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Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

Wang J, Zhong Y, Ma X, Xiao X, Cheng C, Chen Y, Iwuchukwu I, Gaines KJ, Zhao B, Liu S, Travers JB, Bihl JC, Chen Y - Sci Rep (2016)

Bottom Line: Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging.Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA).In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, 45435, USA.

ABSTRACT
Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma, and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

No MeSH data available.


Related in: MedlinePlus

Characterization of EC-MVs and EPC-MVs by NTA, TEM and western blot.(a) representative NTA plots show size/concentration distribution of MVs isolated from cell cultures. (b) representative western blot bands showing specific expression of Annexin V in MVs. (c) TEM micrographs of MVs. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. TEM: transmission electron microscopy. Annexin V: specific for MVs.
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f1: Characterization of EC-MVs and EPC-MVs by NTA, TEM and western blot.(a) representative NTA plots show size/concentration distribution of MVs isolated from cell cultures. (b) representative western blot bands showing specific expression of Annexin V in MVs. (c) TEM micrographs of MVs. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. TEM: transmission electron microscopy. Annexin V: specific for MVs.

Mentions: According to NTA and TEM analysis, the size of both EC-MVs and EPC-MVs ranged from 120–700 nm in diameter (Fig. 1a,b). The western blot results confirmed the expression of MV specific marker Annexin V in EC-MVs and EPC-MVs (Fig. 1c).


Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-dot Based Nanoparticle Tracking Analysis.

Wang J, Zhong Y, Ma X, Xiao X, Cheng C, Chen Y, Iwuchukwu I, Gaines KJ, Zhao B, Liu S, Travers JB, Bihl JC, Chen Y - Sci Rep (2016)

Characterization of EC-MVs and EPC-MVs by NTA, TEM and western blot.(a) representative NTA plots show size/concentration distribution of MVs isolated from cell cultures. (b) representative western blot bands showing specific expression of Annexin V in MVs. (c) TEM micrographs of MVs. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. TEM: transmission electron microscopy. Annexin V: specific for MVs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837394&req=5

f1: Characterization of EC-MVs and EPC-MVs by NTA, TEM and western blot.(a) representative NTA plots show size/concentration distribution of MVs isolated from cell cultures. (b) representative western blot bands showing specific expression of Annexin V in MVs. (c) TEM micrographs of MVs. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. TEM: transmission electron microscopy. Annexin V: specific for MVs.
Mentions: According to NTA and TEM analysis, the size of both EC-MVs and EPC-MVs ranged from 120–700 nm in diameter (Fig. 1a,b). The western blot results confirmed the expression of MV specific marker Annexin V in EC-MVs and EPC-MVs (Fig. 1c).

Bottom Line: Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging.Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA).In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, 45435, USA.

ABSTRACT
Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma, and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

No MeSH data available.


Related in: MedlinePlus