Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization
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...
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| Veröffentlicht in: | Nature protocols 2019-04, Vol.14 (4), p.1027-1053 |
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| description | 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 ( |
| doi_str_mv | 10.1038/s41596-019-0126-x |
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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.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/s41596-019-0126-x</identifier><identifier>PMID: 30833697</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/2196 ; 631/1647/2230 ; 631/57/2282 ; 631/61/350/877 ; Analytical Chemistry ; Animals ; Atomic properties ; Biological Techniques ; Biomedical and Life Sciences ; Cell culture ; Cell fractionation ; Cell separation ; Cell-Derived Microparticles - chemistry ; Channel flow ; Composition ; Computational Biology/Bioinformatics ; Conditioning ; Cross flow ; Culture Media, Conditioned - chemistry ; Exosomes ; Exosomes - chemistry ; Fractionation ; Fractionation, Field Flow - instrumentation ; Fractionation, Field Flow - methods ; Humans ; Hydrodynamics ; Life Sciences ; Light scattering ; Melanoma, Experimental - chemistry ; Melanoma, Experimental - pathology ; Membranes, Artificial ; Methods ; Mice ; Microarrays ; Micrometers ; Molecular biology ; Nanoparticles ; Nanoparticles - chemistry ; Nanoparticles - ultrastructure ; Optimization ; Organelles ; Organic Chemistry ; Particle Size ; Photon correlation spectroscopy ; Protocol ; Separation ; Tumor Cells, Cultured ; Ultracentrifugation ; Ultraviolet radiation</subject><ispartof>Nature protocols, 2019-04, Vol.14 (4), p.1027-1053</ispartof><rights>The Author(s), under exclusive licence to Springer Nature Limited 2019</rights><rights>COPYRIGHT 2019 Nature Publishing Group</rights><rights>2019© The Author(s), under exclusive licence to Springer Nature Limited 2019</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c673t-c13e0b863ea30bbc4cb97480efd0e8350f17d0193b14b87fd19c4821ead676e93</citedby><cites>FETCH-LOGICAL-c673t-c13e0b863ea30bbc4cb97480efd0e8350f17d0193b14b87fd19c4821ead676e93</cites><orcidid>0000-0003-0193-4131 ; 0000-0002-7158-2373</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/s41596-019-0126-x$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/s41596-019-0126-x$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,780,784,885,27923,27924,41487,42556,51318</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30833697$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhang, Haiying</creatorcontrib><creatorcontrib>Lyden, David</creatorcontrib><title>Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>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.</description><subject>631/1647/2196</subject><subject>631/1647/2230</subject><subject>631/57/2282</subject><subject>631/61/350/877</subject><subject>Analytical Chemistry</subject><subject>Animals</subject><subject>Atomic properties</subject><subject>Biological Techniques</subject><subject>Biomedical and Life Sciences</subject><subject>Cell culture</subject><subject>Cell fractionation</subject><subject>Cell separation</subject><subject>Cell-Derived Microparticles - chemistry</subject><subject>Channel flow</subject><subject>Composition</subject><subject>Computational Biology/Bioinformatics</subject><subject>Conditioning</subject><subject>Cross flow</subject><subject>Culture Media, Conditioned - chemistry</subject><subject>Exosomes</subject><subject>Exosomes - chemistry</subject><subject>Fractionation</subject><subject>Fractionation, Field Flow - instrumentation</subject><subject>Fractionation, Field Flow - methods</subject><subject>Humans</subject><subject>Hydrodynamics</subject><subject>Life Sciences</subject><subject>Light scattering</subject><subject>Melanoma, Experimental - chemistry</subject><subject>Melanoma, Experimental - pathology</subject><subject>Membranes, Artificial</subject><subject>Methods</subject><subject>Mice</subject><subject>Microarrays</subject><subject>Micrometers</subject><subject>Molecular biology</subject><subject>Nanoparticles</subject><subject>Nanoparticles - chemistry</subject><subject>Nanoparticles - ultrastructure</subject><subject>Optimization</subject><subject>Organelles</subject><subject>Organic Chemistry</subject><subject>Particle Size</subject><subject>Photon correlation spectroscopy</subject><subject>Protocol</subject><subject>Separation</subject><subject>Tumor Cells, Cultured</subject><subject>Ultracentrifugation</subject><subject>Ultraviolet 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field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization</title><author>Zhang, Haiying ; Lyden, David</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c673t-c13e0b863ea30bbc4cb97480efd0e8350f17d0193b14b87fd19c4821ead676e93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>631/1647/2196</topic><topic>631/1647/2230</topic><topic>631/57/2282</topic><topic>631/61/350/877</topic><topic>Analytical Chemistry</topic><topic>Animals</topic><topic>Atomic properties</topic><topic>Biological Techniques</topic><topic>Biomedical and Life Sciences</topic><topic>Cell culture</topic><topic>Cell fractionation</topic><topic>Cell separation</topic><topic>Cell-Derived Microparticles - chemistry</topic><topic>Channel flow</topic><topic>Composition</topic><topic>Computational Biology/Bioinformatics</topic><topic>Conditioning</topic><topic>Cross flow</topic><topic>Culture Media, Conditioned - chemistry</topic><topic>Exosomes</topic><topic>Exosomes - chemistry</topic><topic>Fractionation</topic><topic>Fractionation, Field Flow - instrumentation</topic><topic>Fractionation, Field Flow - methods</topic><topic>Humans</topic><topic>Hydrodynamics</topic><topic>Life Sciences</topic><topic>Light scattering</topic><topic>Melanoma, Experimental - chemistry</topic><topic>Melanoma, Experimental - pathology</topic><topic>Membranes, Artificial</topic><topic>Methods</topic><topic>Mice</topic><topic>Microarrays</topic><topic>Micrometers</topic><topic>Molecular biology</topic><topic>Nanoparticles</topic><topic>Nanoparticles - chemistry</topic><topic>Nanoparticles - ultrastructure</topic><topic>Optimization</topic><topic>Organelles</topic><topic>Organic Chemistry</topic><topic>Particle Size</topic><topic>Photon correlation spectroscopy</topic><topic>Protocol</topic><topic>Separation</topic><topic>Tumor Cells, Cultured</topic><topic>Ultracentrifugation</topic><topic>Ultraviolet radiation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zhang, Haiying</creatorcontrib><creatorcontrib>Lyden, David</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Middle School</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical 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Protoc</addtitle><date>2019-04-01</date><risdate>2019</risdate><volume>14</volume><issue>4</issue><spage>1027</spage><epage>1053</epage><pages>1027-1053</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>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.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>30833697</pmid><doi>10.1038/s41596-019-0126-x</doi><tpages>27</tpages><orcidid>https://orcid.org/0000-0003-0193-4131</orcidid><orcidid>https://orcid.org/0000-0002-7158-2373</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | 631/1647/2196 631/1647/2230 631/57/2282 631/61/350/877 Analytical Chemistry Animals Atomic properties Biological Techniques Biomedical and Life Sciences Cell culture Cell fractionation Cell separation Cell-Derived Microparticles - chemistry Channel flow Composition Computational Biology/Bioinformatics Conditioning Cross flow Culture Media, Conditioned - chemistry Exosomes Exosomes - chemistry Fractionation Fractionation, Field Flow - instrumentation Fractionation, Field Flow - methods Humans Hydrodynamics Life Sciences Light scattering Melanoma, Experimental - chemistry Melanoma, Experimental - pathology Membranes, Artificial Methods Mice Microarrays Micrometers Molecular biology Nanoparticles Nanoparticles - chemistry Nanoparticles - ultrastructure Optimization Organelles Organic Chemistry Particle Size Photon correlation spectroscopy Protocol Separation Tumor Cells, Cultured Ultracentrifugation Ultraviolet radiation |
| title | Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization |
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