Nhp01srm

Endothelial cells (50,000 cells per well) were seeded in a 96-well plate in serum dilution buffer (SDB) (1× annexin V buffer (51-66121E, BD Pharmingen), 1 mM MgCl₂ (68475, Sigma) and 1% BSA (A9576, Sigma)). Pooled normal human serum (NHS, Complement Technology) or cynomolgus monkey serum (NHP01SRM, BioIVT) diluted in SDB were added to appropriate wells at a final concentration of 25% and incubated for 30 min at 37 °C. For negative controls, cells were treated with 25% serum containing 10 mM EDTA (15575038, Thermo Fisher) to inactivate complement. After incubation, cells were washed and stained with phycoerythrin-conjugated anti-C3b antibodies at a 1:100 dilution (846104, BioLegend) and Ghost Dye Red 780 viability dye at a 1:500 dilution (13-0865, Tonbo Biosciences) for 30 min at 4 °C in the dark. Cells were washed twice, immediately acquired on a BD FACSymphony A3 cytometer and analysed in Flow Jo. C3b deposition was plotted as MFI and statistics were calculated using GraphPad Prism v8.2.0.

Endothelial cells (50,000 cells per well) were seeded in a 96-well plate in serum dilution buffer (SDB) (1× annexin V buffer (51-66121E, BD Pharmingen), 1 mM MgCl₂ (68475, Sigma) and 1% BSA (A9576, Sigma)). Pooled normal human serum (NHS, Complement Technology) or cynomolgus monkey serum (NHP01SRM, BioIVT) diluted in SDB were added to appropriate wells at a final concentration of 25% and incubated for 30 min at 37 °C. For negative controls, cells were treated with 25% serum containing 10 mM EDTA (15575038, Thermo Fisher) to inactivate complement. After incubation, cells were washed and stained with phycoerythrin-conjugated anti-C3b antibodies at a 1:100 dilution (846104, BioLegend) and Ghost Dye Red 780 viability dye at a 1:500 dilution (13-0865, Tonbo Biosciences) for 30 min at 4 °C in the dark. Cells were washed twice, immediately acquired on a BD FACSymphony A3 cytometer and analysed in Flow Jo. C3b deposition was plotted as MFI and statistics were calculated using GraphPad Prism v8.2.0.