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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 RNA extraction and RT-qPCR analysis of HUVEC immediately after the TLA assay. Fold relative COX-2 mRNA to GAPDH expression in VEGF-treated HUVEC undergoing differentiation in 24-well plates in the TLA assay. RNA was directly extracted from the cells and targets measured by Taqman RT-qPCR; n = 4 separate experiments.
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Fig8: Direct RNA extraction and RT-qPCR analysis of HUVEC immediately after the TLA assay. Fold relative COX-2 mRNA to GAPDH expression in VEGF-treated HUVEC undergoing differentiation in 24-well plates in the TLA assay. RNA was directly extracted from the cells and targets measured by Taqman RT-qPCR; n = 4 separate experiments.

Mentions: Total RNA was extracted from HUVEC differentiated into tubes using the TLA assay following exposure to VEGF (25 ng/ml) without prior cell isolation according to the manufacturer’s recommended protocol (Qiagen Total RNA Extraction kit). Subsequent standard RT-qPCR analysis readily detected GAPDH and COX-2 expression (previously shown to increase in HUVEC in response to VEGF treatment [6]). COX-2 expression in the TLA was not significantly induced relative to GAPDH at 16 h in VEGF stimulated tube-forming HUVEC (Figure 8 and Additional file 6: Figure S2).Figure 8


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 RNA extraction and RT-qPCR analysis of HUVEC immediately after the TLA assay. Fold relative COX-2 mRNA to GAPDH expression in VEGF-treated HUVEC undergoing differentiation in 24-well plates in the TLA assay. RNA was directly extracted from the cells and targets measured by Taqman RT-qPCR; n = 4 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig8: Direct RNA extraction and RT-qPCR analysis of HUVEC immediately after the TLA assay. Fold relative COX-2 mRNA to GAPDH expression in VEGF-treated HUVEC undergoing differentiation in 24-well plates in the TLA assay. RNA was directly extracted from the cells and targets measured by Taqman RT-qPCR; n = 4 separate experiments.
Mentions: Total RNA was extracted from HUVEC differentiated into tubes using the TLA assay following exposure to VEGF (25 ng/ml) without prior cell isolation according to the manufacturer’s recommended protocol (Qiagen Total RNA Extraction kit). Subsequent standard RT-qPCR analysis readily detected GAPDH and COX-2 expression (previously shown to increase in HUVEC in response to VEGF treatment [6]). COX-2 expression in the TLA was not significantly induced relative to GAPDH at 16 h in VEGF stimulated tube-forming HUVEC (Figure 8 and Additional file 6: Figure S2).Figure 8

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