Abstract
BackgroundBasement 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.ResultsGeltrex™ 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users.
Highlights
Basement matrices such as MatrigelTM and GeltrexTM are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays
We show that spreading low volumes of GeltrexTM basement membrane thinly onto glass or tissue culture plastic enables the use of high-resolution imaging and direct RNA extraction from endothelial cells that are actively engaged in the tube-forming process
human umbilical vein endothelial cells (HUVEC) or EA.hy926 formed tubelike structures under non-stimulated conditions and the number of tubes formed was significantly increased in response to the distinct angiogenesis inducers vascular endothelial growth factor (VEGF; 25 ng/ml) and GW0742 (1 μM), a selective PPARβ/δ agonist [3,6] (Figure 1)
Summary
Basement matrices such as MatrigelTM and GeltrexTM are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. We show that spreading low volumes of GeltrexTM basement membrane thinly onto glass or tissue culture plastic enables the use of high-resolution imaging and direct RNA extraction from endothelial cells that are actively engaged in the tube-forming process. This simple assay generates comparable data to those derived from large volume matrix assays, and has a far greater utility in terms of imaging and transcriptional analysis accompanied by a 25–30 fold reduction in relative cost
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