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

To pre-clear the collected conditioned medium, centrifuge it at 500 x g for 5 minutes at 4 degrees Celsius. Transfer the supernatant into a new tube and discard the pellet. After repeating this centrifugation step with the recovered supernatant, recover the supernatant again and discard the pellet.

Then, centrifuge the recovered supernatant at 2,000 x g for 15 minutes and 4 degrees Celsius. Keep the supernatant and discard the pellet. Filter the supernatant once through 0.22-micrometer filter attached to a 20-milliliter syringe into a fresh centrifuge tube.

Meanwhile, to prepare 25% sucrose solution in deuterium oxide, accurately weigh out 1.9 grams of sucrose in a universal tube. Then, add deuterium oxide until the weight reaches 7.6 grams. Then, fill up an ultracentrifuge tube with 22.5 milliliters of pre-cleared conditioned medium.

Place a glass pipette in the tube. Add 3 milliliters of sucrose solution through the pipette so that the solution forms a separate layer beneath the conditioned medium. Carefully place the tube containing layered conditioned medium sucrose solution into the bucket of a swing-out rotor.

Secure the bucket into the rotor. Place the rotor into the ultracentrifuge and spin it 100,000 x g at 4 degrees Celsius for 1.5 hours. Collect 2 milliliters of the sucrose layer from the tube, 1 milliliter at a time, using a P1000 pipette with its tip attached to a 10-microliter tip and add it to an ultracentrifuge bottle containing 20 milliliters of filtered PBS for washing.

Place the tube into a fixed-angle rotor and centrifuge it at 100,000 x g at 4 degrees Celsius for 1.5 hours. Use a 10-milliliter serological pipette to carefully remove the supernatant. Resuspend the pellet with 400 microliters of filtered PBS.