Extracellular Vesicle Isolation from Cell and Plasma

EVs were extracted from HAEC cell culture medium or plasma samples using an ExoQuick precipitation kit (SBI, System Biosciences, Mountain view, CA) according to the manufacturer’s instructions. Briefly, the culture medium and plasma were thawed on ice and centrifuged at 3000×g for 15 min and 10,000×g for 30 min. For plasma EV isolation, 250 μl of the supernatant was mixed with 67 μl of the ExoQuick precipitation reagent and incubated at 4°Cfor 30 min, followed by centrifugation at 3000×g for 10 min. For the isolation of EVs in cell medium, an Amicon Ultra Centrifugal Filter Unit (100 kDa, Millipore) was used to concentrate the supernatant. The ultrafiltration supernatant was mixed with the ExoQuick precipitation reagent at the ratio of 5:1, and incubated at 4 °C overnight, followed by centrifugation at 1500×g for 30 min. The EV pellet was subsequently resuspended in 200 μl phosphate buffered saline (PBS). This isolation method has been well validated with other techniques including electron microscopy [20 (link)]. EV concentrations and size distribution were measured by nanoparticle tracking analysis (NTA) (NanoSight, NanoSight Ltd., UK).