Limits...
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

Identification of cEC-MVs and cEPC-MVs from human plasma by using anti-CD105 or anti-CD34 conjugated-microbeads and Q-dots combined with fluorescence NTA.(a1,b1) the proportions of CD105+ cMVs or CD34+ cMVs in plasma that were isolated by anti-CD105 or anti-CD34 conjugated-microbeads. (a2,b2) the proportion of CD105+ cMVs or CD34+ cMVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots. (a3,b3) The absolute number of CD105+ cMVs or CD34+ cMVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml day 1 ischemic stroke patient plasma. *p < 0.05, vs. flow cytometry. N = 8/group. 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
getmorefigures.php?uid=PMC4837394&req=5

f4: Identification of cEC-MVs and cEPC-MVs from human plasma by using anti-CD105 or anti-CD34 conjugated-microbeads and Q-dots combined with fluorescence NTA.(a1,b1) the proportions of CD105+ cMVs or CD34+ cMVs in plasma that were isolated by anti-CD105 or anti-CD34 conjugated-microbeads. (a2,b2) the proportion of CD105+ cMVs or CD34+ cMVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots. (a3,b3) The absolute number of CD105+ cMVs or CD34+ cMVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml day 1 ischemic stroke patient plasma. *p < 0.05, vs. flow cytometry. N = 8/group. cEC-MVs: circulating microvesicles released from endothelial cells. cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.

Mentions: Since one specific surface antigen cannot accurately define these MVs are EC-MVs and EPC-MVs1012, we incubated those microbeads captured MVs with the other cell-origin marker, CD144 (EC marker) or KDR (EPC marker). Results revealed that the detection rate of CD105+CD144+ EC-MVs was approximately 76% and of CD34+KDR+ EPC-MVs was approximately 73% (Fig. 2a2,b2). When taken isolation and detection together, the overall efficiencies of detecting CD105+CD144+ EC-MVs and CD34+KDR+ EPC-MVs were over 70% (Fig. 2a3,b3). In addition, the results showed that over 76% of CD105+ MVs and CD34+ MVs co-labeled with the MV universal marker Annexin V (a positive control) and only a small portion was positive for EX specific marker CD63 (a negative control), suggesting a very low amount of EXs in MV samples (Fig. 2a3,b3). When compared to the results from flow cytometery, the MV concentrations detected by NTA were almost two-orders of magnitude higher (Fig. 2a4,b4). Of note, analysis of the CD105+ MVs (white curve) and Q-dots labeled CD105+MVs (yellow curve) showed overlap size profiles (Fig. 2c), indicating that Q-dots binding did not change the physical characters of MVs.


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)

Identification of cEC-MVs and cEPC-MVs from human plasma by using anti-CD105 or anti-CD34 conjugated-microbeads and Q-dots combined with fluorescence NTA.(a1,b1) the proportions of CD105+ cMVs or CD34+ cMVs in plasma that were isolated by anti-CD105 or anti-CD34 conjugated-microbeads. (a2,b2) the proportion of CD105+ cMVs or CD34+ cMVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots. (a3,b3) The absolute number of CD105+ cMVs or CD34+ cMVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml day 1 ischemic stroke patient plasma. *p < 0.05, vs. flow cytometry. N = 8/group. 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

f4: Identification of cEC-MVs and cEPC-MVs from human plasma by using anti-CD105 or anti-CD34 conjugated-microbeads and Q-dots combined with fluorescence NTA.(a1,b1) the proportions of CD105+ cMVs or CD34+ cMVs in plasma that were isolated by anti-CD105 or anti-CD34 conjugated-microbeads. (a2,b2) the proportion of CD105+ cMVs or CD34+ cMVs co-labeled with CD144-, or KDR-, or Annexin V-, or CD63- conjugated Q-dots. (a3,b3) The absolute number of CD105+ cMVs or CD34+ cMVs that were positive for CD144, or KDR, or Annexin V, or CD63 in per ml day 1 ischemic stroke patient plasma. *p < 0.05, vs. flow cytometry. N = 8/group. cEC-MVs: circulating microvesicles released from endothelial cells. cEPC-MVs: circulating microvesicles released from endothelial progenitor cells.
Mentions: Since one specific surface antigen cannot accurately define these MVs are EC-MVs and EPC-MVs1012, we incubated those microbeads captured MVs with the other cell-origin marker, CD144 (EC marker) or KDR (EPC marker). Results revealed that the detection rate of CD105+CD144+ EC-MVs was approximately 76% and of CD34+KDR+ EPC-MVs was approximately 73% (Fig. 2a2,b2). When taken isolation and detection together, the overall efficiencies of detecting CD105+CD144+ EC-MVs and CD34+KDR+ EPC-MVs were over 70% (Fig. 2a3,b3). In addition, the results showed that over 76% of CD105+ MVs and CD34+ MVs co-labeled with the MV universal marker Annexin V (a positive control) and only a small portion was positive for EX specific marker CD63 (a negative control), suggesting a very low amount of EXs in MV samples (Fig. 2a3,b3). When compared to the results from flow cytometery, the MV concentrations detected by NTA were almost two-orders of magnitude higher (Fig. 2a4,b4). Of note, analysis of the CD105+ MVs (white curve) and Q-dots labeled CD105+MVs (yellow curve) showed overlap size profiles (Fig. 2c), indicating that Q-dots binding did not change the physical characters of MVs.

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