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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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Gallery of Anx5-positive extracellular vesicles (EVs) observed by EM. (A-E) Representative images of Anx5-positive EVs observed on EM grids after sedimentation. Most EVs present a near-circular shape. Very rare gold-nanoparticles (NPs) are present in the background, illustrating the high specificity of Anx5-gold-NP labeling. Some EVs devoid of Anx5-gold-NPs are labeled with arrows, including 50-nm spherical EVs in (B) and a 2-μm tubular EV in (C). The size of the Anx5-positive EVs is indicated on the images (in nm). Considering that a spherical EV, of radius R and area 4πR², transforms upon sedimentation into a flattened circular pancake, of radius R' and area 2πR'², the radius of the flattened pancake is related to the radius of the original sphere by the relationship R  =  R'√2. The size indicated in the images for the circular EVs is the diameter of the equivalent spheres. Scale bars: (A-D) 200 nm; (E) 2 μm. (F) Size histogram of Anx5-positive EVs. About 80% Anx5-positive EVs are comprised between 100 and 400 nm. Histogram determined over 500 images of EVs recorded on three EM grids. Error bars correspond to SD between the three individual datasets [28].
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fig05: Gallery of Anx5-positive extracellular vesicles (EVs) observed by EM. (A-E) Representative images of Anx5-positive EVs observed on EM grids after sedimentation. Most EVs present a near-circular shape. Very rare gold-nanoparticles (NPs) are present in the background, illustrating the high specificity of Anx5-gold-NP labeling. Some EVs devoid of Anx5-gold-NPs are labeled with arrows, including 50-nm spherical EVs in (B) and a 2-μm tubular EV in (C). The size of the Anx5-positive EVs is indicated on the images (in nm). Considering that a spherical EV, of radius R and area 4πR², transforms upon sedimentation into a flattened circular pancake, of radius R' and area 2πR'², the radius of the flattened pancake is related to the radius of the original sphere by the relationship R  =  R'√2. The size indicated in the images for the circular EVs is the diameter of the equivalent spheres. Scale bars: (A-D) 200 nm; (E) 2 μm. (F) Size histogram of Anx5-positive EVs. About 80% Anx5-positive EVs are comprised between 100 and 400 nm. Histogram determined over 500 images of EVs recorded on three EM grids. Error bars correspond to SD between the three individual datasets [28].

Mentions: A gallery of EV images obtained by this procedure is presented in Fig.5. Most EVs (Fig.5A–D) present a near-circular shape, as expected from spherical EVs projected onto a flat support. Some EVs present a tubular morphology (Fig.5C,D), while a minor number consists of large fragments and erythrocyte ghosts (Fig.5E).


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)

Gallery of Anx5-positive extracellular vesicles (EVs) observed by EM. (A-E) Representative images of Anx5-positive EVs observed on EM grids after sedimentation. Most EVs present a near-circular shape. Very rare gold-nanoparticles (NPs) are present in the background, illustrating the high specificity of Anx5-gold-NP labeling. Some EVs devoid of Anx5-gold-NPs are labeled with arrows, including 50-nm spherical EVs in (B) and a 2-μm tubular EV in (C). The size of the Anx5-positive EVs is indicated on the images (in nm). Considering that a spherical EV, of radius R and area 4πR², transforms upon sedimentation into a flattened circular pancake, of radius R' and area 2πR'², the radius of the flattened pancake is related to the radius of the original sphere by the relationship R  =  R'√2. The size indicated in the images for the circular EVs is the diameter of the equivalent spheres. Scale bars: (A-D) 200 nm; (E) 2 μm. (F) Size histogram of Anx5-positive EVs. About 80% Anx5-positive EVs are comprised between 100 and 400 nm. Histogram determined over 500 images of EVs recorded on three EM grids. Error bars correspond to SD between the three individual datasets [28].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Gallery of Anx5-positive extracellular vesicles (EVs) observed by EM. (A-E) Representative images of Anx5-positive EVs observed on EM grids after sedimentation. Most EVs present a near-circular shape. Very rare gold-nanoparticles (NPs) are present in the background, illustrating the high specificity of Anx5-gold-NP labeling. Some EVs devoid of Anx5-gold-NPs are labeled with arrows, including 50-nm spherical EVs in (B) and a 2-μm tubular EV in (C). The size of the Anx5-positive EVs is indicated on the images (in nm). Considering that a spherical EV, of radius R and area 4πR², transforms upon sedimentation into a flattened circular pancake, of radius R' and area 2πR'², the radius of the flattened pancake is related to the radius of the original sphere by the relationship R  =  R'√2. The size indicated in the images for the circular EVs is the diameter of the equivalent spheres. Scale bars: (A-D) 200 nm; (E) 2 μm. (F) Size histogram of Anx5-positive EVs. About 80% Anx5-positive EVs are comprised between 100 and 400 nm. Histogram determined over 500 images of EVs recorded on three EM grids. Error bars correspond to SD between the three individual datasets [28].
Mentions: A gallery of EV images obtained by this procedure is presented in Fig.5. Most EVs (Fig.5A–D) present a near-circular shape, as expected from spherical EVs projected onto a flat support. Some EVs present a tubular morphology (Fig.5C,D), while a minor number consists of large fragments and erythrocyte ghosts (Fig.5E).

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