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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

The dynamic change of cEC-MVs and cEPC-MVs in stroke patient plasma on days 1, 3 and 5 after admission.  (a) the dynamic change of cEC-MVs and cEPC-MVs in per ml plasma on days 1, 3 and 5 after stroke patient admission upon analyzed by NTA. cEC-MVs: circulating microvesicles released from endothelial cells; cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.
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f5: The dynamic change of cEC-MVs and cEPC-MVs in stroke patient plasma on days 1, 3 and 5 after admission. (a) the dynamic change of cEC-MVs and cEPC-MVs in per ml plasma on days 1, 3 and 5 after stroke patient admission upon analyzed by NTA. cEC-MVs: circulating microvesicles released from endothelial cells; cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.

Mentions: We assessed the dynamic changes of CD105+CD144+ cEC-MVs and CD34+KDR+ cEPC-MVs in patients at days 1, 3 and 5 after admission. As shown in Fig. 5, there were significant elevated levels of CD105+CD144+ cEC-MVs on days 3 and 5‚ÄČas compared with that in day 1, indicating that the levels of cEC-MVs could serve as indicators for endothelium damage under the ischemic condition. Meanwhile, the level of CD34+KDR+ cEPC-MVs on day 5 was elevated as compared to that on day 1, but there was no difference between days 1 and 3.


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)

The dynamic change of cEC-MVs and cEPC-MVs in stroke patient plasma on days 1, 3 and 5 after admission.  (a) the dynamic change of cEC-MVs and cEPC-MVs in per ml plasma on days 1, 3 and 5 after stroke patient admission upon analyzed by NTA. cEC-MVs: circulating microvesicles released from endothelial cells; cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: The dynamic change of cEC-MVs and cEPC-MVs in stroke patient plasma on days 1, 3 and 5 after admission. (a) the dynamic change of cEC-MVs and cEPC-MVs in per ml plasma on days 1, 3 and 5 after stroke patient admission upon analyzed by NTA. cEC-MVs: circulating microvesicles released from endothelial cells; cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.
Mentions: We assessed the dynamic changes of CD105+CD144+ cEC-MVs and CD34+KDR+ cEPC-MVs in patients at days 1, 3 and 5 after admission. As shown in Fig. 5, there were significant elevated levels of CD105+CD144+ cEC-MVs on days 3 and 5‚ÄČas compared with that in day 1, indicating that the levels of cEC-MVs could serve as indicators for endothelium damage under the ischemic condition. Meanwhile, the level of CD34+KDR+ cEPC-MVs on day 5 was elevated as compared to that on day 1, but there was no difference between days 1 and 3.

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