Isolation and Purification of Extracellular Vesicles

We previously developed a method for isolating EVs from plasma samples (Karimi et al., 2018 (link)), and this was applied in the current study for comparing the composition of EVs in serum and plasma. Briefly, a 50% iodixanol (OptiPrep™, Sigma Aldrich) working solution was prepared and used to further prepare 30% and 10% iodixanol solutions. Next, 6‐ml plasma or serum was layered on top of 2‐ml 50%, 2‐ml 30% and 2‐ml 10% iodixanol solutions (13.2 mL, Open‐Top Thinwall Ultra‐Clear Tube, product no: 344059, Beckman Coulter) before being ultracentrifuged at 178,000 × g_avg for 2 h at 4°C (SW 41 Ti rotor, k‐factor 143.9, Beckman Coulter). A visible EV‐enriched band with a volume of 1 ml was collected from the 30% and 10% interface and then loaded onto a home‐made SEC column packed with Sepharose CL‐2B (GE Healthcare, Uppsala, Sweden) in a Telos SPE column (Kinesis, Cambridgeshire, UK) as previously described in Ax et al. (2020 (link)) and Karimi et al. (2018 (link)). The collection of 0.5‐ml fractions immediately begun when the sample was added. In total, 30 fractions of 0.5 ml were eluted and collected using 0.2‐μm‐filtered PBS as the elution buffer.