Measurement of ROS

The level of intracellular ROS was measured using a ROS detection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's guidelines. ROS levels were measured with a FACSCalibur™ flow cytometer. To examine the role of intracellular ROS in the cytotoxicity and apoptosis induced by vitamin C, 5 mM antioxidant N-acetylcysteine (NAC; Tocris Bioscience) was incubated prior to co-incubation with vitamin C. Cytotoxicity was determined by MTT assays, and apoptosis was analyzed by flow cytometry following double staining with annexin V-FITC and PI.