Nonyl acridine orange nao

The treated cells (5 x 10⁴ cells/mL) were collected by centrifugation (450 x g/5 minutes at 4°C). The supernatant was discarded and pellet was resuspended with 1 mL fresh prewarmed FBS-free MEM with addition of 1 μL of 50 μM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific, Waltham, MA, USA), 5 mM nonyl acridine orange (NAO; Sigma-Aldrich, St. Louis, MO, USA), 10 mM hydroethidine (HE; Thermo Fisher Scientific, Waltham, MA, USA) or 10 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA; Thermo Fisher Scientific, Waltham, MA, USA). The cells stained with TMRE or NAO were incubated for 15 minutes at 37°C whereas cells stained with HE and DCFH-DA were incubated for 30 minutes at 37°C in the dark. After incubation, the cells were centrifuged (450 x g/5 minutes at 4°C) and pellet was washed with 1 mL chilled PBS solution. The supernatant was discarded and 500 μL of chilled PBS was used to resuspend the pellets. The stained cell suspension was transferred to flow tubes and analyzed using FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA). [dx.doi.org/10.17504/protocols.io.bdqii5ue; dx.doi.org/10.17504/protocols.io.bdqji5un; dx.doi.org/10.17504/protocols.io.bdqki5uw; dx.doi.org/10.17504/protocols.io.bdqmi5u6]

Myocytes were loaded with dyes for >20 min on a glass-bottom Petri dishes, incubated in HEPES-buffered solution (same composition as the storage solution) at 23 °C, and imaged with a LSM-510 inverted confocal microscope using a Plan-Neofluar 63 ×/1.25N.A. oil immersion lens (Carl Zeiss Inc., Jena, Germany). Scans were recorded in a single channel mode with excitation at 543 nm (for tetramethyl rhodamine methyl ester (TMRM; Molecular Probes, Inc., Eugene, OR, USA), nonyl acridine orange (NAO; Sigma-Aldrich, St. Louis, MI, USA), and mitotracker Deep Red (MTDR; ThermoFisher Scientific, Waldham, MA, USA)), collecting simultaneous fluorescence emission with LP560, LP 505, and LP650 nm, respectively. The confocal pinhole was set to obtain spatial resolutions of 0.4 μm in the horizontal plane and 1.0 μm in the axial dimension. Image processing was performed using Fiji software (U.S. National Institutes of Health, Bethesda, MD, USA). Frame scan along z-direction was performed to cover the entire space occupied with nuclei, which was identified as space of a spheroid shape (usually two per cell) poor in mitochondria. Time series mode along a single x–y plane was performed with 5 second intervals between scans.

The ROS level, mitochondrial mass and glucose uptake were determined by staining with 1 μM dihydrodichlorofluorescein diacetate (H₂DCFDA, Sigma-Aldrich, St. Louis, MO, USA), 10 nM nonyl acridine orange (NAO, Sigma-Aldrich, St. Louis, MO, USA) and 150 μM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, Life Technologies, Gaithersburg, MD, USA), respectively, for 30 min at 37 °C. The results were acquired by fluorescence microscopy (Olympus, Hamburg, Germany) and BD Accuri™ C6 (BD Biosciences, San Jose, CA, USA) with a minimum of 5000 events per sample. ROS inhibition was produced using 100 μM butylated hydroxyanisole (BHA, Sigma-Aldrich, St. Louis, MO, USA).