Transmission Electron Microscopy and MVB Quantification

Cells were harvested and fixed overnight at 4°C in 0.1 M sodium phosphate buffer containing 2.5% glutaraldehyde. Then, the cell pellets were washed in phosphate buffer and incubated with 1% OsO₄ for 90 min at 4°C. Samples were dehydrated, embedded in Spurr’s resin and sectioned. Ultrathin sections (50–70 nm) were then fixed with 2% uranyl acetate for 10 min and with a lead-staining solution for 5 min. A Tecnai G2 Spirit Bio TWIN transmission electron microscope (FEI, Hillsboro, United States) was used to analyze the intracellular morphological changes. MVBs, atypical MVB Ⅱ and empty vacuoles inside the cells were identified and counted based on their morphological characteristics. A minimum of 20 MVBs in each of at least 20 cells from each experiment were analyzed for quantifying intraluminal vesicles (ILVs). Data were collected from two independent experiments.

The morphology of exosomes prepared from culture supernatants was examined by transmission electron microscopy (TEM). The exosome pellets were fixed in 2% glutaraldehyde overnight at 4°C. A drop of 10 μL exosome suspension was loaded onto Formvar/Carbon–coated TEM copper grids and allowed to stand for 20 min. Excess fluid was drawn off. The sample was post-fixed with 1% uranyl acetate for 5 min, washed with double distilled water for 8 times, and allowed to dry under an electric incandescent lamp for 10 min before being examined with a Tecnai G2 Spirit Bio TWIN transmission electron microscope (FEI, Hillsboro, United States) operated at 120 kV.