Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

1. Density Gradient Purification

1. Add 1.5 mL of 60% iodixanol (stock solution in water) to the 1.5 mL sucrose/Tris buffer containing EVs to create a final solution containing 30% iodixanol. Pipette up and down several times to mix solution thoroughly.
   NOTE: Be cautious to avoid losing solution in the pipette tip, as the high iodixanol concentration is quite viscous.
2. Transfer the 30% iodixanol buffer solution containing EVs to the bottom of a 5.5 mL ultracentrifugation tube.
3. Prepare at least 1.5 mL of 20% and 10% iodixanol solutions per sample in ultrapure water from the 60% iodixanol stock solution.
4. Measure 1.3 mL of 20% iodixanol and carefully layer on top of the bottom gradient layer using a syringe and an 18 G needle. To avoid mixing the layers at the density interface, keep the bevel of the needle in contact with the inside of the ultracentrifugation tube just above the meniscus and add the solution dropwise.
5. Layer 1.2 mL of 10% iodixanol solution on top of the 20% iodixanol layer using the same technique as above.
6. Carefully balance and load the ultracentrifugation tubes into rotor buckets and centrifuge in a swing-bucket rotor at 268,000 x g for 50 min at 4 °C. Set the acceleration and deceleration speeds of the centrifuge to the minimum rates allowed to avoid disruption of the density layers.
7. Label ten 1.5 mL microcentrifuge tubes for each sample to correspond to fractions 1 through 10 of the density gradients. Fraction 1 is designated as the topmost fraction that will be first removed, while fraction 10 is the bottom fraction last removed.
8. Once the gradient ultracentrifugation has completed, gently remove the tubes from the rotor buckets and place in a stable holder. A visible band of vesicles will often be seen in the fraction of interest. Pipette ten serial fractions of 490 µL from the top of the gradient into the corresponding tubes. There will be a small amount of fluid remaining at the bottom of the tube that likely contains contaminating proteins. This sample can be discarded or retained as fraction 11, if desired.
9. Measure the refractive indices of fractions using a refractometer. This can also be performed with a control gradient run in parallel if sample conservation is necessary. Pipette 20–30 µL of each fraction containing iodixanol onto the refractometer surface and estimate the density by comparing the refractive indices to known iodixanol densities.
   NOTE: Fractions can be stored overnight in the iodixanol-containing solution if needed before proceeding to the next ultracentrifugation wash step.
10. Transfer each fraction to a clean 12 mL ultracentrifugation tube. Add 5 mL of 1x phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) to the tube and mix by pipetting slowly up and down.
    NOTE: PBS added to the samples should be particle-free, ensured by filtering sterile PBS through a 0.22 µm filter and storing at room temperature to avoid salt precipitation.
11. Add an additional 6 mL of 1x PBS to the top and again mix carefully.
12. Ultracentrifuge the tubes at 100,000 x g for 2 h at 4 °C to re-pellet small vesicles. Decant the supernatant and tap the tubes dry before lysis of vesicles for protein analysis or re-suspension of EVs for morphologic analysis.