Detection of Intracellular Reactive Oxygen Species (ROS)

The accumulation of intracellular ROS was examined by a ROS assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with usage of 2, 7-dichlorofluorescein diacetate (DCFH-DA, Invitrogen, USA) as a fluorescence probe. After appropriate treatment, the HCT116 cells were collected and washed with ice-cold phosphate buffered saline (PBS) thrice, followed by incubation in DMEM supplemented with 10 μM DCFH-DA for 20 min at 37°C in the dark. After centrifugation at 800 g, 4°C for 5 min, the fluorescence was evaluated by a fluorimeter excited at 488 nm and emitted at 525 nm. The images were photographed by a confocal microscope.