mAb-induced ROS and cell death is mediated by an NADPH oxidase. (A) Pharmacologic inhibition of NADPH oxidase blocks mAb-induced ROS production. Raji cells were pretreated with the NADPH oxidase inhibitor, diphenylene iodonium (DPI; 50μM) 1 hour before the addition of mAb (10 μg/mL). Generation of ROS was assessed 4 hours later using carboxy-H₂DCFDA staining (25μM, 30 minutes, 37°C), and analyzed by flow cytometry. Representative histograms from 3 independent experiments are shown. (B) The effect of NADPH oxidase inhibition on cell death. Raji cells were treated with DPI and mAb as in (A) and cell death quantified 4 hours post-mAb treatment using PI staining analyzed by flow cytometry. Bars represent mean + SEM from 3 separate experiments. (C) The effects of NADPH oxidase inhibition on mAb-induced cell death and (D) ROS were confirmed using a second pharmacologic inhibitor. Raji cells were pretreated with mycophenolic acid (MPA; 10μM) for 16 hours in the presence or absence of guanosine (20μM) followed by addition of anti-CD20 mAb (10 μg/mL) for 4 hours. Cell death (C) and ROS (D) were then assessed by flow cytometry using PI and HE, respectively. Bars represent mean + SEM from 3 separate experiments. (E) Raji cells were nucleofected with siRNA against NOX2 and incubated for 48 hours. Cells were then treated with mAbs (10 μg/mL) and assessed for cell death after 4 hours using PI staining (left panel). Knockdown of NOX2 was confirmed by Western blotting (right panel). Bars represent mean + SEM from 3 separate experiments.