Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Catherine DeMarino; Robert  A. Barclay; Michelle  L. Pleet; Daniel  O. Pinto; Heather Branscome; Siddhartha Paul; Benjamin Lepene; Nazira El-Hage; Fatah Kashanchi

doi:10.3791/59876

Purificación de preparaciones de vesícula extracelular de alto rendimiento lejos del virus

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Purificación de preparaciones de vesícula extracelular de alto rendimiento lejos del virus

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07:15 min

September 12, 2019

DOI: 10.3791/59876-v

Catherine DeMarino*¹, Robert A. Barclay*¹, Michelle L. Pleet¹, Daniel O. Pinto¹, Heather Branscome^1,2, Siddhartha Paul³, Benjamin Lepene⁴, Nazira El-Hage⁵, Fatah Kashanchi¹

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

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Overview

This protocol is significant because it concentrates extracellular vesicles (EVs) through a combination of technologies, allowing for the separation of virions away from EVs. It maximizes EV recovery above the current gold standard of ultracentrifugation, making it ideal for the study of EVs and viral infections.

Key Study Components

Area of Science

Extracellular vesicles
Viral infections
Biological protocols

Background

This method incorporates EV precipitation, density gradient ultracentrifugation, and particle capture.
It allows for a streamlined workflow and a reduction of starting volume requirements.
Reproducible preparations are essential for all EV research.
The protocol can be adapted to other virus systems, such as HTLV, Ebola, and Zika.

Purpose of Study

To isolate extracellular vesicles with high efficiency and yield.
To provide a method for virus-free EV preparations.
To facilitate multiple downstream analysis and characterization of EVs.

Methods Used

EV precipitation
Density gradient ultracentrifugation
Particle capture techniques
Optimization of nanoparticle preparation

Main Results

High efficiency in isolating EVs from virions.
Improved recovery rates compared to traditional methods.
Adaptability to various viral systems.
Potential challenges for first-time users in nanoparticle preparation.

Conclusions

This protocol offers a reliable method for studying EVs in the context of viral infections.
It sets a new standard for EV isolation techniques.
Careful adherence to specifications is recommended for optimal results.

Frequently Asked Questions

What are extracellular vesicles?

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play a role in cell communication.

How does this protocol improve EV isolation?

This protocol combines multiple technologies to enhance the efficiency and yield of EV isolation compared to traditional methods.

Can this method be used for different viruses?

Yes, the protocol can be adapted for various viruses, including HTLV, Ebola, and Zika.

What challenges might a first-time user face?

First-time users may struggle with nanoparticle preparation, so following the specifications closely is advised.

What is the significance of separating virions from EVs?

Separating virions from EVs is crucial for obtaining pure EV preparations for accurate analysis and characterization.

Is optimization necessary for this protocol?

Some optimization may be necessary depending on the specific system being used.

Este protocolo aísla las vesículas extracelulares (EV) lejos de los viriones con alta eficiencia y rendimiento mediante la incorporación de precipitación EV, ultracentrifugación de gradiente de densidad y captura de partículas para permitir un flujo de trabajo simplificado y una reducción del inicio requisitos de volumen, lo que resulta en preparaciones reproducibles para su uso en todas las investigaciones de vehículos eléctrico.

Este protocolo es significativo porque concentra vesículas extracelulares, vehículos eléctricos, a través de una combinación de tecnologías, al tiempo que permite la separación de viriones lejos de los vehículos eléctricos. Este método maximiza la recuperación de EV por encima del estándar de oro actual de ultracentrifugación para múltiples análisis aguas abajo y caracterización de preparaciones EV libres de virus. Este protocolo es ideal para el estudio de los vehículos eléctricos y las infecciones virales.

Este método se puede adaptar a otros sistemas de virus, como la HTLV, el ébola, el zika y más. Yo esperaría que un usuario por primera vez de este método para luchar con la preparación de nanopartículas, por lo que recomendamos seguir cuidadosamente nuestras especificaciones. Puede ser necesaria alguna optimización, dependiendo del sistema.

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