Dcfh da fluorescent probe

NRK52E intracellular ROS was measured with a DCFH-DA fluorescent probe (Nanjing jiancheng Bioengineering Institude, Nanjing, China). The drug-incubated cells were incubated with DCFH-DA (10 μM) for 30 min in a humidified chamber in the dark. Fluorescence signals were acquired and quantified using Image-Pro Plus software (version 3.0, Media Cybernetics, Bethesda, MD, USA) by using the same parameters.

MDA assay was performed according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Seventy -two h after transfection, a specified extraction solvent was added and cells were harvested after centrifugation and the protein was quantified. Glacial acetic acid and other reagents in the kit were added before heating for 40 min in a 95 °C water bath. The absorbance was measured at 530 nm wavelength. ROS assay was conducted using a DCFH-DA fluorescent probe (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and the final concentration was 10 μM. After incubation at 37 °C for 60 min, the fluorescence intensity was detected. The excitation wavelength was set at 485 nm while the emission wavelength was 525 nm.