Mitotracker green

For the determination of mitochondrial mass, ROS, and mitochondrial membrane potential (MMP), PK-15 cells were cultured in 24-well plates. Briefly, after removing the culture medium, cells were washed with HBSS. The mitochondrial mass was probed using 0.1 μM MitoTracker Green (excitation—490 nm; emission—516 nm) (Dojindo), ROS levels were probed using 10 μM of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (excitation—480 nm; emission—525 nm) (Sigma-Aldrich, St. Louis, MO, USA), and MMP was probed using 0.1 μM JC-1 (excitation—490 nm; the monomeric form: emission—527 nm; J-aggregates: emission—590 nm.) (BD Biosciences, San Diego, CA, USA) for 30 min at 37 °C in the dark following the manufacturer’s instructions. After staining, the cells were trypsinized, collected using centrifugation (3 min, 500× g), and re-suspended in HBSS for flow cytometric analysis. All flow cytometric experiments were performed on the BD FACSCalibur™ Flow Cytometer (BD Biosciences, San Diego, CA, USA). Data were analyzed using FlowJo software, version 10 (Treestar, San Carlos, CA, USA).

IR780 was purchased from Sigma Corporation. Griess assay, H₂DCFDA, Mitotracker Red and DAF-FM DA were obtained by Beyotime Institute of Biotechnology (China). Singlet Oxygen Sensor Green (SOSG) was purchased from Thermo Fisher Scientific. Cytotoxicity ROS-ID Hypoxia/Oxidative Stress Detection Kit was obtained from Enzo Life Sciences. The 2-iminothiolane, isopentylnitrite, perfluorodecalin (FDC) and fluorescein isothiocyanate (FITC) were obtained from Aladdin Industrial Corporation (Shanghai, China). Mitotracker Green, Mitochondrial Membrane Potential Detection Kit (JC-1), and ATP assay kit were purchased from Dojindo Laboratories (Japan). All of the chemicals used in this study were of analytical grade and used without further purification.