Isolation and Characterization of Extracellular Vesicles

For electron microscopy, EVs were isolated using the ExoQuick method (System Biosciences, Palo Alto, CA, USA), as described earlier.⁶² (link) Briefly, plasma was passed through a 0.8 μm filter, and ExoQuick was added as described by the manufacturer. Purified EVs were re-suspended in PBS then placed on a formvar-coated copper grid and incubated for 30 min as described.⁶³ (link) The grid was washed with 1× PBS 3 times, and samples were fixed for 10 min by placing the grid onto 2% paraformaldehyde. Fixation was followed by several washes with deionized water, and samples were contrasted by adding 2% uranyl acetate for 15 min. Samples were embedded by adding a drop of 0.13% methyl cellulose and 0.4% uranyl acetate for 10 min before subsequent examination in a Philips CM10 transmission electron microscope and imaging via Gatan CCD digital camera. The purity of EVs was determined by western blot analysis for CD63 expression as described.⁴¹ (link)