Mitotracker staining and transmission electron microscopy
analysis
Cells were placed in 35 mm glass bottom dish with 10 mm micro-well
(Cellvis, Mountain View, CA, USA) and treated with or without GS for 48
h. After staining with Mitotracker (Beyotime Biotechnology, Shanghai,
China) at 200 nM diluted in pre-warmed culture media for 30 min, cells
were stained with Hoechst 33342 (Sigma-Aldrich) to mark the nuclei as
previously described (Lin et al., 2015).
Twenty visual fields in each well from three dishes at 400×
magnification were observed by
the
Nikon C2 confocal microscope with ZEN software (Nikon, Tokyo, Japan),
and mitochondria quantity was analzyed. For transmission electron
microscopy (TEM) analysis, cells treated with or without GS were fixed
in 2.5% glutaraldehyde for 1 h and then incubated with 1% osmium
tetroxide. After dehydration and embedding in Durcupan Water-Soluble
Embedding Medium, the specimens were sectioned into 60 nm by a diamond
knife and mounted on copper grids. Micrographs were viewed by TEM (FEI
Tecnai G2 20; TWIN, Hillsboro, OR, USA) to analyze and quantify the
mitochondrial size and amount in each cell from four chambers by ImageJ
software (NIH, Bethesda, MD, USA) (Liu et
al., 2018).