1. Collect ~200 ml culture medium, centrifuge at 1,000 g for 10 min at 4°C to remove intact cells. 2. Transfer supernatant, centrifuge at 2,000 g for 20 min to remove debris. 3. Centrifuge at 10,000 g for 30 min to remove microvesicles. 4. Ultracentrifuge at 100,000 g for 2 hr to pellet crude exosomes; resuspend pellet in 100 μl PBS. 5. Prepare 10-90% sucrose solutions, mix crude exosome preparation with 90% sucrose solution to 82% final concentration, layer remaining sucrose solutions from 70% to 10% on top, ultracentrifuge at 100,000 g for 16 hr. 6. Collect 2 ml fractions, add 9 ml PBS to each, centrifuge at 100,000 g for 1 hr, resuspend pellets in 50 μl PBS. The third fraction (34%-40% interface) contains exosomes, the sixth fraction (70%-82% interface) contains aggregates.