induced ROS-generation and apoptosis in human neuroblastoma cell. Representative flow cytometric analysis of intracellular ROS-generation in SH-SY5Y cells by 25 μM CdCl2 or 100 μM H2O2 treated for the indicated times (control;dot line, CdCl2 and H2O2; solid line) (upper panel). ROS-generation was measured using H2DCFDA as described in the materials and method and quantified (lower panel). (A). CdCl2-induced apoptosis measured by annexin V binding for various dose (upper panel) and time (lower panel) of CdCl2. Apoptotic cells were quantified. (B). SH-SY5Y cells were treated with or without a ROS scavenger, NAC (1 μM) for 1 hr and then incubated with CdCl2 (25 μM) or H2O2 for 12 hr. Apoptosis was measured by annexin V binding (upper panel) and DAPI staining (lower panel) (C). Annexin V binding apoptotic cells were quantified. Arrows indicated DAPI-positive cells shown fragmentation or nuclei condensation were observed in CdCl2 treated cells. SH-SY5Y cells were pretreated with a calcium chelater, BAPTA-AM (10 μM) for 30 min and then incubated with CdCl2 (25 μM, 12 hr). ROS-generation was quantified (D) and apoptosis was measured by DAPI staining (E). Data are presented as mean ± SEM, from at least three independent experiments. *p < 0.05, **p < 0.01 or ***p < 0.001 vs. control.