Iodixanol gradients (Optiprep, Sigma-Aldrich D1556) were made by layering 2.5 ml each of 18, 14, 10, 6% iodixanol (from 60% iodixanol at 1.320 ± 0.001 g/ml) diluted in PBS. extracellular vesicle enriched fractions in 1.6 ml PBS were loaded onto the gradient. After ultracentrifugation at 200 000 × g (60 min, 4°C, TH-641 rotor, 13.2 ml thinwall polypropylene tubes, acceleration/deceleration 9), 12 fractions (0.96 ml/each) were collected from the top. Fraction densities were measured by absorbance at 340 nm. Fractions were diluted in 5 ml PBS and washed by ultracentrifugation at 200 000 × g (60 min, 4°C, AH-650 rotor, 5 ml Beckman Ultra-Clear tubes, acceleration/deceleration 9). Pellets were pipetted up and down 10 times and vortexed for 5 s in 120 μl PBS. Tubes were placed on ice (20 min) followed by another round of pipetting/vortexing as above. Resuspensions were stored at –80°C.