Ns300 nanoparticle characterization system

EVs were purified by sequential ultracentrifugation. Cells and debris were removed by centrifugation at 1500× g for 20 min. Large microvesicles were then removed by centrifuging the supernatants at 12,000× g for 20 min, and the supernatant was then filtered through 0.22 µm filters. Finally, EVs were collected by ultracentrifugation in 39 mL ultracentrifugation tubes at 120,000× g for 120 min in a 70 Ti fixed-angle rotor. EVs were resuspended in PBS. Protein concentration was measured by BCA (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). EV size and particle number were analyzed using the NS300 nanoparticle characterization system (NanoSight, Malvern Instruments, Malvern, Worcestershire, UK) and atomic force microscopy (vide infra).

The exosomes were purified by sequential centrifugation as previously described [44 (link),62 (link),63 (link)]. In brief, cells were cultured in media supplemented with 10% exosome-free FBS. After 72 h, cells were digested and counted. The supernatants were centrifuged at 500× g at 4 °C for 10 min to remove living cells. To remove dead cells and large cell debris, the supernatants were then centrifuged at 2000× g at 4 °C for 10 min. To remove large vesicles, the supernatants were centrifuged at 10,000× g at 4 °C for 30 min. Finally, the exosomes were harvested by centrifugation at 100,000× g at 4 °C for 70 min (Beckman 32 Ti rotor, Germany). The exosomes were washed with PBS (Thermo, USA) and pelleted again through ultracentrifugation at 100,000× g at 4 °C for 70 min. The exosome pellets were resuspended in the corresponding volume of PBS according to cell numbers to ensure that the same volume of PBS contained exosomes from the same cell number. Exosome size and particle concentration were analyzed using an NS300 nanoparticle characterization system (NanoSight, Malvern Instruments, Malvern, UK) equipped with a red laser (580 nm). The exosomes were lysed with denaturing RIPA buffer (plus the cocktail), and their markers were analyzed by WB. The exosomes were stored short-term at 4 °C and long-term at −80 °C.

Circulating small EVs were isolated from 1 mL of pre-filtered plasma using a procedure described in the exoEasy Plasma Handbook (QIAGEN) [11 (link)]. Briefly, plasma was mixed with binding buffer (XBP) and added to the exoEasy membrane affinity column for binding. After centrifugation, the flow-through was discarded and wash buffer (XWP) was added to the column to wash off nonspecifically retained materials. Subsequently, the spin column membrane was incubated with elution buffer (XE) for 5 min and centrifuged for 5 min at 500 × g to collect the eluted circulating small EVs. Circulating small EV preparations were verified by electron microscopy (JEM-2100 Plus) and further analyzed for vesicle size and particle number using the NS300 nanoparticle characterization system (NanoSight, Malvern Instruments) equipped with a blue laser (488 nm). For protein analysis, circulating small EVs were concentrated using ultracentrifugation at 120,000 × g for 4 h and subjected to Western blot analysis of known vesicle-enriched proteins, including CD9 (Invitrogen, 10626D), Syntenin-1 (Proteintech, 22,399–1-AP), and a negative marker of cellular contamination, Calnexin (Proteintech, 10,427–2-AP).