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Tube-Forming Assays

  • Ryan M. Brown
  • Christopher J. Meah
  • Victoria L. Heath
  • Iain B. Styles
  • Roy BicknellEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1430)

Abstract

Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

Key words

Angiogenesis Endothelial cell Matrigel Tube formation Sprouting ImageJ 

References

  1. 1.
    Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8:464–478CrossRefPubMedGoogle Scholar
  2. 2.
    Heath VL, Bicknell R (2009) Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 6:395–404CrossRefPubMedGoogle Scholar
  3. 3.
    Auerbach R, Lewis R, Shinners B et al (2003) Angiogenesis assays: a critical overview. Clin Chem 49:32–40CrossRefPubMedGoogle Scholar
  4. 4.
    Bikfalvi A, Cramer EM, Tenza D et al (1991) Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays. Biol Cell 72:275–278CrossRefPubMedGoogle Scholar
  5. 5.
    Connolly JO, Simpson N, Hewlett L et al (2002) Rac regulates endothelial morphogenesis and capillary assembly. Mol Cell Biol 13:2474–2485CrossRefGoogle Scholar
  6. 6.
    Kaur S (2011) Characterisation of the expression profile and endothelial function of Rho GTPase RhoJ. PhD Thesis. University of BirminghamGoogle Scholar
  7. 7.
    Donovan D, Brown NJ, Bishop ET et al (2001) Comparison of three in vitro human “angiogenesis” assays with capillaries formed in vivo. Angiogenesis 4:113–121CrossRefPubMedGoogle Scholar
  8. 8.
    Staton CA, Reed MWR, Brown NJ (2009) A critical analysis of current in vitro and in vivo angiogenesis assays. Int J Exp Pathol 90:195–221CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nguyen DHT, Stapleton SC, Yang MT et al (2013) Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc Natl Acad Sci U S A 110:6712–6717CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Kang H, Bayless KJ, Kaunas R (2008) Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices. Am J Physiol Heart Circ Physiol 3120:2087–2097CrossRefGoogle Scholar
  11. 11.
    Khoo CP, Micklem K, Watt SM (2011) A comparison of methods for quantifying angiogenesis in the Matrigel assay in vitro. Tissue Eng Part C Methods 17:895–906CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Landini G (2008) Advanced shape analysis with ImageJ. In: Second ImageJ user developer conference proceedings. Luxembourg. p 116–121Google Scholar
  13. 13.
    Carpentier G, Martinelli M, Courty J et al (2012) Angiogenesis analyzer for ImageJ. In: Fourth ImageJ user developer conference proceedings. Mondorf-les-Bains, Luxembourg, p 198–201Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ryan M. Brown
    • 1
  • Christopher J. Meah
    • 2
  • Victoria L. Heath
    • 2
  • Iain B. Styles
    • 3
  • Roy Bicknell
    • 2
    Email author
  1. 1.Centre for Doctoral Training in Physical Sciences of Imaging in the Biomedical Sciences and School of ChemistryUniversity of BirminghamBirminghamUK
  2. 2.School of Immunity and Infection and Cancer Studies, Institute of Biomedical Research, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK
  3. 3.School of Computer ScienceUniversity of BirminghamBirminghamUK

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