Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

作者：Christina M. Coughlan, Kimberley D. Bruce, Olivier Burgy, Timothy D. Boyd, Cole R. Michel, Josselyn E. Garcia‐Perez, Vanesa Adame, Paige E. Anton, Brianne Magouirk Bettcher, Heidi J. Chial, Mélanie Königshoff, Elena W.Y. Hsieh, Michael W. Graner, Huntington Potter · 发表于：Current Protocols in Cell Biology · 年份：2020 · DOI：10.1002/cpcb.110 · 被引用次数：332 · 研究领域：Extracellular vesicles in disease、MicroRNA in disease regulation、Viral Infections and Vectors

Abstract Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation‐ and column‐based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek ® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream ® ). High‐performance liquid chromatography followed by nano‐flow liquid chromatography–mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile...