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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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Determination of the number of Anx5-F* molecules needed to detect individual extracellular vesicles (EVs) by fluorescence (FL) triggering. (A-C) FS triggering analysis of a mixture of Quantum™ fluorescein isothiocyanate (FITC)-5 MESF particles made of six particle populations containing, respectively, 0, 1484, 9828, 47 640, 188 438 and 756 101 MESF. (A) The 7.7-μm fluorescent particles form a homogeneous cluster, located in the upper right corner of the FS vs. SS color dot plot. (B) Histogram of fluorescence intensities, showing four resolved peaks (A,B,C,D) corresponding to particles containing 9828 fluorophores and above, while the 1484 MESF particles form a peak (E) overlapping the blank particles (F), which present some auto-fluorescence. (C) Calibration curve correlating the MESF-FITC of the particles to their mean fluorescence intensity, taken from (B). (D) Enlarged view from the low-fluorescence values of the calibration curve (C). The minimal fluorescence intensity of 2 arbitrary units measured by FL triggering is indicated (horizontal arrow), together with its corresponding MESF value, namely 2500 MESF (vertical arrow). (E) FL1 triggering analysis of the Quantum™ FITC-5 MESF particles. Four peaks (A,B,C,D) corresponding to particles containing 9828 MESF and above are detected, while the population of 1484 MESF particles is not detected. The threshold is represented by a solid blue line.
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fig04: Determination of the number of Anx5-F* molecules needed to detect individual extracellular vesicles (EVs) by fluorescence (FL) triggering. (A-C) FS triggering analysis of a mixture of Quantum™ fluorescein isothiocyanate (FITC)-5 MESF particles made of six particle populations containing, respectively, 0, 1484, 9828, 47 640, 188 438 and 756 101 MESF. (A) The 7.7-μm fluorescent particles form a homogeneous cluster, located in the upper right corner of the FS vs. SS color dot plot. (B) Histogram of fluorescence intensities, showing four resolved peaks (A,B,C,D) corresponding to particles containing 9828 fluorophores and above, while the 1484 MESF particles form a peak (E) overlapping the blank particles (F), which present some auto-fluorescence. (C) Calibration curve correlating the MESF-FITC of the particles to their mean fluorescence intensity, taken from (B). (D) Enlarged view from the low-fluorescence values of the calibration curve (C). The minimal fluorescence intensity of 2 arbitrary units measured by FL triggering is indicated (horizontal arrow), together with its corresponding MESF value, namely 2500 MESF (vertical arrow). (E) FL1 triggering analysis of the Quantum™ FITC-5 MESF particles. Four peaks (A,B,C,D) corresponding to particles containing 9828 MESF and above are detected, while the population of 1484 MESF particles is not detected. The threshold is represented by a solid blue line.

Mentions: Next, we focused on the determination of the sensitivity of the FL triggering approach, which can be defined as the minimal number of Anx5-F* molecules needed for detecting one single Anx5-positive EV. The fluorescence intensities of a mixture of MESF-FITC calibration particles containing from 0 to 756 000 MESF were measured by FS triggering (Fig.4A,B), from which a calibration curve relating the number of fluorophores of the particles to their fluorescence intensity was drawn (Fig.4C).


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)

Determination of the number of Anx5-F* molecules needed to detect individual extracellular vesicles (EVs) by fluorescence (FL) triggering. (A-C) FS triggering analysis of a mixture of Quantum™ fluorescein isothiocyanate (FITC)-5 MESF particles made of six particle populations containing, respectively, 0, 1484, 9828, 47 640, 188 438 and 756 101 MESF. (A) The 7.7-μm fluorescent particles form a homogeneous cluster, located in the upper right corner of the FS vs. SS color dot plot. (B) Histogram of fluorescence intensities, showing four resolved peaks (A,B,C,D) corresponding to particles containing 9828 fluorophores and above, while the 1484 MESF particles form a peak (E) overlapping the blank particles (F), which present some auto-fluorescence. (C) Calibration curve correlating the MESF-FITC of the particles to their mean fluorescence intensity, taken from (B). (D) Enlarged view from the low-fluorescence values of the calibration curve (C). The minimal fluorescence intensity of 2 arbitrary units measured by FL triggering is indicated (horizontal arrow), together with its corresponding MESF value, namely 2500 MESF (vertical arrow). (E) FL1 triggering analysis of the Quantum™ FITC-5 MESF particles. Four peaks (A,B,C,D) corresponding to particles containing 9828 MESF and above are detected, while the population of 1484 MESF particles is not detected. The threshold is represented by a solid blue line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Determination of the number of Anx5-F* molecules needed to detect individual extracellular vesicles (EVs) by fluorescence (FL) triggering. (A-C) FS triggering analysis of a mixture of Quantum™ fluorescein isothiocyanate (FITC)-5 MESF particles made of six particle populations containing, respectively, 0, 1484, 9828, 47 640, 188 438 and 756 101 MESF. (A) The 7.7-μm fluorescent particles form a homogeneous cluster, located in the upper right corner of the FS vs. SS color dot plot. (B) Histogram of fluorescence intensities, showing four resolved peaks (A,B,C,D) corresponding to particles containing 9828 fluorophores and above, while the 1484 MESF particles form a peak (E) overlapping the blank particles (F), which present some auto-fluorescence. (C) Calibration curve correlating the MESF-FITC of the particles to their mean fluorescence intensity, taken from (B). (D) Enlarged view from the low-fluorescence values of the calibration curve (C). The minimal fluorescence intensity of 2 arbitrary units measured by FL triggering is indicated (horizontal arrow), together with its corresponding MESF value, namely 2500 MESF (vertical arrow). (E) FL1 triggering analysis of the Quantum™ FITC-5 MESF particles. Four peaks (A,B,C,D) corresponding to particles containing 9828 MESF and above are detected, while the population of 1484 MESF particles is not detected. The threshold is represented by a solid blue line.
Mentions: Next, we focused on the determination of the sensitivity of the FL triggering approach, which can be defined as the minimal number of Anx5-F* molecules needed for detecting one single Anx5-positive EV. The fluorescence intensities of a mixture of MESF-FITC calibration particles containing from 0 to 756 000 MESF were measured by FS triggering (Fig.4A,B), from which a calibration curve relating the number of fluorophores of the particles to their fluorescence intensity was drawn (Fig.4C).

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