Cd80 fc

50,000 cells were seeded in a microfluidic channel (#80666, μ-Slide VI 0.1, Ibidi). and incubated at 5% CO₂ and 37 °C for 24 hours. Micron-sized particles (#10003D, 2.8-μm Dynabeads, ThermoFischer) decorated with CD80-Fc (#555404, Biolegend) were flowed into the microfluidic channel to bind to the cells. A 14-pN hydrodynamic force was generated by flowing the medium through the microfluidic channel with at appropriate flow rate using a syringe pump (#NE-1002X, Pump System Inc.). The cell-bound particles were then imaged at 10-second interval for 5 minutes at 10x magnification. The particle displacement and velocity were quantified using TrackMate, a plugin function of FIJI-ImageJ. The forces generated by cells were estimated by summing the applied hydrodynamic force and interfacial drag (Park et al., 2019 (link)).

Flat-bottom 96-well plates were coated with the described amounts of anti-CD3 (OTK3; eBioscience) and human recombinant PD-L1-Fc (Biolegend), CD80-Fc (Biolegend), or IgG1-Fc (Sino Biological) diluted in PBS overnight at 4 °C. Mixture was aspirated, and PBMCs were cultured with soluble anti-CD28 (CD28.2; BD Biosciences) for the described amounts of time.

Single-cell suspensions of splenocytes were CD8 T-cell-enriched with MagniSort mouse CD8 T-cell enrichment kit (Invitrogen). Flat-bottom 96-well plates were coated with the described amounts of anti-CD3ε (145–2C11; BD Biosciences) and mouse recombinant PD-L1-Fc (Biolegend), CD80-Fc (Biolegend), or IgG1-Fc (Sino Biological) diluted in PBS overnight at 4 °C. The mixture was aspirated, and CD8 T-cells were cultured with soluble anti-CD28 (37.51; BD Biosciences) for the described amounts of time.