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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

High contrast images of differentiated HUVEC in the TLA assay by Differential Interference Contrast (DIC) and confocal fluorescence microscopy. HUVEC stimulated to form tube-like structures with 25 ng/ml VEGF on a 35 mm glass-bottom dish covered with 10 μl matrix is compatible with DIC (x20 objective) (A) and confocal microscopy (x63 objective); Scale bar = 25 μm (B). Mitochondria (green) were stained using MitoTraker green (200nM). For 3D imaging (C) of the mitochondrial network, z-stacks were obtained at x40 magnification and re-constructed using Volocity software.
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Fig6: High contrast images of differentiated HUVEC in the TLA assay by Differential Interference Contrast (DIC) and confocal fluorescence microscopy. HUVEC stimulated to form tube-like structures with 25 ng/ml VEGF on a 35 mm glass-bottom dish covered with 10 μl matrix is compatible with DIC (x20 objective) (A) and confocal microscopy (x63 objective); Scale bar = 25 μm (B). Mitochondria (green) were stained using MitoTraker green (200nM). For 3D imaging (C) of the mitochondrial network, z-stacks were obtained at x40 magnification and re-constructed using Volocity software.

Mentions: Unlike cell monolayers, the traditional tube formation assay with its large matrix volume has greatly reduced microscopic working distances, making single cell analysis highly challenging. Using HUVEC stimulated with VEGF (25 ng/ml; 16 h) in 35 mm glass-bottom plates coated with 10 μl of Geltrex™, differential interference contrast (DIC) images were acquired using a Zeiss Axiovert 135 microscope (×20 objective). As shown in Figure 6A the TLA is clearly compatible with use of this high contrast technique.Figure 6


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)

High contrast images of differentiated HUVEC in the TLA assay by Differential Interference Contrast (DIC) and confocal fluorescence microscopy. HUVEC stimulated to form tube-like structures with 25 ng/ml VEGF on a 35 mm glass-bottom dish covered with 10 μl matrix is compatible with DIC (x20 objective) (A) and confocal microscopy (x63 objective); Scale bar = 25 μm (B). Mitochondria (green) were stained using MitoTraker green (200nM). For 3D imaging (C) of the mitochondrial network, z-stacks were obtained at x40 magnification and re-constructed using Volocity software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: High contrast images of differentiated HUVEC in the TLA assay by Differential Interference Contrast (DIC) and confocal fluorescence microscopy. HUVEC stimulated to form tube-like structures with 25 ng/ml VEGF on a 35 mm glass-bottom dish covered with 10 μl matrix is compatible with DIC (x20 objective) (A) and confocal microscopy (x63 objective); Scale bar = 25 μm (B). Mitochondria (green) were stained using MitoTraker green (200nM). For 3D imaging (C) of the mitochondrial network, z-stacks were obtained at x40 magnification and re-constructed using Volocity software.
Mentions: Unlike cell monolayers, the traditional tube formation assay with its large matrix volume has greatly reduced microscopic working distances, making single cell analysis highly challenging. Using HUVEC stimulated with VEGF (25 ng/ml; 16 h) in 35 mm glass-bottom plates coated with 10 μl of Geltrex™, differential interference contrast (DIC) images were acquired using a Zeiss Axiovert 135 microscope (×20 objective). As shown in Figure 6A the TLA is clearly compatible with use of this high contrast technique.Figure 6

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