A simple flow cytometry method improves the detection of phosphatidylserine‐exposing extracellular vesicles
Summary Background Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in...
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| Published in: | Journal of thrombosis and haemostasis Vol. 13; no. 2; pp. 237 - 247 |
|---|---|
| Main Authors: | , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
England
Elsevier Limited
01.02.2015
Wiley BlackWell Publishing Ltd |
| Subjects: | |
| ISSN: | 1538-7933, 1538-7836, 1538-7836 |
| Online Access: | Get full text |
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| Abstract | Summary
Background
Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).
Objectives
Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)‐exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.
Methods
The concentration of PS‐exposing EVs in platelet‐free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin‐5 (Anx5) as the specific label. In addition, PS‐exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.
Results
We show that about 50× more Anx5‐positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5‐positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering.
Conclusion
This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV‐based diagnosis assays. |
|---|---|
| AbstractList | Summary
Background
Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).
Objectives
Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)‐exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.
Methods
The concentration of PS‐exposing EVs in platelet‐free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin‐5 (Anx5) as the specific label. In addition, PS‐exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.
Results
We show that about 50× more Anx5‐positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5‐positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering.
Conclusion
This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV‐based diagnosis assays. Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM). Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders. The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids. We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000-2500 Anx5 molecules. Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering. This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays. Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).BACKGROUNDPlasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.OBJECTIVESOur objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.METHODSThe concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000-2500 Anx5 molecules. Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering.RESULTSWe show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000-2500 Anx5 molecules. Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering.This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays.CONCLUSIONThis study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays. Background: Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).Objectives: Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.Methods: The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.Results: We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering.Conclusion: This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays. Summary Background Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM). Objectives Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders. Methods The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids. Results We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per µL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000-2500 Anx5 molecules. Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering. Conclusion This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays. |
| Author | Brisson, A. R. Arraud, N. Gounou, C. Linares, R. |
| Author_xml | – sequence: 1 givenname: N. surname: Arraud fullname: Arraud, N. organization: UMR‐5248‐CBMN CNRS‐University of Bordeaux‐IPB – sequence: 2 givenname: C. surname: Gounou fullname: Gounou, C. organization: UMR‐5248‐CBMN CNRS‐University of Bordeaux‐IPB – sequence: 3 givenname: R. surname: Linares fullname: Linares, R. organization: UMR‐5248‐CBMN CNRS‐University of Bordeaux‐IPB – sequence: 4 givenname: A. R. surname: Brisson fullname: Brisson, A. R. organization: UMR‐5248‐CBMN CNRS‐University of Bordeaux‐IPB |
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| Copyright | 2014 The Authors. published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis. Copyright © 2015 International Society on Thrombosis and Haemostasis licence_http://creativecommons.org/publicdomain/zero 2014 The Authors. published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis 2014 |
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| Keywords | electron microscopy phosphatidylserines flow cytometry cell-derived microparticles blood plasma |
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Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications... Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease... Summary Background Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications... Background: Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as... |
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| SubjectTerms | Annexin A5 Biochemistry Biochemistry, Molecular Biology Bioengineering Biomarkers - blood blood plasma Cell-Derived Microparticles - chemistry Cell-Derived Microparticles - ultrastructure cell‐derived microparticles Coagulation electron microscopy Flow Cytometry Fluorescent Dyes Humans Life Sciences Male Microscopy, Electron Phenotype phosphatidylserines Phosphatidylserines - blood Predictive Value of Tests |
| Title | A simple flow cytometry method improves the detection of phosphatidylserine‐exposing extracellular vesicles |
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