Intracellular Reactive Oxygen Species (ROS) in HT-29 Cells

Intracellular levels of ROS were detected using the 2',7'-dichloro fluorescein diacetate (DCFH-DA) to fluorescent 2',7'-dichlorofluorescin molecular probe according to the Reactive Oxygen Species Assay Kit Protocol (Beyotime Biotechnology, Shanghai, China). HT-29 cells with an initial density of 5 × 10⁵ cells/ml were treated with 500 μg/ml TBs-E for 48 h in a 6-well plate. The cells were incubated with 10 μM DCFH-DA for 20 min at 37°C in the dark. After being washed two times in cold PBS, the cells were analyzed with a fluorescence microscope (ZEISS, Axio vert. A1) to determine the ROS fluorescence intensity.