Exosome Isolation and Characterization Protocol

The supernatants were collected to isolate exosomes via ultracentrifugation according to a previous study [52 (link)]. The supernatants were centrifuged at 800 g for 15 min followed by 10,000 rpm for 30 min at 4 °C to remove cells and debris and then ultrafilter and concentrated at 2000 rpm for 20 min using ultrafiltration centrifugal tube (Millipore, USA). Concentrated supernatants were centrifuged at 140,000g for 90 min at 4 °C in a Type Ti100 rotor using an XL-100K ultracentrifuge (Beckman). After resuspension in PBS, the exosome pellet was ultracentrifuged again for 90 min at 140,000g. Finally, the exosomes were resuspended in PBS, filtered using a 0.22-μm filter (Millipore, USA), and analyzed with a Enhance BCA Protein Assay kit (Beyotime, China). Approximately 6 mg of exosomes are obtained per 500 ml of supernatant. The purified exosomes were resuspended in PBS (200 µl) and further diluted by 1- to 10- hundred folds for analysis. The samples were fixed with 2.5% glutaraldehyde overnight at 4 °C. Ten microliters of the mixture were applied to copper grids and stained with 1% phosphotungstic acid for 1 min. The dried grid was observed by a Tecnai G2 TEM (FEI, USA). NTA was conducted using a Zeta View system (NanoSight, UK) to automatically track the Brownian motion and size distribution data of exosomes in real time.