Jc 10

Mitochondrial membrane potential (MMP) was determined using JC-10 (Enzo Life Sciences, Farmingdale, NY, USA) [45 ]. For this, 20 000 cells per well were cultured in 96-well clear plates. Nuclei were stained with Hoechst (Invitrogen, #H3570) in culture medium and cells were incubated with 3 μg/mL JC-10 in KRBH buffer for 15 min. Live cells were imaged with a 10× objective using ImageXpress Confocal (Molecular Device) and images were segmented to identify the cells. The ratio of aggregated to monomer JC-10 (590 nm/525 nm) was calculated as a readout of the mitochondrial membrane potential, using KNIME software. Fluorescence ratio was calculated in energized mitochondria (in standard culture medium) and after depolarization of the organelles, obtained by adding 1 μM FCCP (Sigma Aldrich, #C2920).
To measure mitochondrial membrane potential in primary human myotubes, 8000 cells per well were seeded in a black 96-well clear-bottom plate, coated for 1 h at RT with human fibronectin (Corning, #CLS356008) at 20 μg/mL and washed twice with PBS (Thermo Fisher, #10010023). Then Skeletal Muscle Cell Growth Medium (AmsBio, #SKM-M medium) was added and cells were incubated overnight and subjected to adenoviral infection with MCU or scrambled shRNA as described in section 2.1. JC-10 sensor was used as described above for HAP1 cells.

The dye JC-10 (Enzo Life Sciences, Farmingdale, NY, USA) was used to evaluate mitochondrial membrane potential. The third instar larval eye discs were dissected and labeled with JC-10 as described.^{49 (link)} Images were obtained on a Nikon A1 confocal microscope. The ratio of fluorescence emissions at 525 and 590 nm was used for quantification analysis.

For both ROS and mitochondrial ROS (mito-ROS) experiments, 2×10⁵ cells/ml were seeded in black 96 well plates (Greiner Bio One, UK) and allowed to settle overnight. Cells were exposed to QD for 4, 8 and 18h, washed three times with PBS and incubated for 30min in full culture media supplemented with 10 µM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA; Molecular Probes, Invitrogen, UK) for ROS or 20 μM JC-10 (Enzo Life Sciences, UK) for mito-ROS assessment. Cells were washed twice with PBS and fluorescence readings were taken in an Omega microplate reader (BMG Labtech, UK) at 480 and 520nm excitation and 540 and 590nm emission for ROS and mito-ROS analyses respectively, according to the manufacturer’s instructions. For mito-ROS experiments, results were expressed as the ratio of damaged over healthy mitochondria (green/red). For both ROS and mito-ROS experiments, data were expressed as relative to the control level. All experiments were conducted in triplicate and were accompanied with negative diluent only controls and positive control treatments of 0.33M (1%) H₂O₂ for 2h prior to chemical staining.