Increased ROS in neuroblastoma and rhabdomyosarcoma cells under hypoxia. The generation of intracellular oxidative activities was evaluated by measuring intracellular peroxide-dependent oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Sigma D6883) to form the fluorescent compound, 2′,7′-dichlorofluorescein (DCF) (50). Tumor cells were cultured in 96-well culture plates. After culturing overnight under normoxic or hypoxic (1%O2) conditions, the cells were incubated with 20μM of DCFDA at 37{degree sign}C for 30min. The culture medium was then removed, and the cells were washed twice with Hank's buffered salt solution. The fluorescence intensity of the cells in each well was determined using a Spectromax plate reader with excitation at 495nm and emission at 529nm. Thereafter cell viability was determined by AlamarBlue assay and DCF fluorescence was normalized to the viability. Results are expressed as a percentage of fluorescence intensity with respect to the cells under normoxic conditions. Error bars indicate SEM from three independent experiments. *P