Background: Basement matrices such as Matrigelââ??¢ and Geltrexââ??¢ are used in a variety of cell culture assays of\nanchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix\nrecommended for these assays (approximately 150 ?l/cm2) are costly, limit working distances for microscopy, and require\ncell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer\nangiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.\nResults: Geltrexââ??¢ basement matrix at 5 ?l/cm2 in 24-well (10 ?l) or 96-well (2 ?l) plates supports endothelial cell\ndifferentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working\ndistances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily.\nUsing MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the\n3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial\ntotal RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial\ncells without the need to initially detach cells from their supporting matrix.\nConclusions: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation\nof endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with\nthe added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and\nhigh-resolution microscopy.
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