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

Samenvatting

Source: Urwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)

This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

Protocol

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 solutio....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
0.45 µm filter	VWR	28145-505
12 mL ultracentrifuge tubes	Beckman Coulter	331372
5.5 mL ultracentrifuge tubes	Beckman Coulter	344057
FA-45-6-30 rotor	Eppendorf	5820715006
MLS-50 swinging-bucket rotor	Beckman Coulter	367280
Optima MAX-XP Benchtop Ultracentrifuge	Beckman Coulter	393315
Optima XE-100 ultracentrifuge	Beckman Coulter	A94516
Optiprep	Sigma	D1556	60% iodixanol in sterile water solution
Refracto 30PX (refractometer)	Mettler Toledo	51324650
S-4-104 rotor	Eppendorf	5820759003
SW 41 Ti swinging-bucket Rotor	Beckman Coulter	333790
Tabletop 5804R centrifuge	Eppendorf	22623508