Alexa 647 conjugated secondary antibody

Cells were washed with PBS and stained with anti-C5b-9 monoclonal antibody (Santa Cruz Biotechnology, Inc; dilution at 1/100) for 30 minutes on ice. Then, cells were washed with PBS and stained with Alexa 647–conjugated secondary antibody (1/500 dilution; Abcam) and Alexa 488–conjugated anti-C3c antibody (1/150 dilution; Abcam) for another 30 minutes on ice. The cells were also labeled with anti-C4d biotinylated monoclonal antibody (1/50 dilution; Quidel) and phycoerythrin-streptavidin (1/500 dilution; BD Pharmingen). Ten thousand events per sample were collected by a BD FACSCalibur and data were analyzed using FlowJo software version 10.5.3 (FlowJo Inc).

Purified factor H proteins (10 µg; Complement Technology, Inc) and 20 µg/mL S1 or S2 were added to 20 µL of NHS and incubated for 15 minutes at 37°C. TF1PIGAnull cells (120 000 cells per sample) in 80 µL of GVB⁰ MgEGTA buffer (pH 6.4) were then added to the serum mixture followed by incubation for 15 minutes at 37°C with constant shaking. After the reaction, the cells were washed and stained with an anti-C5b-9 monoclonal antibody (1/100 dilution; Santa Cruz Biotechnology, Inc) on ice for 30 minutes followed by Alexa 647–conjugated secondary antibody (1/500 dilution; Abcam). Cells were also stained with Alexa 488–conjugated anti-C3c antibody (1/150 dilution; Abcam). C5b-9 and C3c depositions on the cell surface were measured by a BD FACSCalibur.