Mitosox red fluorescent probe

Mitochondrial superoxide anion levels in early- and late-passage C2C12 myoblasts and differentiated C2C12 cells were evaluated using a MitoSOX^TM Red fluorescent probe (M36008, Invitrogen) that permeates live cells, selectively accumulates in mitochondria, and is rapidly oxidized by superoxide. Cells were incubated with 1.25 μM MitoSOX in HBSS Ca/Mg for 10 min in dark at 37 °C with a 5% CO₂ atmosphere. Then, cell nuclei were stained by incubating with 1 µg/mL Hoechst (HY-15559A, MedChem Express) in HBSS, for 10 min at 37 °C in the dark, with a 5% CO₂ atmosphere. Afterwards, samples were examined by confocal microscopy (Leica TCS SP8 X)), images were acquired (Leica Application Suite X version 1.8.1) and analysis was performed using Image J software (NIH, Frederick, MD, USA).

High-glucose Duldecco’s modified Eagle’s medium (DEMEM) (D5648, Sigma-Aldrich, MO, USA); Fetal Bovine Serum (FBS) (102700-106, Gibco, MA, USA); Sodium bicarbonate (S5761, Sigma-Aldrich); Antibiotic/antimycotic solution (A5955, Sigma-Aldrich); Horse Serum (16050122, Gibco); Trypan Blue dye (T8154, Sigma-Aldrich); 2’,7’-diclorofluorescin diacetate with a thiol-reactive chloromethyl group (CM-H2DCFDA, C6827, Invitrogen, CA, USA); HBSS (14025092, Gibco); Hydrogen peroxide (H2O2) (Sigma Aldrich; 216763); MitoSOXTM Red fluorescent probe (M36008, Invitrogen); Hoechst (HY-15559A, MedChem Express); Glucose (Sigma, 7021); Glutamine (Sigma; G8540); Oligomycin (Sigma; 75351); Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma; C2920); Rotenone (Sigma; R8875)/antimycin (Sigma; A8674); RIPA buffer (R0278, Sigma-Aldrich); Dithiothreitol, protease inhibitor cocktail (P8340, Sigma-Aldrich); Laemmli buffer (1610737, BioRad); Nonfat dry milk (170-6404, BioRad); TBS (50 mM Tris-HCl, (pH 7.5); Chemiluminescent horseradish peroxidase substrate (WBKLS0500, Millipore Corp., Darmstadt, Germany); Bafilomycin A1 (11038, Cayman, MI, USA); Atg7 siRNA (si-Atg7) (SI00900529, Qiagen; Hilden, Germany); Shc1 siRNA (si-Shc1) (1027418, Qiagen); scrambled siRNA (si-SCR) (D-001810-03-20; Dharmacon, Bucks, UK); Lipofectamine 2000 (11668-030; Invitrogen).

To measure mitochondrial ROS, HK-2 cells were seeded on slides in 12-well culture plates. After treatment, live HK-2 cells were stained with 10 μM DCFH-DA and 50 nM MitoTracker Red for 30 min at 37°C or incubated with MitoSOX™ Red fluorescent probe (5 μM, M36008, Invitrogen, USA) at 37°C for 20 min. Moreover, 4-μm-thick frozen sections were stained with 5 μM MitoSOX™ Red fluorescent probe for 20 min at 37°C in the dark. After being washed three times with phosphate-buffered saline (PBS), the stained HK-2 cells and frozen kidney sections were immediately visualized under a fluorescence microscope. The mitochondrial membrane potential was evaluated by using JC-1 dye (Beyotime, China), and live HK-2 cells or frozen kidney sections were incubated with JC-1 staining solution for 20 min at 37°C in the dark. After the cells were washed three times with PBS, images were subsequently acquired with a fluorescence microscope.