Dihydroethidium dhe staining

Dihydroethidium (DHE) staining (Molecular Probes, Eugene, OR, USA) was carried out to detect the level of reactive oxygen species (ROS) levels in H9C2 cells and frozen heart sections according to the previous description (Dai et al., 2017 (link)). To evaluate the levels of ROS in H9C2 cells, H9C2 cells were seeded into 24-well plates and exposed to palmitate with or without metformin and atorvastatin for 48 h. Then, H9C2 cells were twice rinsed with phosphate-buffered saline (PBS) and incubated with 5 μmol/L of DHE for 15 min at 37°C. After that, the fluorescent images of H9C2 cells were captured by a fluorescence microscope (BX63; Olympus, Tokyo, Japan), and the fluorescence intensity was detected by a microplate reader (SpectraMax M3, Molecular Devices Inc., Sunnyvale, CA, USA) under specific wave length conditions (excitation's wavelength = 518 nm; fluorescence's wavelength = 605 nm). The levels of ROS in heart tissues were determined as follows: left ventricles were excised from mice and immediately embedded into optimal cutting temperature (OCT) compound; the tissues were cut into 10-μm-thick sections and then were incubated with DHE (5 μmol/L) in PBS in a dark and humidified container at 37°C for 15 min. Finally, the fluorescence images were observed under a fluorescence microscope.

To evaluate the effect of dapagliflozin on ROS production, superoxide anion radicals were detected by dihydroethidium (DHE) staining (Molecular Probes, Eugene, OR, USA). Briefly, the kidney sections were incubated with DHE (2 µmol/l) at 37°C in a humidified chamber protected from light for 45 min. The DHE fluorescence intensity was analyzed using BIOZERO software (Keyence) in 10 intestitia per animal.