Exosome Isolation: A Density-based Ultracentrifugation Technique Using Sucrose Cushion to Separate Exosomes from Conditioned Media of Cultured Cells

1. Exosome Isolation onto a Sucrose Cushion

1. Pre-clear the harvested conditioned media (CM) by differential centrifugation and filtration as follows.
   1. Centrifuge at 500 x g for 5 min at 4 °C. Transfer the supernatant into a new tube and discard the pellet. Repeat this centrifugation step once more, recovering the supernatant and discarding the pellet.
   2. Centrifuge the supernatant from step 1.1. at 2,000 x g for 15 min and 4 °C and then discard pellet. Filter the recovered supernatant once through 0.22 µm filters.
2. During pre-clearing, prepare 25% (w/w) sucrose solution in deuterium oxide by accurately weighing out 1.9 g (± 0.001 g) of sucrose in a universal tube, and then topping up with deuterium oxide until the weight reaches 7.6 g (± 0.001 g).
3. Fill up an ultracentrifuge tube (see Table of Materials) with 22.5 mL of pre-cleared CM. Make up the CM to 22.5 mL with 0.22 µm-filtered PBS if the current volume is less than that.
4. Place a glass pipette (see Table of Materials) in the tube and, through it, add 3 mL of sucrose solution so that the solution forms a separate layer beneath the CM.
5. Carefully place the tube containing layered CM/sucrose solution into the bucket of a swing-out rotor (see Table of Materials) and secure the bucket into the rotor.
6. Place the rotor into the ultracentrifuge (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4 °C.
7. Collect 2 mL of the sucrose layer and add this to an ultracentrifuge bottle (see Table of Materials) containing 20 mL of filtered PBS for a washing step.
8. Place the tubes into a fixed-angle rotor (see Table of Materials) and spin at 100,000 x g for 1.5 h at 4°C.
9. Carefully remove the supernatant with a 10 mL serological pipette and resuspend the pellet with 400 µL filtered PBS. Keep this exosome stock at 4 °C or -80 °C for short-term and long-term storage respectively.