Mitotracker deep red 633

Preliminary experiments were performed to evaluate the amount of mitochondria in control and treated cells to exclude that possible changes in mitochondrial function could be due to uneven mitochondrial numbers. Briefly, H9c2 cells seeded on glass coverslips were loaded with MitoTracker Deep Red 633 mitochondrial fluorescent dye (0.5 μM, Life Technologies, Carlsbad, CA, USA) dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v), added to the culture medium for 20 min at 37 °C. Cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and red fluorescence was analyzed using a Leica TCS SP5 confocal scanning microscope (Leica, Mannheim, Germany) equipped with an argon laser source (excitation λ 633 nm) and a x63 oil immersion objective. Mitochondrial amount was also measured by flow cytometry. Single-cell suspensions were incubated with MitoTracker Deep Red 633 (200 nM) for 20 min at 37 °C and immediately analysed with a FACSCanto flow cytometer (Becton–Dickinson, San Jose, CA). Data were analyzed using FACSDiva software (Becton–Dickinson). Since both methods detected no substantial differences among the different experimental groups, the results of the subsequent experiments to assess mitochondrial function were deemed reliable.

MitoTracker Deep Red 633 (Life Technologies, Carlsbad, CA, USA) was used to stain mitochondria by confocal microscopy as previously described [21 (link)]. Mitochondrial number was also monitored by flow cytometry. Cells were incubated with MitoTracker Deep Red 633 (200 nM) for 20 min at 37 °C and immediately analysed by a FACSCanto flow cytometer (Becton-Dickinson).