Abstract
Exosomes and microvesicles are extracellular vesicles (EVs) released by most cell types. The role of EVs as a method of intercellular communication has led to these vesicles becoming a major area of interest in a variety of scientific fields including neuroscience. Emerging evidence is now demonstrating that the biomolecular composition of EVs, especially exosomes, can play a role in the progression of disease including various neurodegenerative diseases and cancer. In addition to the miRNA profiles of EVs, these vesicles also show interesting changes in protein expression profiles under different physiological and pathological conditions. Characterization of these profiles could prove valuable for both understanding disease pathogenesis and for the discovery of new biomarkers of disease. In this chapter, we describe a protocol for isolation of exosomes and microvesicles from immortalized HT22 cells and primary cortical neurons with sufficient yield and low serum contamination required for downstream analysis and label-free relative quantitation by mass spectrometry.
*These authors have contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zeringer E, Barta T, Li M, Vlassov AV (2015) Strategies for isolation of exosomes. Cold Spring Harb Protoc 2015(4):319–323. doi:10.1101/pdb.top074476. PubMed PMID: 25834266
Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383. doi:10.1083/jcb.201211138. PubMed PMID: 23420871; PubMed Central PMCID: PMC3575529
Muralidharan-Chari V, Clancy JW, Sedgwick A, D’Souza-Schorey C (2010) Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci 123(Pt 10):1603–1611. doi:10.1242/jcs.064386. PubMed PMID: 20445011; PubMed Central PMCID: PMC2864708
Gupta A, Pulliam L (2014) Exosomes as mediators of neuroinflammation. J Neuroinflammation 11:68. doi:10.1186/1742-2094-11-68. PubMed PMID: 24694258; PubMed Central PMCID: PMC3994210
Livshits MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM (2015) Isolation of exosomes by differential centrifugation: theoretical analysis of a commonly used protocol. Sci Rep 5:17319. doi:10.1038/srep17319. PubMed PMID: 26616523; PubMed Central PMCID: PMC4663484
Properzi F, Logozzi M, Fais S (2013) Exosomes: the future of biomarkers in medicine. Biomark Med 7(5):769–778. doi:10.2217/bmm.13.63. PubMed PMID: 24044569
Thery C, Amigorena S, Raposo G, Clayton A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr protoc Cell Biol Chapter 3:Unit 3.22. doi: 10.1002/0471143030.cb0322s30. PubMed PMID: 18228490
Woo JA, Zhao X, Khan H, Penn C, Wang X, Joly-Amado A, Weeber E, Morgan D, Kang DE (2015) Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Abeta ligation to beta1-integrin conformers. Cell Death Differ 22(6):921–934. doi:10.1038/cdd.2015.5. PubMed PMID: 25698445; PubMed Central PMCID: PMC4423195
Woo JA, Boggess T, Uhlar C, Wang X, Khan H, Cappos G, Joly-Amado A, De Narvaez E, Majid S, Minamide LS, Bamburg JR, Morgan D, Weeber E, Kang DE (2015) RanBP9 at the intersection between cofilin and Abeta pathologies: rescue of neurodegenerative changes by RanBP9 reduction. Cell Death Dis 6:1676. doi:10.1038/cddis.2015.37. PubMed PMID: 25741591; PubMed Central PMCID: PMC4385917
Dahlgren KN, Manelli AM, Stine WB Jr, Baker LK, Krafft GA, LaDu MJ (2002) Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. J Biol Chem 277(35):32046–32053. doi:10.1074/jbc.M201750200. PubMed PMID: 12058030
Meberg PJ, Miller MW (2003) Culturing hippocampal and cortical neurons. Methods Cell Biol 71:111–127. PubMed PMID: 12884689
Acknowledgment
Richard Witas, Dale Chaput, and Hirah Khan all contributed equally to this work.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Witas, R., Chaput, D., Khan, H., Stevens, S.M., Kang, D. (2017). Isolation and Proteomic Analysis of Microvesicles and Exosomes from HT22 Cells and Primary Neurons. In: Kobeissy, F., Stevens, Jr., S. (eds) Neuroproteomics. Methods in Molecular Biology, vol 1598. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6952-4_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6952-4_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6950-0
Online ISBN: 978-1-4939-6952-4
eBook Packages: Springer Protocols