Dihydroethidium kit

Superoxide anion and ROS levels were measured using the dihydroethidium kit (Beyotime Biotechnology, Shanghai, China) and the Reactive Oxygen Species Detection kit (Beyotime Biotechnology, Shanghai, China), respectively. According to the manufacturer’s instruction, HK-2 cells were incubated with dihydroethidium (DHE, 1 μM) or dichlorodihydrofluorescein diacetate (DCF, 10 μM) at 37 °C for 30 min in the medium with DAPI (1 μg/mL, Beyotime Biotechnology, Shanghai, China) in the dark. Then, the cells were observed and photographed under a fluorescence microscope (IX71, OLYMPUS, Tokyo, Japan) after washing.

For kidney tissues, reactive oxygen species levels in the kidney were measured using the Dihydroethidium kit (Beyotime Biotechnology, S0063, China). Briefly, mice kidney frozen sections were incubated with Dihydroethidium (DHE) for 30 min at 37°C in the dark, and nuclei were stained with DAPI before sealing the sections. For HK-2 cells, ROS levels in HK-2 cells were assessed using a reactive oxygen species detection kit (Beyotime Biotechnology, S0033S, China). HK-2 cells were stained with Hoechst 33342 (Beyotime Biotechnology, C1027, China), and dichlorodihydrofluorescein diacetate (DCFH-DA) were incubated together for 20 min at 37°C in the dark. The fluorescence intensity was observed using a fluorescence microscope with a randomly selected field of view.

Intracellular ROS levels were measured according to the instructions of the Reactive Oxygen Species Detection Kit (Beyotime Biotechnology, S0033S, China). Briefly, HK-2 cells pretreated with different reagents were incubated with 10 μM of dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 minutes at 37°C in the dark. For tissues, fresh tissues were frozen sectioned, and then intratissue ROS levels were measured using the Dihydroethidium kit (Beyotime Biotechnology, S0063, China). Frozen sections of fresh kidneys from differently treated mice were incubated with 10 μM of Dihydroethidium at 37°C for 30 minutes in the dark. Cell nuclei were labeled by using DAPI 5 minutes before observation.