A protocol for exosome isolation and characterization: Evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods

David W. Greening, Rong Xu, Hong Ji, Bow J. Tauro, Richard J. Simpson

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

247 Citations (Scopus)

Abstract

Exosomes are 40–150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40–150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

Original languageEnglish
Title of host publicationProteomic Profiling
Subtitle of host publicationMethods and Protocols
EditorsAnton Posch
Place of PublicationUnited States
PublisherHumana Press
Pages179-209
Number of pages31
ISBN (Electronic)9781493925506
ISBN (Print)9781493925490
DOIs
Publication statusPublished - 2015
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1295
ISSN (Print)1064-3745

Keywords

  • Density
  • EpCAM
  • Exosome
  • Extracellular vesicle
  • Immunoaffinity capture
  • Isolation
  • Proteomics
  • Protocol
  • Purification
  • Shed microvesicles

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