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A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space

  • Rocío Pérez-González
  • Sebastien A. Gauthier
  • Asok Kumar
  • Mitsuo Saito
  • Mariko Saito
  • Efrat LevyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)

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.

Key words

Extracellular vesicles Exosomes Brain Extracellular space Differential ultracentrifugation Sucrose step gradient Delipidation 

Notes

Acknowledgments

This work was supported by the National Institutes of Health (AG017617) and the Alzheimer’s Association (NIRG-14-316622).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Rocío Pérez-González
    • 1
  • Sebastien A. Gauthier
    • 1
  • Asok Kumar
    • 1
  • Mitsuo Saito
    • 2
  • Mariko Saito
    • 3
    • 4
  • Efrat Levy
    • 1
    • 4
    • 5
    Email author
  1. 1.Center for Dementia ResearchNathan S. Kline Institute for Psychiatric ResearchOrangeburgUSA
  2. 2.Division of Analytical PshycopharmacologyNathan S. Kline Institute for Psychiatric ResearchOrangeburgUSA
  3. 3.Division of NeurochemistryNathan S. Kline Institute for Psychiatric ResearchOrangeburgUSA
  4. 4.Department of PsychiatryNew York University Langone Medical CenterNew YorkUSA
  5. 5.Department of Biochemistry and Molecular PharmacologyNew York University Langone Medical CenterNewYorkUSA

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