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A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles.

Arraud N, Gounou C, Linares R, Brisson AR - J. Thromb. Haemost. (2014)

Bottom Line: We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN CNRS-University of Bordeaux-IPB, Pessac, France.

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Phenotyping of Anx5-positive extracellular vesicles (EVs) detected by fluorescence (FL) triggering. A platelet-free plasma (PFP) sample was double labeled with Anx5-Cy5 and either anti-mouse-IgG-PE (A), anti-CD41-mAb-PE (B) or anti-CD235a-mAb-PE (C). FL6 fluorescence was used as a trigger, which means that all the detected events are Anx5-positive EVs. Anti-mouse-IgG-PE was used as an isotypic antibody to create a PE positivity gate on the FL6 vs. FL2 color dot plots. Events positive for only Anx5 are colored blue, while events positive for both Anx5 and a PE-conjugated mAb are colored red. On the bottom row, events labeled with Anx5 and/or a PE-conjugated antibody are backgated and displayed on FS vs. SS color dot plots. (C, top row) A small population of erythrocyte ghosts (EG) is observed in the top right corner (colored green). EG are characterized by their high intensity for both Anx5 and anti-CD235a labeling.
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fig06: Phenotyping of Anx5-positive extracellular vesicles (EVs) detected by fluorescence (FL) triggering. A platelet-free plasma (PFP) sample was double labeled with Anx5-Cy5 and either anti-mouse-IgG-PE (A), anti-CD41-mAb-PE (B) or anti-CD235a-mAb-PE (C). FL6 fluorescence was used as a trigger, which means that all the detected events are Anx5-positive EVs. Anti-mouse-IgG-PE was used as an isotypic antibody to create a PE positivity gate on the FL6 vs. FL2 color dot plots. Events positive for only Anx5 are colored blue, while events positive for both Anx5 and a PE-conjugated mAb are colored red. On the bottom row, events labeled with Anx5 and/or a PE-conjugated antibody are backgated and displayed on FS vs. SS color dot plots. (C, top row) A small population of erythrocyte ghosts (EG) is observed in the top right corner (colored green). EG are characterized by their high intensity for both Anx5 and anti-CD235a labeling.

Mentions: Finally, we attempted to phenotype the Anx5-positive EVs detected by FL triggering. We focused on the populations of EVs derived from platelets and erythrocytes, which were identified by labeling with anti-CD41-mAb-PE and anti-CD235a-mAb-PE, respectively. As shown in Fig.6, when PFP was double labeled with Anx5-Cy5 and either anti-CD41-PE (Fig.6B) or anti-CD235a-PE (Fig.6C), a population of events positive for both markers was observed in the FL6 vs. FL2 color dot plots. We found that, for this PFP sample, 23% and 8% of the Anx5-positive EVs detected by FL triggering derived from platelets and erythrocytes, respectively.


A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles.

Arraud N, Gounou C, Linares R, Brisson AR - J. Thromb. Haemost. (2014)

Phenotyping of Anx5-positive extracellular vesicles (EVs) detected by fluorescence (FL) triggering. A platelet-free plasma (PFP) sample was double labeled with Anx5-Cy5 and either anti-mouse-IgG-PE (A), anti-CD41-mAb-PE (B) or anti-CD235a-mAb-PE (C). FL6 fluorescence was used as a trigger, which means that all the detected events are Anx5-positive EVs. Anti-mouse-IgG-PE was used as an isotypic antibody to create a PE positivity gate on the FL6 vs. FL2 color dot plots. Events positive for only Anx5 are colored blue, while events positive for both Anx5 and a PE-conjugated mAb are colored red. On the bottom row, events labeled with Anx5 and/or a PE-conjugated antibody are backgated and displayed on FS vs. SS color dot plots. (C, top row) A small population of erythrocyte ghosts (EG) is observed in the top right corner (colored green). EG are characterized by their high intensity for both Anx5 and anti-CD235a labeling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359678&req=5

fig06: Phenotyping of Anx5-positive extracellular vesicles (EVs) detected by fluorescence (FL) triggering. A platelet-free plasma (PFP) sample was double labeled with Anx5-Cy5 and either anti-mouse-IgG-PE (A), anti-CD41-mAb-PE (B) or anti-CD235a-mAb-PE (C). FL6 fluorescence was used as a trigger, which means that all the detected events are Anx5-positive EVs. Anti-mouse-IgG-PE was used as an isotypic antibody to create a PE positivity gate on the FL6 vs. FL2 color dot plots. Events positive for only Anx5 are colored blue, while events positive for both Anx5 and a PE-conjugated mAb are colored red. On the bottom row, events labeled with Anx5 and/or a PE-conjugated antibody are backgated and displayed on FS vs. SS color dot plots. (C, top row) A small population of erythrocyte ghosts (EG) is observed in the top right corner (colored green). EG are characterized by their high intensity for both Anx5 and anti-CD235a labeling.
Mentions: Finally, we attempted to phenotype the Anx5-positive EVs detected by FL triggering. We focused on the populations of EVs derived from platelets and erythrocytes, which were identified by labeling with anti-CD41-mAb-PE and anti-CD235a-mAb-PE, respectively. As shown in Fig.6, when PFP was double labeled with Anx5-Cy5 and either anti-CD41-PE (Fig.6B) or anti-CD235a-PE (Fig.6C), a population of events positive for both markers was observed in the FL6 vs. FL2 color dot plots. We found that, for this PFP sample, 23% and 8% of the Anx5-positive EVs detected by FL triggering derived from platelets and erythrocytes, respectively.

Bottom Line: We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN CNRS-University of Bordeaux-IPB, Pessac, France.

Show MeSH
Related in: MedlinePlus