Induction of neutrophil ROS production by αCD20 mAbs. PMNs were isolated by dextran sedimentation followed by standard density gradient centrifugation. (A) PMNs from healthy donors were assessed for ROS production by isoluminol-enhanced chemiluminescence in the presence of purified CLL cells (CLL:PMN ratio, 1:2) and the presence or absence of soluble RTX (10 μg/mL; Roche) or OFA (10 μg/mL; GlaxoSmithKline) and DPI (3μM; Sigma-Aldrich; n = 5). (B) Individual kinetic graph from 1 experiment shown in panel A. (C) PMNs derived from patients with CLL in the presence or absence of plate-bound RTX or OFA (10 μg/mL; n = 4). (D) Individual kinetic graph from 1 experiment shown in panel C. (E) NK cell death in coculture experiments. PMNs and NK cells were added to 96-well plates, previously coated with RTX or OFA, at a ratio of 1:1. After 16 hours at 37°C, cells were washed and stained with the live/dead fixable dead cell stain kit (Invitrogen) and NK cell viability was assessed by flow cytometry (n = 4-9). Error bars represent SEM. Data in panels A and C display total ROS production (area under curve). Statistical analyses were performed using 1-way ANOVAs with Bonferroni post hoc test for panels A and C and the Mann-Whitney U test for panel E. *P ≤ .05, **P ≤ .01, ***P ≤ .001. ANOVA, analysis of variance; PMN, polymorphonuclear cell; RLU, relative light units.