Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

Abstract

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

This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

Protocol

1. Tissue Dissociation and Differential Centrifugation

1. Prepare 10 mL of dissociation buffer (10 mg of papain; 5.5 mM L-cysteine; 67 µM 2-mercaptoethanol; 1.1 mM EDTA) in Hibernate-E medium for approximately 0.4–1.0 g of tissue.
   NOTE: All the solutions used for EV enrichment and purification should be diluted in ultrapure filtered water.
2. Add whole fresh or frozen tissue to dissociation buffer in a 50 mL conical tube and incubate in a warm water bath at 37 °C for 20 min. Tissue may be cut into smaller fragments before incubation if needed.
3. Following the incubation, add protease and ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 µm filter	VWR	28145-505
12 mL ultracentrifuge tubes	Beckman Coulter	331372
5.5 mL ultracentrifuge tubes	Beckman Coulter	344057
HALT phosphatase inhibitor (100x solution)	ThermoFisher Scientific	78420
HALT protease inhibitor (100x solution)	ThermoFisher Scientific	78438
Hibernate E medium	ThermoFisher Scientific	A1247601
MLS-50 swinging-bucket rotor	Beckman Coulter	367280
Optima MAX-XP Benchtop Ultracentrifuge	Beckman Coulter	393315