Cells were grown in glass-bottom dishes (Matsunami Glass, Kishiwada, Japan).
After pharmacological treatments, the cells were washed twice with the
serum-free medium. Mitochondrial staining was performed using MitoBright Red
(300 nM, #MT07, Dojindo Molecular Technology, Kumamoto, Japan) at 37°C for
30 min. Thereafter, the culture media was changed, and serial confocal
images were acquired using a TCS-SP5 confocal laser scanning microscope
(Leica Microsystems, Mannheim, Germany) with excitation and emission
wavelengths set at 561 nm and 600 nm, respectively. The numbers of dots
representing mitochondria per cell were counted from 15 randomly chosen
cells. Fluorescence was quantified in the regions of interest under a
nonsaturated excitation condition, using the z-stack imaging and the
associated analysis software. The Mtphagy Dye® (#MD01, Dojindo Molecular
Technology) was used to detect mitophagy in cells. Briefly, cells were
incubated with Mtphagy Dye (200 nM) at 37°C for 45 min. After washing twice
with serum-free media, the cells were subjected to pharmacological
treatment, and images were captured using the TCS-SP5 confocal laser
scanning microscope. The proportions of cells bearing Mtphagy Dye®-positive
dots were calculated by an examiner blinded to the identity of
pharmacological treatments. All cells within the five randomly chosen visual
fields (100–300 cells/field) were examined.
After pharmacological treatments, the cells were washed twice with the
serum-free medium. Mitochondrial staining was performed using MitoBright Red
(300 nM, #MT07, Dojindo Molecular Technology, Kumamoto, Japan) at 37°C for
30 min. Thereafter, the culture media was changed, and serial confocal
images were acquired using a TCS-SP5 confocal laser scanning microscope
(Leica Microsystems, Mannheim, Germany) with excitation and emission
wavelengths set at 561 nm and 600 nm, respectively. The numbers of dots
representing mitochondria per cell were counted from 15 randomly chosen
cells. Fluorescence was quantified in the regions of interest under a
nonsaturated excitation condition, using the z-stack imaging and the
associated analysis software. The Mtphagy Dye® (#MD01, Dojindo Molecular
Technology) was used to detect mitophagy in cells. Briefly, cells were
incubated with Mtphagy Dye (200 nM) at 37°C for 45 min. After washing twice
with serum-free media, the cells were subjected to pharmacological
treatment, and images were captured using the TCS-SP5 confocal laser
scanning microscope. The proportions of cells bearing Mtphagy Dye®-positive
dots were calculated by an examiner blinded to the identity of
pharmacological treatments. All cells within the five randomly chosen visual
fields (100–300 cells/field) were examined.