title: A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.
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.
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. afaulkner@rvc.ac.uk.
authors.forename: Ashton
authors.initials: A
authors.lastname: Faulkner
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. rpurcell@rvc.ac.uk.
authors.forename: Robert
authors.initials: R
authors.lastname: Purcell
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. ahibbert@rvc.ac.uk.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hibbert
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. slatham@rvc.ac.uk.
authors.forename: Sally
authors.initials: S
authors.lastname: Latham
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. sthomson@rvc.ac.uk.
authors.forename: Scott
authors.initials: S
authors.lastname: Thomson
authors.affiliation: Diabetes and Nutritional Sciences Division, School of Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK. wendy.hall@kcl.ac.uk.
authors.forename: Wendy L
authors.initials: WL
authors.lastname: Hall
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. cwheeler@rvc.ac.uk.
authors.forename: Caroline
authors.initials: C
authors.lastname: Wheeler-Jones
authors.affiliation: Comparative Biomedical Sciences, Royal Veterinary College, University of London Royal College Street, London, NW1 0TU, UK. dbishopbailey@rvc.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Bishop-Bailey
mesh_terms: D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology*; D016326:Extracellular Matrix Proteins; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D008928:Mitochondria; D018919:Neovascularization, Physiologic
chemical_list: D016326:Extracellular Matrix Proteins