Nanosight tracking analysis system

A Nanosight Tracking Analysis system (Brookhaven Instruments Corp, USA) was employed to determine the particle size and particle concentration per milliliter.

For particle size determination, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were performed in accordance with the protocols. Briefly, the collected exosomes were fixed with 1% glutaraldehyde at 4 °C overnight. After washing, the vesicles were loaded onto formvar/carbon-coated nickel TEM grids and incubated for 30 min. After removing the excess fluid, our samples were then stained with aqueous phosphotungstic acid for 60 s and finally imaged by TEM. The size and concentration of vesicles were determined by a NanoSight tracking analysis system (Brookhaven Instruments Corp, USA).

ASC-Exos were extracted by differential ultracentrifugation. Briefly, ASCs (passage 3) were cultured with complete DMEM containing 10% FBS (depletion of exosomes by ultracentrifugation) for 2 days. The cell supernatants was centrifuge at 300 × g for 10 min at 4 °C, 1200×g for 20 min at 4 °C, and 10,000×g for 30 min at 4 °C to eliminate dead cells and large cell debris. II The supernatants were then collected and ultracentrifuged (Hitachi, CP100NX, Japan) at 100,000×g for 70 min at 4 °C, followed by being washed in PBS, and ultracentrifuged at 100,000×g for 70 min at 4 °C to eliminate contaminating proteins. Final pellets were resuspended in sterile 100 μl PBS and characterized by nanosight tracking analysis system (Brookhaven Instruments Corp, USA), transmission electron microscopy (JEM-1400FLASH; JEOL Ltd., Tokyo, Japan), and Western blot.