Cell death induced by mAbs correlates with generation of ROS. (A) Raji cells were incubated with a panel of mAbs (10 μg/mL) for 4 hours, and assessed for the induction of cell death (PI) and ROS (HE) by flow cytometry. Bars represent mean + SEM from at least 3 separate experiments. (B) mAb induced cell death was directly correlated with production of ROS by flow cytometric analysis of Raji cells treated with anti-CD20 mAbs as before and dual-stained with HE (to label ROS) and annexin V Cy5.5 (to label dead cells). H₂O₂ (0.1%) was used as a positive control. Representative data are shown. (C) Live cell imaging to asses the production of ROS after mAb treatment of Raji cells. Cells were prelabeled with carboxy-H₂DCFDA, and then treated with GA101 (10 μg/mL). Phase-contrast and green fluorescence images were captured and overlaid with a representative image captured 40 minutes after mAb treatment shown. Scale bar: 20 μm. mAb-induced ROS is generated within cellular aggregates toward the cell periphery (D) The amount of ROS production as measured by carboxy-H₂DCFDA staining was quantified by flow cytometry. Representative histogram data are shown. (E) Cell death (PI) and ROS (HE) induced by GA101 IgG and F(ab)′₂ fragments were compared in Raji cells treated as before. Bars represent mean + SEM from at least 3 separate experiments. Together these data demonstrate that cell death induced by mAb to CD20 and HLA-DR correlates with the generation of ROS.