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Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation

  • Karine Laulagnier
  • Charlotte Javalet
  • Fiona J. Hemming
  • Rémy SadoulEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)

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.

Key words

Exosomes Mature neurons Primary neuronal culture Regulated secretion Glutamatergic synapses Bicuculline 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Karine Laulagnier
    • 1
    • 2
  • Charlotte Javalet
    • 1
    • 2
  • Fiona J. Hemming
    • 1
    • 2
  • Rémy Sadoul
    • 1
    • 2
    Email author
  1. 1.Univ. Grenoble AlpesGrenoble Institut des Neurosciences, GINF-38000 GrenobleFrance
  2. 2.INSERM U1216F-38000 GrenobleFrance

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