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Crude EVs are loaded on top of a discontinuous iodixanol gradient and centrifuged for 17 h at 100,000 × g, 4 °C. Following centrifugation, three EV-containing fractions are isolated. These fractions are washed and re-pelleted to remove the iodixanol. All three samples of EVs are then combined into a single tube, washed and pelleted to create one purified EV sample.
