Fig. 1

Illustrative overview of the EV separation protocol. A Separation of EV from [1] PBS spiked with recombinant EV (rEV), and from body fluids [2] blood plasma and [3] urine by orthogonal biophysical methods. B OptiPrep density gradient centrifugation of rEV-spiked PBS (both top-down (TD) and bottom-up (BU) approach), crude blood plasma extracts (TD approach) and concentrated urine (BU approach). C Manual preparation of a density gradient with the addition of 100 µL 0.4% (wt/vol) trypan blue solution to the 20% and 5% (wt/vol) iodixanol solutions. The centrifuge tube is tilted to 70° and the 20% iodixanol solution is carefully, dropwise transferred to the surface of the liquid. Manual fraction collection in which the tube is being held upright and fractions are carefully collected by slowly pipetting 1 mL from the central bottom of the concave meniscus at the liquid surface. D Visualization of the Biomek 4000 automated workstation setup for density gradient preparation. Automated fraction collection with liquid-level sensing enabling precise collection of the upper 1 mL volume on the liquid surface. E Characterization of rEV-enriched fractions by fluorescent nanoparticle tracking analysis (fNTA) and p24 ELISA, and of EV preparations obtained from blood plasma and urine by LC-MS/MS and transmission electron microscopy (TEM)