Purification of exosomes 1. Grow Burkitt’s Lymphoma cell lines from 1 x 10^7 (in one 10 cm dish) to 2 x 10^8 cells (in twenty 10 cm dishes) in 200 mL RPMI 1640 medium containing 10% exosome-depleted FBS in the 5% CO2 incubator at 37 °C. 2. Harvest culture medium containing exosomes and centrifuge in 50 mL conical tubes at 1,500 RPM for 10 min at room temperature to remove cells. 3. Centrifuge the supernatant in 50 mL conical tubes at 3,500 RPM for 15 min at room temperature to remove cell debris. 4. Ultracentrifuge the supernatant in polyallomer centrifuge tubes at 25,000 RPM for 1 h at 4 °C with an SW28 rotor. 5. Resuspend the pelleted exosomes in 100 µL TNE buffer overnight at 4 °C. 6. Fractionate the exosomes using a 0.25-2.5 M sucrose gradient in TNE buffer in polyallomer centrifuge tubes at 25,000 RPM for 4 h at 4 °C with an SW40Ti rotor. The exosome fraction usually locates around the 6th fraction from the top. 7. Collect the band (about 1 mL) carefully with an autopipette. 8. Ultracentrifuge fractionated exosomes at 25,000 RPM for 1 h at 4 °C with an SW40Ti rotor. 9. Resuspend the pelleted exosomes in 100 ~ 200 µL TNE buffer overnight at 4 °C. 10. Determine the total protein concentration in the fractions by the Bradford protein assay. 11. Confirm the exosome fraction (4 µg each) by western blot analysis with anti-CD63 monoclonal antibody (1:1,000 dilution). Fluorescent labeling of exosomes 1. Incubate 1 mL of fractionated exosomes (100 ng/mL) with 6 µL of 10 µM stock solution of 1, 1'-Dioctadecyl-3, 3, 3', 3'-Tetramethylindocarbocyanine Perchlorate (DiI) in methanol for 1 h in the dark at room temperature with gentle agitation. 2. Confirm the efficiency of labeling with a small amount of exosomes under a fluorescent or confocal laser scanning microscope. 3. Aliquot and store at -80 °C.