Mitoview red

For microscopic visualization of metabolically active mitochondria, mitochondrial membrane potential, and mitochondrial ROS, cells were cultured in 96-well plates—black/clear Sterile Imaging Plate (BD 353219, Falcon Corning, Glendale, Arizona, USA). After inducing insulin resistance and NMN treatment, the culture medium was aspirated from each well, followed by two washes with PBS. Subsequently, cells were separately incubated with 100 µL per well of MitoView Red (25 nM), JC-1 (2 µg/mL), and DHR123 (2.8 µg/mL) (GeneCopoeia, Rockville, MD, USA) diluted in serum-free culture medium at 37 °C in the dark. After 30 min, the staining solution was removed, and the cell surface was washed twice with PBS. Following staining, cells were maintained in phenol red-free culture medium (normoglycemic or hyperglycemic) and were visualized using an Olympus IX73 inverted fluorescence microscope.

H9c2 cells were seeded onto coverslips in clear-bottom confocal dishes at a density of 50,000 cells per dish. Following treatment with LPS, the cells were incubated with 30 nM MitoView Red (GeneCopoeia, United States) at 37°C for 30 min. The cells were then washed with PBS three times. Mitochondrial morphology was visualized using a confocal microscope equipped with a 60× oil immersion objective. Red fluorescence represented the mitochondria, and the lengths of the mitochondria were measured.

H9c2 cells were seeded into a μ-Slide 8-well glass bottom plate (#80826, ibidi, Germany) at a total number of 7500 per well. After treatments of D-galactose (0, 10, 20, and 40 g/l, Sigma, USA) or Mdivi-1 (40 μM, Sigma, USA), the cells were incubated with 50 nM MitoView Red (GeneCopoeia, USA) at 37°C for 30 min. Then, the cells were washed with PBS for three times. Mitochondrial morphology in each group was captured using a confocal microscope (Leica TCS SP8, Germany) equipped with a 63x oil immersion objective. Red fluorescence represents the mitochondria stained by MitoView Red.