Abstract
Exosomes are here defined as extracellular vesicles (EVs) in the approximate size range of 30–100 nm in diameter, and are observed in most body fluids containing typical exosomal markers such as CD9, CD63, and CD81. Potential subpopulations of exosomes can be captured by targeting these markers using magnetic beads. Magnetic beads are versatile tools for exosome isolation and downstream analysis. Here, we describe the workflow of immuno magnetic isolation and analysis of exosomes by flow cytometry, Western immunoblotting, and electron microscopy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579
Janowska-Wieczorek A, Wysoczynski M, Kijowski J, Marquez-Curtis L, Machalinski B, Ratajczak J, Ratajczak MZ (2005) Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer. Int J Cancer 113:752–760
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659
Pegtel DM, van de Garde MD, Middeldorp JM (2011) Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion. Biochem Biophys Acta 1809:715–721
Belting M, Wittrup A (2008) Nanotubes, exosomes, and nucleic acid-binding peptides provide novel mechanisms of intercellular communication in eukaryotic cells: implications in health and disease. J Cell Biol 183:1187–1191
Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, Rak J (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624
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 3:3–22
Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293–304
Cheruvanky H, Zhou T, Pisitkun JB, Kopp MA, Knepper PST, Yuen RA (2007) Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292:4
Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G (1970) Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40:734–744
Adams A (1973) Concentration of Epstein-Barr virus from cell culture fluids with polyethylene glycol. J Gen Virol 20:391–394
Lewis GD, Metcalf TG (1988) Polyethylene glycol precipitation for recovery of pathogenic viruses, including Hepatitis A virus and human Rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol 4:5
Yoshioka Y, Konishi Y, Kosaka N, Katsuda T, Kato T, Ochiya T (2013) Comparative marker analysis of extracellular vesicles in different human cancer types. J Extracell Vesicles 2
Clayton A, Court J, Navabi H, Adams M, Mason MD, Hobot JA, Newman GR, Jasani B (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163–174
Mitchell JP, Court J, Mason MD, Tabi Z, Clayton A (2008) Increased exosome production from tumour cell cultures using the Integra CELLine Culture System. J Immunol Methods 335:98–105
Acknowledgement
Bente Kierulf, Anette Kullman, and Malin Karlsson (Life Technologies) for expert advice and critically reading the manuscript, Sasha Vlassov and Mu Li (Life Technologies) for NanoSight® data, Antje Hoenen and Norbert Roos (University of Oslo, Norway) for technical assistance in electron microscopy.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Oksvold, M.P., Neurauter, A., Pedersen, K.W. (2015). Magnetic Bead-Based Isolation of Exosomes. In: Sioud, M. (eds) RNA Interference. Methods in Molecular Biology, vol 1218. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1538-5_27
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1538-5_27
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1537-8
Online ISBN: 978-1-4939-1538-5
eBook Packages: Springer Protocols