2 7 dichlorofluorescin diacetate dcf da

The presence of ROS in myocardial frozen sections was detected using the oxidative fluorescent dye dihydroethidium (DHE; Sigma-Aldrich, MO, USA). Each heart was perfusion-fixed with 4% paraformaldehyde, 5% sucrose, and 20 mM EDTA (pH 7.4) for 10 min; then excised and embedded in optimal-cutting-temperature compound; flash frozen in liquid nitrogen; and stored at −80°C. Serial 10-μm-thick sections of each heart were stained with DHE according to the manufacturer's instructions. Cellular mitochondrial ROS production was measured using a fluorescent microplate reader with 2′,7′-dichlorofluorescin diacetate (DCFDA; Beyotime Biotechnology, Shanghai, China), which is de-esterified intracellularly and is converted to the highly fluorescent molecule 2′,7′-dichlorofluorescin (DCF) in the presence of ROS. Mitochondrial ROS levels were determined as the intensity of DCF fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

The ROS detection assay was performed by using 2′,7′-dichlorofluorescin diacetate (DCFDA) staining (Beyotime Corporation). Briefly, DCFDA was diluted to 10 μM with DMEM medium and added to the cells. After incubation for 1 hour, cells were exposed with different agent treatments at 37°C for 10 hours. Finally, fluorescence signal intensities indicating ROS levels were recorded by flow cytometry (Beckman Coulter) using excitation and emission spectra of 488/525 nm.