ROS are required for mAb-induced cell death. (A) Before the addition of mAbs, cells were treated with PBS or the ROS scavenger, 4,5-dihydroxyl-1,3-benzededisulfonic acid (tiron; 10mM) for 2 hours. ROS production was then determined 4 hours later using HE staining, and analyzed by flow cytometry. Representative data from at least 3 independent experiments are shown. Bars represent mean ROS production ± SEM. (B) Cells were treated with tiron as before and the amount of mAb-induced cell death quantified 4 hours later using PI staining analyzed by flow cytometry. Bars represent mean cell death ± SEM from at least 3 separate experiments. (C) Cells were prelabeled with ⁵¹Cr for 1 hour at 37°C before treatment with tiron (10mM) for 2 hours. Cells were the treated with mAbs as before and ⁵¹Cr-release determined after 4 hours as described in “Cell death assays.” Bars represent mean + SEM from quintuplicate samples representative of 3 independent experiments.