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.
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Acknowledgment
K.L. was supported by “Fondation Plan Alzheimer.” C.J. and M.C. were supported by the “Ministère de l’Enseignement Supérieur et de la Recherche.” This work was funded by INSERM, Université Grenoble Alpes, and ANR (08-Blanc-0271 to R.S.).
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Laulagnier, K., Javalet, C., Hemming, F.J., Sadoul, R. (2017). Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation. In: Hill, A. (eds) Exosomes and Microvesicles. Methods in Molecular Biology, vol 1545. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6728-5_9
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DOI: https://doi.org/10.1007/978-1-4939-6728-5_9
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6728-5
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