Chapter title |
A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.
|
---|---|
Chapter number | 10 |
Book title |
Exosomes and Microvesicles
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6728-5_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6726-1, 978-1-4939-6728-5
|
Authors |
Rocío Pérez-González, Sebastien A. Gauthier, Asok Kumar, Mitsuo Saito, Mariko Saito, Efrat Levy, Pérez-González, Rocío, Gauthier, Sebastien A., Kumar, Asok, Saito, Mitsuo, Saito, Mariko, Levy, Efrat |
Editors |
Andrew F Hill |
Abstract |
Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins. |
X Demographics
Geographical breakdown
Country | Count | As % |
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Unknown | 2 | 100% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 91 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 14 | 15% |
Researcher | 13 | 14% |
Student > Bachelor | 10 | 11% |
Student > Master | 8 | 9% |
Student > Doctoral Student | 7 | 8% |
Other | 15 | 16% |
Unknown | 24 | 26% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 18 | 20% |
Medicine and Dentistry | 10 | 11% |
Neuroscience | 9 | 10% |
Agricultural and Biological Sciences | 9 | 10% |
Engineering | 4 | 4% |
Other | 12 | 13% |
Unknown | 29 | 32% |