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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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Comparison of manual versus automated quantitation of angiogenic parameters. A comparison of (A) average number of branches/field and (B) average number of junctions/field obtained from the 24-well data acquired from the HUVEC TLA experiments reported in Figure 1 when quantified manually or with the use of the angiogenesis macro for ImageJ. n = 5 independent donors. *p < 0.05 **p < 0.01 ***p < 0.001 vs control as determined by one-way ANOVA followed by Dunnett’s post-analysis test.
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Fig4: Comparison of manual versus automated quantitation of angiogenic parameters. A comparison of (A) average number of branches/field and (B) average number of junctions/field obtained from the 24-well data acquired from the HUVEC TLA experiments reported in Figure 1 when quantified manually or with the use of the angiogenesis macro for ImageJ. n = 5 independent donors. *p < 0.05 **p < 0.01 ***p < 0.001 vs control as determined by one-way ANOVA followed by Dunnett’s post-analysis test.

Mentions: A common goal for those routinely using the basement matrix assay for assessing angiogenic potential is the use of automated quantification systems. The use of an automated quantification system (ImageJ Angiogenesis Analyzer) provides qualitatively but not always quantitatively identical data to manual counting (Figure 4; Additional file 1: Table S1).Figure 4


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)

Comparison of manual versus automated quantitation of angiogenic parameters. A comparison of (A) average number of branches/field and (B) average number of junctions/field obtained from the 24-well data acquired from the HUVEC TLA experiments reported in Figure 1 when quantified manually or with the use of the angiogenesis macro for ImageJ. n = 5 independent donors. *p < 0.05 **p < 0.01 ***p < 0.001 vs control as determined by one-way ANOVA followed by Dunnett’s post-analysis test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Comparison of manual versus automated quantitation of angiogenic parameters. A comparison of (A) average number of branches/field and (B) average number of junctions/field obtained from the 24-well data acquired from the HUVEC TLA experiments reported in Figure 1 when quantified manually or with the use of the angiogenesis macro for ImageJ. n = 5 independent donors. *p < 0.05 **p < 0.01 ***p < 0.001 vs control as determined by one-way ANOVA followed by Dunnett’s post-analysis test.
Mentions: A common goal for those routinely using the basement matrix assay for assessing angiogenic potential is the use of automated quantification systems. The use of an automated quantification system (ImageJ Angiogenesis Analyzer) provides qualitatively but not always quantitatively identical data to manual counting (Figure 4; Additional file 1: Table S1).Figure 4

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