ROS activity in cells and mitochondria was measured as previously described (24 (link)). In brief, grouped cells were loaded with 5 µmol/mL of Cell Rox probe (C10444, Thermo Fisher Scientific) or Mito Sox probe (M36008, Thermo Fisher Scientific) for 30 min or 10 min at 37 ℃ in the dark, after which the probes were discarded. Next, the cells fixed by paraformaldehyde (4%, 15 min) were stained by DAPI (1 µg/mL) for 15 min in the dark and then treated with an anti-fluorescence quencher, followed by fluorescence microscope (Olympus Corporation, Japan) observation. The average fluorescence intensity was analyzed by ImageJ software.
To determine the ROS activity in myocardial tissues, the following steps were performed using a ROS kit (mlbio). Specifically, the myocardial tissues were mixed in a certain amount of PBS solution (pH =7.4), followed by 20-min centrifugation (2,000–3,000 rpm/min). The supernatant was gathered and diluted at a ratio of 1:1. Subsequently, 50 µL samples were added to reaction wells and cultured for 1-h at 37 ℃. Afterwards, the plates were washed for 3 times with 30 s for each time. After color development, 50 µL of stop buffer was dropped immediately, and the OD value of each well was determined at 450 nm wavelength.
To determine the ROS activity in myocardial tissues, the following steps were performed using a ROS kit (mlbio). Specifically, the myocardial tissues were mixed in a certain amount of PBS solution (pH =7.4), followed by 20-min centrifugation (2,000–3,000 rpm/min). The supernatant was gathered and diluted at a ratio of 1:1. Subsequently, 50 µL samples were added to reaction wells and cultured for 1-h at 37 ℃. Afterwards, the plates were washed for 3 times with 30 s for each time. After color development, 50 µL of stop buffer was dropped immediately, and the OD value of each well was determined at 450 nm wavelength.
