Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material
Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles an...
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2019-12-01
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Online Access: | http://dx.doi.org/10.1080/20013078.2019.1587567 |
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doaj-1fe916b741a541b1809dc001f6b39d592020-11-25T01:55:53ZengTaylor & Francis GroupJournal of Extracellular Vesicles2001-30782019-12-018110.1080/20013078.2019.15875671587567Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference materialAndré Görgens0Michel Bremer1Rita Ferrer-Tur2Florian Murke3Tobias Tertel4Peter A. Horn5Sebastian Thalmann6Joshua A. Welsh7Christine Probst8Coralié Guerin9Chantal M. Boulanger10Jennifer C. Jones11Helmut Hanenberg12Uta Erdbrügger13Joanne Lannigan14Franz L. Ricklefs15Samir El-Andaloussi16Bernd Giebel17University Hospital Essen, University of Duisburg-EssenUniversity Hospital Essen, University of Duisburg-EssenUniversity Hospital Essen, University of Duisburg-EssenUniversity Hospital Essen, University of Duisburg-EssenUniversity Hospital Essen, University of Duisburg-EssenUniversity Hospital Essen, University of Duisburg-EssenLuminex B.VNational Cancer Institute, National Institutes of HealthAmnis/LuminexParis Descartes UniversityParis Descartes UniversityNational Cancer Institute, National Institutes of HealthUniversity Children’s Hospital Essen, University Duisburg-EssenUniversity of VirginiaUniversity of Virginia School of MedicineUniversity Medical Center Hamburg EppendorfClinical Research Center, Karolinska InstitutetUniversity Hospital Essen, University of Duisburg-EssenExtracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.http://dx.doi.org/10.1080/20013078.2019.1587567extracellular vesiclesexosomesmicrovesiclesimaging flow cytometryflow cytometryreference materialstandardisationsubmicron particle analysiscd63 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
André Görgens Michel Bremer Rita Ferrer-Tur Florian Murke Tobias Tertel Peter A. Horn Sebastian Thalmann Joshua A. Welsh Christine Probst Coralié Guerin Chantal M. Boulanger Jennifer C. Jones Helmut Hanenberg Uta Erdbrügger Joanne Lannigan Franz L. Ricklefs Samir El-Andaloussi Bernd Giebel |
spellingShingle |
André Görgens Michel Bremer Rita Ferrer-Tur Florian Murke Tobias Tertel Peter A. Horn Sebastian Thalmann Joshua A. Welsh Christine Probst Coralié Guerin Chantal M. Boulanger Jennifer C. Jones Helmut Hanenberg Uta Erdbrügger Joanne Lannigan Franz L. Ricklefs Samir El-Andaloussi Bernd Giebel Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material Journal of Extracellular Vesicles extracellular vesicles exosomes microvesicles imaging flow cytometry flow cytometry reference material standardisation submicron particle analysis cd63 |
author_facet |
André Görgens Michel Bremer Rita Ferrer-Tur Florian Murke Tobias Tertel Peter A. Horn Sebastian Thalmann Joshua A. Welsh Christine Probst Coralié Guerin Chantal M. Boulanger Jennifer C. Jones Helmut Hanenberg Uta Erdbrügger Joanne Lannigan Franz L. Ricklefs Samir El-Andaloussi Bernd Giebel |
author_sort |
André Görgens |
title |
Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
title_short |
Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
title_full |
Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
title_fullStr |
Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
title_full_unstemmed |
Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
title_sort |
optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material |
publisher |
Taylor & Francis Group |
series |
Journal of Extracellular Vesicles |
issn |
2001-3078 |
publishDate |
2019-12-01 |
description |
Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases. |
topic |
extracellular vesicles exosomes microvesicles imaging flow cytometry flow cytometry reference material standardisation submicron particle analysis cd63 |
url |
http://dx.doi.org/10.1080/20013078.2019.1587567 |
work_keys_str_mv |
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