Exoquick ev precipitation solution

EVs were extracted from 50 µL of plasma using ExoQuick EV precipitation solution (System Biosciences, Palo Alto, CA, USA), as previously described [36 (link)], employing an additional purification step. Following the manufacturer’s instructions, 13 μL of ExoQuick buffer (System Biosciences) was added to 50 μL of plasma and incubated at 4 °C for 30 min. It was then centrifuged at 1500× g for 30 min to sediment EVs. Lastly, the supernatant was removed, and the pellet was resuspended in 100 μL of sterile PBS.

EVs were isolated from the conditioned medium of either untreated or CSE-treated HBE cells. The conditioned medium was first centrifuged at 3000 g for 15 min, and the supernatant was filtered through a 0.22 μm PVDF filter (Millipore). ExoQuick-TC EV precipitation solution (System Biosciences) was added to the filtered medium in a 1:5 ratio. After mixing and refrigeration for at least 12 h, it was centrifuged at 1500 g for 30 min. For serum EV isolation, 63 µL of ExoQuick EV precipitation solution (System Biosciences) was added to 250 µL of serum. After freezing overnight, the mixture was centrifuged at 1500 g for 30 min. EV pellets were resuspended in 1×PBS. The size distribution and concentration of the EVs were analyzed using a ZetaView particle tracker (ParticleMetrix, Germany).