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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 efficiencies and specificities of the methods by combining microbeads with NTA for purifying and detecting EC-MVs and EPC-MVs. (a1,b1) the purification efficiencies and specificities of EC-MVs and EPC-MVs in the total MVs, which were collected from EC or EPC culture medium by ultra-centrifuge and isolated by various microbeads- conjugated antibodies against EC and EPC specific markers (CD105 and CD34 as well as negative controls, CD41, CD235a). (a2,b2) the detection efficiencies and specificities of EC-MVs in the total CD105+ MVs, or EPC-MVs in the total CD34+ MVs, that were labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots upon detected by fluorescence NTA. (a3,b3) the overall efficiency for measuring the CD105+ MVs or CD34+ MVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots in the total EC-MVs. (a4,b4) the absolute number of CD105+ MVs or CD34+ MVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml EC or EPC culture medium. (c) representative plots showing the size/concentration distribution of the CD105+ beads isolated MVs under fluorescence/non-fluorescence modes. White curve: CD105+ MVs measured under light scatter (non-fluorescence) mode. Yellow curve: CD105+ Q-dots+ MVs measured under fluorescence mode. CD41: specific for platelets; CD235a: specific for erythrocytes; CD34: specific for endothelial progenitor cells; CD105: specific for endothelial cells; CD144: vascular endothelial antigen; KDR: EPC antigen; Annexin V: MV specific antigen; CD63: exosomal specific antigen. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. N = 4/group.
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f2: The efficiencies and specificities of the methods by combining microbeads with NTA for purifying and detecting EC-MVs and EPC-MVs. (a1,b1) the purification efficiencies and specificities of EC-MVs and EPC-MVs in the total MVs, which were collected from EC or EPC culture medium by ultra-centrifuge and isolated by various microbeads- conjugated antibodies against EC and EPC specific markers (CD105 and CD34 as well as negative controls, CD41, CD235a). (a2,b2) the detection efficiencies and specificities of EC-MVs in the total CD105+ MVs, or EPC-MVs in the total CD34+ MVs, that were labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots upon detected by fluorescence NTA. (a3,b3) the overall efficiency for measuring the CD105+ MVs or CD34+ MVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots in the total EC-MVs. (a4,b4) the absolute number of CD105+ MVs or CD34+ MVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml EC or EPC culture medium. (c) representative plots showing the size/concentration distribution of the CD105+ beads isolated MVs under fluorescence/non-fluorescence modes. White curve: CD105+ MVs measured under light scatter (non-fluorescence) mode. Yellow curve: CD105+ Q-dots+ MVs measured under fluorescence mode. CD41: specific for platelets; CD235a: specific for erythrocytes; CD34: specific for endothelial progenitor cells; CD105: specific for endothelial cells; CD144: vascular endothelial antigen; KDR: EPC antigen; Annexin V: MV specific antigen; CD63: exosomal specific antigen. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. N = 4/group.

Mentions: As described, the specific microbeads captured MVs were analyzed by NTA under light scatter mode. The results showed that anti-CD105 conjugated-microbeads had the highest efficiency (>95%) for purifying EC-MVs (Fig. 2a1), and anti-CD34 conjugated-microbeads had the highest efficiency (>95%) for purifying EPC-MVs (Fig. 2b1), than anti-CD41 (specific for platelets) or anti-CD235a (specific for erythrocytes) conjugated microbeads did.


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 efficiencies and specificities of the methods by combining microbeads with NTA for purifying and detecting EC-MVs and EPC-MVs. (a1,b1) the purification efficiencies and specificities of EC-MVs and EPC-MVs in the total MVs, which were collected from EC or EPC culture medium by ultra-centrifuge and isolated by various microbeads- conjugated antibodies against EC and EPC specific markers (CD105 and CD34 as well as negative controls, CD41, CD235a). (a2,b2) the detection efficiencies and specificities of EC-MVs in the total CD105+ MVs, or EPC-MVs in the total CD34+ MVs, that were labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots upon detected by fluorescence NTA. (a3,b3) the overall efficiency for measuring the CD105+ MVs or CD34+ MVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots in the total EC-MVs. (a4,b4) the absolute number of CD105+ MVs or CD34+ MVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml EC or EPC culture medium. (c) representative plots showing the size/concentration distribution of the CD105+ beads isolated MVs under fluorescence/non-fluorescence modes. White curve: CD105+ MVs measured under light scatter (non-fluorescence) mode. Yellow curve: CD105+ Q-dots+ MVs measured under fluorescence mode. CD41: specific for platelets; CD235a: specific for erythrocytes; CD34: specific for endothelial progenitor cells; CD105: specific for endothelial cells; CD144: vascular endothelial antigen; KDR: EPC antigen; Annexin V: MV specific antigen; CD63: exosomal specific antigen. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. N = 4/group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The efficiencies and specificities of the methods by combining microbeads with NTA for purifying and detecting EC-MVs and EPC-MVs. (a1,b1) the purification efficiencies and specificities of EC-MVs and EPC-MVs in the total MVs, which were collected from EC or EPC culture medium by ultra-centrifuge and isolated by various microbeads- conjugated antibodies against EC and EPC specific markers (CD105 and CD34 as well as negative controls, CD41, CD235a). (a2,b2) the detection efficiencies and specificities of EC-MVs in the total CD105+ MVs, or EPC-MVs in the total CD34+ MVs, that were labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots upon detected by fluorescence NTA. (a3,b3) the overall efficiency for measuring the CD105+ MVs or CD34+ MVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots in the total EC-MVs. (a4,b4) the absolute number of CD105+ MVs or CD34+ MVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml EC or EPC culture medium. (c) representative plots showing the size/concentration distribution of the CD105+ beads isolated MVs under fluorescence/non-fluorescence modes. White curve: CD105+ MVs measured under light scatter (non-fluorescence) mode. Yellow curve: CD105+ Q-dots+ MVs measured under fluorescence mode. CD41: specific for platelets; CD235a: specific for erythrocytes; CD34: specific for endothelial progenitor cells; CD105: specific for endothelial cells; CD144: vascular endothelial antigen; KDR: EPC antigen; Annexin V: MV specific antigen; CD63: exosomal specific antigen. EC-MVs: microvesicles released from endothelial cells. EPC-MVs: microvesicles released from endothelial progenitor cells. N = 4/group.
Mentions: As described, the specific microbeads captured MVs were analyzed by NTA under light scatter mode. The results showed that anti-CD105 conjugated-microbeads had the highest efficiency (>95%) for purifying EC-MVs (Fig. 2a1), and anti-CD34 conjugated-microbeads had the highest efficiency (>95%) for purifying EPC-MVs (Fig. 2b1), than anti-CD41 (specific for platelets) or anti-CD235a (specific for erythrocytes) conjugated microbeads did.

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