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A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

Faulkner A, Purcell R, Hibbert A, Latham S, Thomson S, Hall WL, Wheeler-Jones C, Bishop-Bailey D - BMC Cell Biol. (2014)

Bottom Line: Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily.Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells.Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.

View Article: PubMed Central - PubMed

Affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. afaulkner@rvc.ac.uk.

ABSTRACT

Background: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.

Results: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.

Conclusions: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

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Related in: MedlinePlus

Human endothelial cells readily differentiate on thin layers of basement matrix spread on glass. (A) Representative images of EA.hy926 and HUVEC forming tube-like structures after 24 and 16 hours, respectively, when plated onto 10 μl/2 cm2 of basement matrix in the presence of VEGF (25 ng/ml) or GW0742 (1 μM). Images were acquired using Leica DMIRB microscope (x10 objective). Scale bar = 200 μm. (B) Quantification of tubes formed by HUVEC at 16 hours in the presence of VEGF, GW0742 or DMSO control. Data represent mean (± S.E.M) number of branches/field from n = 5 separate donors. **p < 0.01 vs. DMSO control as determined by repeated measures ANOVA followed by Dunnett’s post analysis.
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Fig1: Human endothelial cells readily differentiate on thin layers of basement matrix spread on glass. (A) Representative images of EA.hy926 and HUVEC forming tube-like structures after 24 and 16 hours, respectively, when plated onto 10 μl/2 cm2 of basement matrix in the presence of VEGF (25 ng/ml) or GW0742 (1 μM). Images were acquired using Leica DMIRB microscope (x10 objective). Scale bar = 200 μm. (B) Quantification of tubes formed by HUVEC at 16 hours in the presence of VEGF, GW0742 or DMSO control. Data represent mean (± S.E.M) number of branches/field from n = 5 separate donors. **p < 0.01 vs. DMSO control as determined by repeated measures ANOVA followed by Dunnett’s post analysis.

Mentions: 10 μl of growth factor-reduced Geltrex™ basement matrix was spread evenly onto glass (13 mm coverslip) using a sterile syringe insert. HUVEC or EA.hy926 formed tube-like structures under non-stimulated conditions (vehicle alone; 0.01% DMSO) and the number of tubes formed was significantly increased in response to the distinct angiogenesis inducers vascular endothelial growth factor (VEGF; 25 ng/ml) and GW0742 (1 μM), a selective PPARβ/δ agonist [3,6] (Figure 1). Similar results were obtained when Geltrex™ matrix was spread directly onto tissue culture plastic with VEGF-stimulated HUVEC showing a clear pro-angiogenic response (Figure 2).Figure 1


A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

Faulkner A, Purcell R, Hibbert A, Latham S, Thomson S, Hall WL, Wheeler-Jones C, Bishop-Bailey D - BMC Cell Biol. (2014)

Human endothelial cells readily differentiate on thin layers of basement matrix spread on glass. (A) Representative images of EA.hy926 and HUVEC forming tube-like structures after 24 and 16 hours, respectively, when plated onto 10 μl/2 cm2 of basement matrix in the presence of VEGF (25 ng/ml) or GW0742 (1 μM). Images were acquired using Leica DMIRB microscope (x10 objective). Scale bar = 200 μm. (B) Quantification of tubes formed by HUVEC at 16 hours in the presence of VEGF, GW0742 or DMSO control. Data represent mean (± S.E.M) number of branches/field from n = 5 separate donors. **p < 0.01 vs. DMSO control as determined by repeated measures ANOVA followed by Dunnett’s post analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4263020&req=5

Fig1: Human endothelial cells readily differentiate on thin layers of basement matrix spread on glass. (A) Representative images of EA.hy926 and HUVEC forming tube-like structures after 24 and 16 hours, respectively, when plated onto 10 μl/2 cm2 of basement matrix in the presence of VEGF (25 ng/ml) or GW0742 (1 μM). Images were acquired using Leica DMIRB microscope (x10 objective). Scale bar = 200 μm. (B) Quantification of tubes formed by HUVEC at 16 hours in the presence of VEGF, GW0742 or DMSO control. Data represent mean (± S.E.M) number of branches/field from n = 5 separate donors. **p < 0.01 vs. DMSO control as determined by repeated measures ANOVA followed by Dunnett’s post analysis.
Mentions: 10 μl of growth factor-reduced Geltrex™ basement matrix was spread evenly onto glass (13 mm coverslip) using a sterile syringe insert. HUVEC or EA.hy926 formed tube-like structures under non-stimulated conditions (vehicle alone; 0.01% DMSO) and the number of tubes formed was significantly increased in response to the distinct angiogenesis inducers vascular endothelial growth factor (VEGF; 25 ng/ml) and GW0742 (1 μM), a selective PPARβ/δ agonist [3,6] (Figure 1). Similar results were obtained when Geltrex™ matrix was spread directly onto tissue culture plastic with VEGF-stimulated HUVEC showing a clear pro-angiogenic response (Figure 2).Figure 1

Bottom Line: Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily.Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells.Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.

View Article: PubMed Central - PubMed

Affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. afaulkner@rvc.ac.uk.

ABSTRACT

Background: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.

Results: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.

Conclusions: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

Show MeSH
Related in: MedlinePlus