Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation
Published: April 30, 2023
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 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. Add whole fresh or frozen tissue to dissociation buffer in a 50 mL conical tube and incubate in a warm water bath…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
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Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation. J. Vis. Exp. (Pending Publication), e20631, doi: (2023).