Advertisement

Specific and Generic Isolation of Extracellular Vesicles with Magnetic Beads

  • Ketil W. PedersenEmail author
  • Bente Kierulf
  • Axl Neurauter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1660)

Abstract

This chapter covers magnetic bead-based isolation and analysis of the smallest members of extracellular vesicles (EVs), the exosomes (30–150 nm), generally regarded to originate from the multivesicular bodies (MVBs). Also included, are descriptions of how to prepare samples prior to isolations. The magnetic bead-based isolation workflow is dramatically shortened both by omitting the pre-enrichment step and providing an option for a very short capture time. Three direct exosome isolation strategies are described: (1) “Specific and Direct,” (2) “Semi Generic and Direct” and (3) “Generic and Direct” as well as exosome release from the magnetic beads. Detailed description of downstream exosome analysis is included covering flow cytometry, Western blot and electron microscopy. Finally, a description of exosome isolation from more complex starting material including urine and serum/plasma is discussed.

Key words

Extracellular vesicles Exosomes Direct isolation Generic isolation Analysis Flow cytometry Western Blot Magnetic beads CD9 CD63 CD81 Dynabeads™ Release Electron microscopy Functionality Serum Plasma Urine 

Notes

Acknowledgment

Anette Kullmann for data and Lisbeth Larsen (ThermoFisher Scientific) for technical assistance; Antje Hoenen and Norbert Roos (University of Oslo, Norway) for technical assistance in electron microscopy.

References

  1. 1.
    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 113:752–760CrossRefPubMedGoogle Scholar
  2. 2.
    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 183:1187–1191CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pegtel DM, van de Garde MD, Middeldorp JM (2011) Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion. Biochim Biophys Acta 1809(11-12):715–721CrossRefPubMedGoogle Scholar
  4. 4.
    Thery C, Zitvogl L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:11CrossRefGoogle Scholar
  5. 5.
    Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9(8):581–593CrossRefPubMedGoogle Scholar
  6. 6.
    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(6):654–659CrossRefPubMedGoogle Scholar
  7. 7.
    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(5):619–624CrossRefPubMedGoogle Scholar
  8. 8.
    Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Thery C (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3:26913CrossRefPubMedGoogle Scholar
  9. 9.
    Oksvold MP, Neurauter A, Pedersen KW (2015) Magnetic bead-based isolation of exosomes. In: Sioud M (ed) Methods in molecular biology: RNA interference challenges and therapeutic opportunities. Springer, New York, NY, p 18Google Scholar
  10. 10.
    Mitchell PJ, Welton J, Staffurth J, Court J, Mason MD, Tabi Z, Clayton A (2009) Can urinary exosomes act as treatment response markers in prostate cancer? J Transl Med 7:4CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Akagi T, Hanamura N, Ichiki T (2015) Measuring of individual nanobioparticles on microfluidic chips by laser dark-field imaging. J Photopolym Sci Tech 28(5):727–730CrossRefGoogle Scholar
  12. 12.
    Marimpietri D, Petretto A, Raffaghello L, Pezzolo A, Gagliani C, Tacchetti C, Mauri P, Meliolo G, Pistoia V (2013) Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression. PLoS One 8(9):e75054CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M, Giebel B (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87(1):146–150CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ketil W. Pedersen
    • 1
    Email author
  • Bente Kierulf
    • 1
  • Axl Neurauter
    • 1
  1. 1.Thermo Fisher ScientificOsloNorway

Personalised recommendations