title: A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.
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.
authors.affiliation: Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA.
authors.forename: Rocío
authors.initials: R
authors.lastname: Pérez-González
authors.affiliation: Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA.
authors.forename: Sebastien A
authors.initials: SA
authors.lastname: Gauthier
authors.affiliation: Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA.
authors.forename: Asok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Division of Analytical Pshycopharmacology, Nathan S. Kline Institute for Psychiatric Research, 140 Old OrangeburgRoad, Orangeburg, NY, 10962, USA.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Saito
authors.affiliation: Division of Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA.|Department of Psychiatry, New York University Langone Medical Center, 550 1st Avenue, New York, NY, 10016, USA.
authors.forename: Mariko
authors.initials: M
authors.lastname: Saito
authors.affiliation: Center for Dementia Research, Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA. elevy@nki.rfmh.org.|Department of Psychiatry, New York University Langone Medical Center, 550 1st Avenue, New York, NY, 10016, USA. elevy@nki.rfmh.org.|Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, 550 1st Avenue, NewYork, NY, 10016, USA. elevy@nki.rfmh.org.
authors.forename: Efrat
authors.initials: E
authors.lastname: Levy
mesh_terms: D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D017077:Culture Media, Conditioned; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D005110:Extracellular Space; D000067128:Extracellular Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D014461:Ultracentrifugation
chemical_list: D015415:Biomarkers; D017077:Culture Media, Conditioned
keywords: Brain; Delipidation; Differential ultracentrifugation; Exosomes; Extracellular space; Extracellular vesicles; Sucrose step gradient