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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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Flow cytometry (FCM) analysis of a platelet-free plasma (PFP) sample by FS triggering (A,B) and FL triggering (C,D), in the absence of Ca2+ (A, C) and in the presence of Ca2+ (B, D). (A-D). Each of the four panels presents on the left an FS vs. SS color dot plot and on the right an FL1 vs. SS color dot plot. Thresholds are represented by a solid blue line, while fluorescence positivity gates are represented by a dashed blue line. The positions of 500-nm and 1-μm polystyrene particles are indicated in each FS vs. SS plot (Fig. S2). (A,B) FS triggering analysis. The acquisition time was 10 min. Events labeled with Anx5-Fluo are separated into two groups: events of high fluorescence intensity forming a well-defined cluster are colored blue, and all the other Anx5-positive events are colored red. (C,D) FL triggering analysis. The acquisition time was 1 min, which explains why the cluster of erythrocyte ghosts (colored blue) contains less events than in (B).
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fig01: Flow cytometry (FCM) analysis of a platelet-free plasma (PFP) sample by FS triggering (A,B) and FL triggering (C,D), in the absence of Ca2+ (A, C) and in the presence of Ca2+ (B, D). (A-D). Each of the four panels presents on the left an FS vs. SS color dot plot and on the right an FL1 vs. SS color dot plot. Thresholds are represented by a solid blue line, while fluorescence positivity gates are represented by a dashed blue line. The positions of 500-nm and 1-μm polystyrene particles are indicated in each FS vs. SS plot (Fig. S2). (A,B) FS triggering analysis. The acquisition time was 10 min. Events labeled with Anx5-Fluo are separated into two groups: events of high fluorescence intensity forming a well-defined cluster are colored blue, and all the other Anx5-positive events are colored red. (C,D) FL triggering analysis. The acquisition time was 1 min, which explains why the cluster of erythrocyte ghosts (colored blue) contains less events than in (B).

Mentions: PFP samples were first analyzed by a conventional FCM approach, in which the trigger was set on the forward scatter (FS) parameter and the detection was limited to events labeled by Anx5-F* (Fig.1A,B). The concentration of Anx5-positive EVs detected by this approach was 457 ± 178 (n = 10) per μL pure PFP. This value is consistent with previous reports on EVs from normal PFP [20,23,28,30]. Displayed on a FS vs. side scatter (SS) color dot plot, most of the Anx5-positive EVs are superposed with the background from the buffer (Fig.1A,B). On an FL1 vs. SS plot, two populations of Anx5-positive EVs can be distinguished. About 30% of them (134 ± 91 per μL) form a distinct cluster of high fluorescence intensity events (colored blue in Fig.1B). As previously reported [28], these objects, which are characterized by a high fluorescence intensity and scattering properties similar to 500-nm polymer particles (Fig.1B left), correspond to empty erythrocytes and are called hereafter erythrocyte ghosts. The rest of the Anx5-positive EVs are colored red in Fig.1B.


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)

Flow cytometry (FCM) analysis of a platelet-free plasma (PFP) sample by FS triggering (A,B) and FL triggering (C,D), in the absence of Ca2+ (A, C) and in the presence of Ca2+ (B, D). (A-D). Each of the four panels presents on the left an FS vs. SS color dot plot and on the right an FL1 vs. SS color dot plot. Thresholds are represented by a solid blue line, while fluorescence positivity gates are represented by a dashed blue line. The positions of 500-nm and 1-μm polystyrene particles are indicated in each FS vs. SS plot (Fig. S2). (A,B) FS triggering analysis. The acquisition time was 10 min. Events labeled with Anx5-Fluo are separated into two groups: events of high fluorescence intensity forming a well-defined cluster are colored blue, and all the other Anx5-positive events are colored red. (C,D) FL triggering analysis. The acquisition time was 1 min, which explains why the cluster of erythrocyte ghosts (colored blue) contains less events than in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Flow cytometry (FCM) analysis of a platelet-free plasma (PFP) sample by FS triggering (A,B) and FL triggering (C,D), in the absence of Ca2+ (A, C) and in the presence of Ca2+ (B, D). (A-D). Each of the four panels presents on the left an FS vs. SS color dot plot and on the right an FL1 vs. SS color dot plot. Thresholds are represented by a solid blue line, while fluorescence positivity gates are represented by a dashed blue line. The positions of 500-nm and 1-μm polystyrene particles are indicated in each FS vs. SS plot (Fig. S2). (A,B) FS triggering analysis. The acquisition time was 10 min. Events labeled with Anx5-Fluo are separated into two groups: events of high fluorescence intensity forming a well-defined cluster are colored blue, and all the other Anx5-positive events are colored red. (C,D) FL triggering analysis. The acquisition time was 1 min, which explains why the cluster of erythrocyte ghosts (colored blue) contains less events than in (B).
Mentions: PFP samples were first analyzed by a conventional FCM approach, in which the trigger was set on the forward scatter (FS) parameter and the detection was limited to events labeled by Anx5-F* (Fig.1A,B). The concentration of Anx5-positive EVs detected by this approach was 457 ± 178 (n = 10) per μL pure PFP. This value is consistent with previous reports on EVs from normal PFP [20,23,28,30]. Displayed on a FS vs. side scatter (SS) color dot plot, most of the Anx5-positive EVs are superposed with the background from the buffer (Fig.1A,B). On an FL1 vs. SS plot, two populations of Anx5-positive EVs can be distinguished. About 30% of them (134 ± 91 per μL) form a distinct cluster of high fluorescence intensity events (colored blue in Fig.1B). As previously reported [28], these objects, which are characterized by a high fluorescence intensity and scattering properties similar to 500-nm polymer particles (Fig.1B left), correspond to empty erythrocytes and are called hereafter erythrocyte ghosts. The rest of the Anx5-positive EVs are colored red in Fig.1B.

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