Mitochondria are not essential for mAb-induced ROS production and cell death. (A) To detect mitochondrial membrane depolarization (loss of ΔΨm) Raji cells were prelabeled with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; 2μM) for 30 minutes at 37°C. Cells were then treated with mAb (10 μg/mL) and assessed 4 hours later by flow cytometry. Representative histograms are shown. (B) The antiapoptotic oncoprotein BCL2 does not protect against mAb-induced loss of ΔΨm. WT Raji or a Raji cells transfected to overexpress BCL2 (Raji-BCL2) were labeled with JC-1 and treated as in panel A. Mitoxantrone (1 μg/mL) was used as positive control for apoptosis. Bars represent mean + SEM from 3 separate experiments. (C) Production of ROS by mAb is independent of BCL2 overexpression. Raji or Raji-BCL2 cells were treated with mAbs as before and assessed for production of ROS using HE analyzed by flow cytometry. Bars represent mean + SEM from 3 separate experiments. (D) Cell death and generation of ROS are independent of mitochondrial respiration. Parental Raji or respiratory deficient sub-clones were treated with mAb (10 μg/mL) then assessed for cell death (annexin V/PI) and ROS generation (HE) after 4 hours by flow cytometry. Bars represent mean + SEM from 3 separate experiments. (E) Respiratory deficient Raji subclone C2 was treated with mAb as before and the degree of HA visualized after 4 hours by light microscopy. Original magnification ×20.