The DOI matches its Crossref record; 49 of the 49 references it cites were found in a registry or an index.
Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization
Open accessAbstract
We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows—channel flow and cross-flow—in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics. Zhang and Lyden describe a protocol for asymmetric-flow field-flow fractionation (AF4) to separate and characterize extracellular nanoparticles for investigation of their biogenesis, function and potential in molecular diagnostics and therapeutics.
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Cites
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Modern isolation and separation techniques for extracellular vesicles
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Technologies and Standardization in Research on Extracellular Vesicles
Gandham, S., Su, X., Wood, J., Nocera, A. L., Alli, S. C., Milane, L., Zimmerman, A., Amiji, M., & Ivanov, A. R. (2020) · Trends in Biotechnology · doi:10.1016/j.tibtech.2020.05.012 · Cited by 494
Metadata
- Authors
- Zhang, H., & Lyden, D.
- Venue
- Nature Protocols
- Published
- 2019-03-04
- Volume
- 14
- Issue
- 4
- Pages
- 1027-1053
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2918972495
- Cited by
- 477
- References
- 49
- FWCI
- 34.17
- Type
- journal article
- Language
- en
- Open access
- green
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