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Abstract

Immunology and Infection

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Published: September 12th, 2019

DOI:

10.3791/59876

1Laboratory of Molecular Virology, School of Systems Biology, George Mason University, 2American Type Culture Collection (ATCC), 3ATCC Cell Systems, 4Ceres Nanosciences, Inc, 5Department of Immunology and Nano-medicine, Herbert Wertheim College of Medicine, Florida International University

* These authors contributed equally

Abstract

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

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