Mitosox red

To quantify cellular and mitochondrial reactive oxygen species (ROS) levels, cells were incubated with 5 μM CellROX Green (for cellular ROS; Thermo Fisher Scientific) or MitoSOX red (for mitochondrial ROS; Thermo Fisher Scientific) for 30 min. The cells were subsequently treated with TrypLE Express (Thermo Fisher Scientific) to detach them from the culture plate. The fluorescence signals of 10,000 cells were measured using a FACSCalibur instrument (BD Bioscience). The geometric means of the fluorescence signals were calculated using the Cell Quest software (BD Bioscience).
Mitochondrial ROS levels were determined using confocal microscopy as previously described.³¹, ³², ³³ In brief, cells were cultured in μ‐dishes (Ibidi) and subsequently incubated with 5 μM MitoSOX red (Thermo Fisher Scientific) and 20 nM MitoTracker Green FM (Thermo Fisher Scientific) for 30 min. Fluorescent images of MitoSOX red and MitoTracker Green FM were acquired using a Nikon C2 confocal microscope. The fluorescence intensity of MitoSOX red and MitoTracker Green FM was measured using the NIS‐Elements software (Nikon). To determine mitochondrial ROS levels, the fluorescence intensities of MitoSOX red were divided by that of MitoTracker Green FM.

In order to measure mitochondrial reactive oxygen species (ROS) levels, the cells were cultured in μ-dishes (ibidi, Munich, Germany) and then incubated with 5 μM MitoSOX Red (Thermo Fisher Scientific) and 1 μM Mitotracker Green (Thermo Fisher Scientific) for 30 min at 37 °C. Fluorescent images of MitoSOX Red and Mitotracker Green signals were acquired using a Nikon C2 confocal microscope (Nikon). The fluorescence intensity of MitoSOX Red and Mitotracker Green were measured using the NIS-Elements software (Nikon). The intensity of MitoSOX Red was divided by that of Mitotracker Green to calculate mitochondrial ROS production per mitochondrion.

C2C12 myotubes were incubated in 2 ml Dulbecco's phosphate-buffered saline (D-PBS) containing 250 nM MitoSOX Red (Invitrogen, Paisley, UK) for 30 min at 37 °C [29] (link). Myotubes were washed twice with D-PBS and were maintained in MEM without Phenol Red during the experimental period. MitoSOX Red is a derivative of dihydroethidium (DHE) designed for the selective detection of superoxide in mitochondria and exhibits fluorescence (MitoSOX Red fluorescence) upon oxidation and subsequent binding to mitochondrial DNA (Fig. 1) [30] (link).
The imaging system consisted of a C1 confocal laser-scanning microscope (Nikon Instruments Europe BV, Surrey, UK) equipped with a 405 nm excitation diode laser, a 488 nm excitation argon laser and a 543 nm excitation helium–neon laser. Emission fluorescence was detected through a set of 450/35, 515/30 and 605/15-emission filters. Using a ×40 objective, fluorescence images were captured and analysed with the EZC1 V.3.9 (12 bit) acquisition software. To quantify the degree of co-localisation of fluorescent probes in C2C12 myotubes, NIH Image J software was used. Co-localisation coefficients: Pearson's correlation (Rr) coefficient, Mander's overlap (R) coefficient and Manders's co-localisation coefficient for each image; channel 1 (Mred) and channel 2 (Mgreen) were calculated over the entire confocal image [29] (link).