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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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Influence of platelet-free plasma (PFP) dilution on the detection of Anx5-positive extracellular vesicles (EVs). Curves representing the concentrations of Anx5-positive EVs in diluted PFP samples detected either by FS (dashed lines) or fluorescence (FL) triggering (plain lines) vs. the reciprocal dilution factor, for two PFP samples (represented by squares and circles, respectively). The calculated linear regression lines are presented. R² values higher than 0.96 were obtained for all regression lines. By extrapolating the regression lines to pure PFP (dilution factor 1 x), Anx5-positive EVs concentrations of 35 666 (squares) and 50 239 (circles) are obtained. Each point represents the mean ± SD of two independent aliquots measured in duplicate.
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fig03: Influence of platelet-free plasma (PFP) dilution on the detection of Anx5-positive extracellular vesicles (EVs). Curves representing the concentrations of Anx5-positive EVs in diluted PFP samples detected either by FS (dashed lines) or fluorescence (FL) triggering (plain lines) vs. the reciprocal dilution factor, for two PFP samples (represented by squares and circles, respectively). The calculated linear regression lines are presented. R² values higher than 0.96 were obtained for all regression lines. By extrapolating the regression lines to pure PFP (dilution factor 1 x), Anx5-positive EVs concentrations of 35 666 (squares) and 50 239 (circles) are obtained. Each point represents the mean ± SD of two independent aliquots measured in duplicate.

Mentions: Figure3 shows the results of two independent experiments in which Anx5-positive EVs were detected using either FS triggering (blue symbols) or FL triggering (red symbols), for dilution factors ranging from 4× to 128×. The concentration of Anx5-positive EVs in diluted PFP samples is observed to decrease linearly with the reciprocal dilution factor. In addition, the mean fluorescence intensity of EVs detected by FL triggering remains constant over the entire dilution range (Fig. S6). These results indicate the absence of coincidence effects on the detection of Anx5-positive EVs. Therefore, as the experiments reported here were performed with 10 ×-diluted PFP samples, we conclude that the 22 000 Anx5-positive EVs determined by FL triggering correspond to individual EVs.


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)

Influence of platelet-free plasma (PFP) dilution on the detection of Anx5-positive extracellular vesicles (EVs). Curves representing the concentrations of Anx5-positive EVs in diluted PFP samples detected either by FS (dashed lines) or fluorescence (FL) triggering (plain lines) vs. the reciprocal dilution factor, for two PFP samples (represented by squares and circles, respectively). The calculated linear regression lines are presented. R² values higher than 0.96 were obtained for all regression lines. By extrapolating the regression lines to pure PFP (dilution factor 1 x), Anx5-positive EVs concentrations of 35 666 (squares) and 50 239 (circles) are obtained. Each point represents the mean ± SD of two independent aliquots measured in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Influence of platelet-free plasma (PFP) dilution on the detection of Anx5-positive extracellular vesicles (EVs). Curves representing the concentrations of Anx5-positive EVs in diluted PFP samples detected either by FS (dashed lines) or fluorescence (FL) triggering (plain lines) vs. the reciprocal dilution factor, for two PFP samples (represented by squares and circles, respectively). The calculated linear regression lines are presented. R² values higher than 0.96 were obtained for all regression lines. By extrapolating the regression lines to pure PFP (dilution factor 1 x), Anx5-positive EVs concentrations of 35 666 (squares) and 50 239 (circles) are obtained. Each point represents the mean ± SD of two independent aliquots measured in duplicate.
Mentions: Figure3 shows the results of two independent experiments in which Anx5-positive EVs were detected using either FS triggering (blue symbols) or FL triggering (red symbols), for dilution factors ranging from 4× to 128×. The concentration of Anx5-positive EVs in diluted PFP samples is observed to decrease linearly with the reciprocal dilution factor. In addition, the mean fluorescence intensity of EVs detected by FL triggering remains constant over the entire dilution range (Fig. S6). These results indicate the absence of coincidence effects on the detection of Anx5-positive EVs. Therefore, as the experiments reported here were performed with 10 ×-diluted PFP samples, we conclude that the 22 000 Anx5-positive EVs determined by FL triggering correspond to individual EVs.

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