Rituximab and milatuzumab cytotoxicity is partially dependent on ROS generation and loss of ΔΨ_m. (A-B) Jeko in panel A (top left) and Mino in panel B (top left) cells were treated with rituximab and milatuzumab in the presence of a cross-linking antibody for the indicated time points. ROS generation was determined by flow cytometric analysis using dihydroethidine dye. ROS generation is indicated by right shift of the dihydroethidine curves. Hydrogen peroxide was used as positive control. Top right panel shows pretreatment of MCL cells with the nonspecific ROS scavenger NAC (10mM) and inhibition of ROS generation induced by antibody treatment. For rescue experiments, Jeko (bottom left panel) and Mino (bottom left panel) cells were incubated with NAC for 4 and 8 hours in the absence or presence of the antibodies, and cell viability was determined by annexin V–PI staining and flow cytometry. Bottom right panel shows ΔΨ_m changes in Jeko and Mino cells treated with rituxmab, milatuzumab, or the combination of both in the presence of a cross-linking antibody, for 4, 8 and 24 hours. The ΔΨ_m changes were quantified by flow cytometry determination using JC-1. Data are shown as the percentage of treated cells with intact mitochondria relative to untreated cells at the same time points.