Quantifying PD-L1 and PD-1 Binding Kinetics

The antibody binding and blockade assays were performed as described previously (41 (link)). Briefly, to measure PD-L1 protein and PD-L1 antibody interaction, we seeded 1 × 10⁴ BT549^cPD-L1 cells per well in 96-well plates and then incubated the plates with cIgG control (Rockland Immunochemicals), or 12C10E4 antibody, and anti-canine Alexa Fluor 488 dye conjugate (SouthernBiotech). Every hour, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). To measure PD-1 protein on the cells, we seeded 1 × 10⁴ BT549^cPD-L1 cells per well in 96-well plates, and then incubated the plates with cIgG control (Rockland Immunochemicals), or 12C10E4 antibody, cPD-1-hFc protein (human Fc protein conjugated; SinoBiological US), and/or anti-human Alexa Fluor 488 dye conjugate (Thermo Fisher Scientific). Every 3 hours, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). The Image analysis was performed according to the manufacturer's protocol.