Specific and Generic Isolation of Extracellular Vesicles with Magnetic Beads
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 wordsExtracellular vesicles Exosomes Direct isolation Generic isolation Analysis Flow cytometry Western Blot Magnetic beads CD9 CD63 CD81 Dynabeads™ Release Electron microscopy Functionality Serum Plasma Urine
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.
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