Quantitative Analysis of Exosomes From Murine Lung Cancer Cells by Flow Cytometry

Rim, Kyung-Taek; Kim, Soo Jin

doi:10.15430/jcp.2016.21.3.194

Cited by 26 publications

(28 citation statements)

References 27 publications

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“…The collected exosomes were further purified with anti-CD9 antibody-coated magnetic beads (CD9 Exo-Flow Exosome Purification Kit, System Biosciences, Cat. EXOTC10A-1) as in 54,55 .…”

Section: Collection and Purification Of Cd9-positive Exosomesmentioning

confidence: 99%

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

Yang

Rossignol

Chang

et al. 2020

Sci Rep

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Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PVinfected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, nonenveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

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“…The collected exosomes were further purified with anti-CD9 antibody-coated magnetic beads (CD9 Exo-Flow Exosome Purification Kit, System Biosciences, Cat. EXOTC10A-1) as in 54,55 .…”

Section: Collection and Purification Of Cd9-positive Exosomesmentioning

confidence: 99%

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

Yang

Rossignol

Chang

et al. 2020

Sci Rep

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“…For instance, Rim at el. developed the FACS method for the analysis of exosomes from murine lung‐cancer cells . In this method, the initial isolation of the exosomes was performed using CD9‐ or CD63‐antibody‐coated magnetic beads.…”

Section: Detection Of Exosomesmentioning

confidence: 99%

“…developed the FACS method for the analysis of exosomes from murine lung-cancer cells. [38] In this method, the initial isolation of the exosomes was performed using CD9-or CD63-antibody-coated magnetic beads. After staining the sample with an exo-fluorescein isothiocyanate exosome staining solution, the analysis of exosomes was performed via FACS.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…In recent years, many detection approaches have been extensively developed to analyze exosomes and exosomal cargo for both research and clinical purposes . For example, nanoparticle tracking analysis (NTA), enzyme‐linked immunosorbent assay (ELISA), flow cytometry, and fluorescence‐activated cell sorting (FACS) have successfully been developed for exosome quantification. ELISA is one of the most widely used exosome detection techniques, relying on a sandwich immunoassay between antibodies against the protein enriched membrane of exosomes (e.g., tetraspanin marker of exosome membrane) and a secondary type of antibody marked with horseradish peroxidase (HRP) .…”

Section: Introductionmentioning

confidence: 99%

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Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles

Boriachek

Islam

Möller

et al. 2017

Small

335

313

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Exosomes are nanoscale (≈30-150 nm) extracellular vesicles of endocytic origin that are shed by most types of cells and circulate in bodily fluids. Exosomes carry a specific composition of proteins, lipids, RNA, and DNA and can work as cargo to transfer this information to recipient cells. Recent studies on exosomes have shown that they play an important role in various biological processes, such as intercellular signaling, coagulation, inflammation, and cellular homeostasis. These functional roles are attributed to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting the physiological and pathological conditions in various diseases, including cancer and neurodegenerative, infectious, and autoimmune diseases (e.g., cancer initiation, progression, and metastasis). Due to these unique characteristics, exosomes are considered promising biomarkers for the diagnosis and prognosis of various diseases via noninvasive or minimally invasive procedures. Over the last decade, a plethora of methodologies have been developed for analyzing disease-specific exosomes using optical and nonoptical tools. Here, the major biological functions, significance, and potential role of exosomes as biomarkers and therapeutics are discussed. Furthermore, an overview of the most commonly used techniques for exosome analysis, highlighting the major technical challenges and limitations of existing techniques, is presented.

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“…For all experiments except RNA-Seq, the collected exosomes were further purified with anti-CD9 antibody-coated magnetic beads (CD9 Exo-Flow Exosome Purification Kit, System Biosciences, Cat. EXOTC10A-1) as in (Rim and Kim, 2016;Smith and Daniel, 2016).…”

Section: Collection and Purification Of Exosomes And Cd9-positive Exomentioning

confidence: 99%

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

Yang

Rossignol

Chang

et al. 2019

Preprint

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AbstractEnteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles secreted from host cells. Using biochemical and biophysical methods we identify multiple components in these secreted vesicles, including PV virions; positive and negative-sense viral RNA; essential viral replication proteins; ribosomal and regulatory cellular RNAs; and numerous host cell proteins, such as regulators of cellular metabolism and structural remodeling. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious extracellular vesicles containing both virions and a unique mat-like structure. Based on our biochemical data (western blot, RNA-Seq, and mass spectrometry), these mat-like structures are expected to be comprised of unencapsidated RNA and proteins. Our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral and host RNAs and proteins.ImportanceThe family of picornaviridae is comprised of small positive-sense RNA viruses, many of which are significant human pathogens. Picornaviruses exploit secreted extracellular vesicles for cell-to-cell viral transmission without cell lysis, and poliovirus serves as a model system for picornaviruses that are not protected by a surrounding membrane (non-enveloped viruses). The structure and contents of these vesicles secreted by virus-infected cells are described here. In addition to mature virions, these vesicles carry negative-sense, ‘template’ viral RNA and essential replication proteins, as well as cellular resources from the host. Their complex contents may comprise an enhanced virulence factor for propagation of infection, and understanding their structure and function is helping elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

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Quantitative Analysis of Exosomes From Murine Lung Cancer Cells by Flow Cytometry

Cited by 26 publications

References 27 publications

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus

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