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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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Direct comparison of the thin layer angiogenesis (TLA) assay with standard thick layer assay. Comparison of the average number of branches/field (manual count) formed by HUVEC (25,000 cells/cm2) in the presence or absence of VEGF (25 ng/ml) in the standard thick layer assay (A) and the TLA assay (B). **p < 0.01 vs. control as determined by student’s paired t-test.
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Fig5: Direct comparison of the thin layer angiogenesis (TLA) assay with standard thick layer assay. Comparison of the average number of branches/field (manual count) formed by HUVEC (25,000 cells/cm2) in the presence or absence of VEGF (25 ng/ml) in the standard thick layer assay (A) and the TLA assay (B). **p < 0.01 vs. control as determined by student’s paired t-test.

Mentions: To make a direct comparison of the TLA approach with the traditional thick layer, HUVEC were stimulated to undergo tubulogenesis in the presence or absence of VEGF (25 ng/ml) on either 2 μl/well or 50 μl/well of Geltrex™ in a 96-well format. VEGF induced a similar pro-angiogenic effect on HUVEC whether on a standard thick or TLA basement matrix (Figure 5; Additional files 2: Movie S1A and S1B).Figure 5


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)

Direct comparison of the thin layer angiogenesis (TLA) assay with standard thick layer assay. Comparison of the average number of branches/field (manual count) formed by HUVEC (25,000 cells/cm2) in the presence or absence of VEGF (25 ng/ml) in the standard thick layer assay (A) and the TLA assay (B). **p < 0.01 vs. control as determined by student’s paired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Direct comparison of the thin layer angiogenesis (TLA) assay with standard thick layer assay. Comparison of the average number of branches/field (manual count) formed by HUVEC (25,000 cells/cm2) in the presence or absence of VEGF (25 ng/ml) in the standard thick layer assay (A) and the TLA assay (B). **p < 0.01 vs. control as determined by student’s paired t-test.
Mentions: To make a direct comparison of the TLA approach with the traditional thick layer, HUVEC were stimulated to undergo tubulogenesis in the presence or absence of VEGF (25 ng/ml) on either 2 μl/well or 50 μl/well of Geltrex™ in a 96-well format. VEGF induced a similar pro-angiogenic effect on HUVEC whether on a standard thick or TLA basement matrix (Figure 5; Additional files 2: Movie S1A and S1B).Figure 5

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