Mitosox red

ROS in the total intracellular and TA muscle were examined using DCFH-DA or MitoSox Red staining (Ye Sen, Shanghai, China). For intracellular and mitochondrial ROS, the indicated cells were incubated with 10 μM DCFH-DA or 5 μM MitoSox Red at 37°C for 30 min, washed three times with PBS and analyzed using flow cytometry. To visualize mitochondrial ROS, the indicated cells were incubated with 100 nM MitoTracker Green FM and 5 μM MitoSox Red at 37°C for 30 min (Ye Sen). The cells were then washed three times with PBS and fixed with PFA for 20 min. Then, they were permeabilized with 0.15% Triton X-100 for 12 min and blocked with 10% FBS for 30 min. Lastly, the cells were stained with DAPI for 10 min and imaged with a confocal microscope (Nikon). For TA muscle ROS, the tissue was minced, and digested with 0.2% collagenase II and 0.05% trypsin for 40 min to obtain single-cell suspension. The cells were resuspended with 10 μM DCFH-DA in PBS for 30 min after the centrifugation, followed by washing three times with PBS and analyzed using flow cytometry. For the DCFH-DA staining of TA muscle, sections were washed with PBS and incubated with DCFH-DA (10 μM) at 37°C for 30 min. Then, sections were washed with PBS and incubated with DAPI at room temperature for 10 min to visualize nuclei. The stained sections were observed with a confocal microscope (Nikon).