Separation and Enrichment of Extracellular Vesicles in the Presence of Virions

Abstract

Source: DeMarino, C., et al. Purification of High Yield Extracellular Vesicle Preparations Away from Virus. J. Vis. Exp. (2019)

The video demonstrates the separation of EVs from virions and other contaminants by density gradient centrifugation, followed by capture and concentration with hydrogel nanoparticles. Washed fractions are then ready for downstream identification of those enriched in EVs.

Protocol

1. Filtration and Precipitation of Extracellular Vesicles (EVs)

1. To prepare the culture supernatant from infected or transfected cells (i.e., cell lines and/or primary cells), culture approximately 10 mL of late-log cells for 5 days at 37 °C and 5% CO₂ in appropriate culture medium (i.e., Roswell Park Memorial Institute medium [RPMI] or Dulbecco’s modified Eagle’s medium [DMEM] with 10% fetal bovine serum [FBS]).
   NOTE: All culture medium reagents should be free of EVs, and can be either purchased (see Table of Materials) or prepared in-house by pre-ultracentrifugation of serum at 100,000 ×....

Results

Figure 1: Construction of an iodixanol density gradient. (A) Relative amounts of iodixanol and PBS utilized to create each density fraction (Fraction #). The Fraction # denotes the percentage of iodixanol .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CEM CD4+ Cells	NIH AIDS Reagent Program	117	CEM
DPBS without Ca and Mg (1x)	Quality Biological	114-057-101
Exosome-Depleted FBS	Thermo Fisher Scientific	A2720801
Fetal Bovine Serum	Peak Serum	PS-FB3	Serum
HIV-1 infected U937 Cells	NIH AIDS Reagent Program	165	U1
Nalgene Syringe Filter 0.2 µm SFCA	Thermo Scientific	723-2520
Nanotrap (NT80)	Ceres Nanosciences	CN1030	Reactive Red 120 core
Nanotrap (NT82)	Ceres Nanosciences	CN2010	Cibacron Blue F3GA core
Optima XE-980 Ultracentrifuge	Beckman Coulter	A94471
OptiPrep Density Gradient Medium	Sigma-Aldrich	D1556-250mL	Iodixanol
SW 41 Ti Swinging-Bucket Rotor	Beckman Coulter	331362
Ultra-Clear Tube, 14 mm x 89 mm	Beckman Coulter	344059