 5.0–5.9 μm (diameter range) were purchased from Spherotech. Biotin-labeled recombinant human Fc (hFc, the Fc portion of human IgG1), human and mouse PD-1 (h/mPD-1, extracellular domain of PD-1 fused with hFc at the C-terminal) were purchased from ACRO Biosystems and BPS Bioscience, respectively. Each protein was certified with >90% purity. PE-conjugated monoclonal antibodies (mAbs) mouse anti-human PD1 (EH12.1) and hamster antimouse PD-1 (J43) (BD Biosciences) were used for flow cytometry analysis. For in vitro and in vivo PD-1 blocking studies, purified mAbs mouse antihuman PD-1 (J116) (BioXCell) and rat anti-mouse PD-1 (29F.1A12) (BioLegend) were used.