Extracellular Vesicle Enrichment Protocol

For the enrichment of EVs, a protocol described by Welton et al. was used with slight modifications^{28 (link)}. In brief, the cell culture supernatant was subjected to centrifugation at 4000 × g at +4 °C for 15 min, followed by passing through a 0.22 µm filter to remove cell debris. The supernatant was then ultra-centrifuged at 200,000 × g for 2 h at +4 °C (QuickSeal tube, 90 Ti Fixed angle rotor, Beckman coulter). The supernatant was discarded and the pellet was resuspended in 100 µL of PBS. The total protein concentration of the resuspended pellet was determined using NanoDrop^TM (measuring absorbance at 280 nm, in duplicates). The isolated EVs were used as a standard preparation for determining assay sensitivities.