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

BMC Cell Biology

David Bishop-Bailey²  Caroline Wheeler-Jones²  Wendy L Hall¹  Scott Thomson²  Sally Latham²  Andrew Hibbert²  Robert Purcell²  Ashton Faulkner² 

[1] Diabetes and Nutritional Sciences Division, School of Medicine, King’s College London, 150 Stamford Street, London SE1 9NH, UK;Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London NW1 0TU, UK

关键词: VEGF;    PPAR;    Mitochondria;    Basement matrix;    Endothelial cell;    Angiogenesis;   

Others  :  1088701
DOI  :  10.1186/s12860-014-0041-5

 received in 2014-07-14, accepted in 2014-10-30,  发布年份 2014

【 摘 要 】

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/cm²) 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/cm² 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.

【 授权许可】

   
2014 Faulkner et al.; licensee BioMed Central Ltd.

【 图 表 】

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