Carbon formvar film

Exosomes were fixed with 2% glutaraldehyde/PBS for 30 min at room temperature. A volume of 6 μl was applied to a glow-discharged 200-mesh Cu grid coated with carbon-Formvar film (ProSciTech, Kirwan, QLD, Australia) and allowed to absorb for 5 min. Grids were washed twice with Milli-Q water and contrasted with 1.5% uranyl acetate. Transmission electron microscopy (TEM) was performed on a Tecnai G2 F30 (FEI, Eindhoven, NL) TEM operating at 300 kV across × 15,000 to × 36,000 magnification. Electron micrographs were captured with a Gatan UltraScan® 1000 2 k × 2 k CCD camera (Gatan, Pleasanton, CA).

Human bone marrow mesenchymal stem cells were processed for exosome extraction and purification using an exosome extraction kit (Umibio, China) following the manufacturer's instructions. The cells were treated for 16 h at 4 °C, and the remaining exosome-containing solution was then transferred to a purification column and centrifuged at 1500×g for 30 min to collect the exosomes. Subsequently, the exosomes in the collection column were eluted with the provided eluent. After extraction, the exosomes were initially rinsed twice with 100 μl of phosphate-buffered solution, fixed using 2 % paraformaldehyde followed by 2 % glutaraldehyde, and then placed on a 200-mesh Cu grid coated with carbon-Formvar film (ProSciTech, Kirwan, QLD, Australia). They were allowed to absorb for 5 min, and any excess solution was removed by blotting the edge of each grid with filter paper. Finally, the exosomes were stained with 20 μl of 2 % uranyl acetate in water at room temperature for 2–3 min and photographed using transmission electron microscopy (TEM, Tecnai G2 Spirit Bio Twin, FEI) at 100 kV.