title: Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation.
abstract: Exosomes are vesicles released by most cells into their environment upon fusion of multivesicular endosomes with the plasma membrane. Exosomes are vesicles of 60-100 nm in diameter, floating in sucrose at a density of ~1.15 g/mL and carrying a number of marker proteins such as Alix, Tsg101, and Flotillin-1. We use dissociated cortical neurons cultured for around two weeks as exosome-releasing cells. In these conditions, neurons make mature synapses and form networks that can be activated by physiological stimuli. Here, we describe methods to culture differentiated cortical neurons, induce exosome release by increasing glutamatergic synapse activity, and purify exosomes by differential centrifugations followed by density separation using sucrose gradients. These protocols allow purification of neuronal exosomes released within minutes of activation of glutamatergic synapses.
authors.affiliation: Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000 Grenoble, France.|INSERM U1216, F-38000 Grenoble, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Laulagnier
authors.affiliation: Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000 Grenoble, France.|INSERM U1216, F-38000 Grenoble, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Javalet
authors.affiliation: Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000 Grenoble, France.|INSERM U1216, F-38000 Grenoble, France.
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Hemming
authors.affiliation: Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000 Grenoble, France. remy.sadoul@ujf-grenoble.fr.|INSERM U1216, F-38000 Grenoble, France. remy.sadoul@ujf-grenoble.fr.
authors.forename: Rémy
authors.initials: R
authors.lastname: Sadoul
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D055354:Exosomes / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*
chemical_list: D005971:Glutamates
keywords: Bicuculline; Exosomes; Glutamatergic synapses; Mature neurons; Primary neuronal culture; Regulated secretion