CDC induced by CD20 mAbs on B-cell lines. Raji (A) and Daudi (B) cells were incubated with increasing concentrations CD20 mAbs in the presence of human complement at 37°C for 4 hours. CDC activity was determined by a standard LDH assay as described in “Cytotoxicity assays.” Data are mean ± SD (n = 5). (C) To detect C1q binding to CD20 mAbs coated on the cell surface, Raji cells were incubated with CD20 mAbs at 2 μg/mL rituximab variants at 37°C for 15 minutes. After washing, the cells were incubated with NHS for 10 minutes at 37°C. Deposition of complement components was assessed by FCM. Points represent mean (n = 3); bars represent SD. (D) Translocation of CD20 into Triton X-100-insoluble membrane fraction. Daudi cells were incubated with FITC-labeled anit-CD20 mAbs (10 μg/mL) for 15 minutes at 37°C. After washing, the cells were resuspended and treated with goat anti–human κ F(ab′)₂ fragment (x-link; Southern Biotechnology Associates Inc) or not for 30 minutes on ice. After chilling on ice, half of each sample was treated with 0.5% Triton X-100 for 15 minutes on ice. All of samples were washed and analyzed by FCM to assess bound FITC-CD20 mAbs. The graphs are representative of at least 3 experiments, each of which showed similar results.