Abstract
Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling—all without the need for cellular dissociation—thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6–14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.
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Acknowledgements
We thank all the members of our laboratory and collaborators who have contributed time, effort, expertise, reagents and equipment to the optimization of these protocols. This work was sponsored by Cancer Research UK, Breast Cancer Campaign and the Medical Research Council (grant no. 93277).
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Contributions
M.B. compiled the protocol with input from the authors, collected collagen time-course and control data and developed the proliferation assay adaptation. S.D.R. performed the optimization experiments, developed the siRNA and lentivirus protocols and provided Matrigel data. T.L. carried out the Imaris analysis and optimized simultaneous protein/RNA extraction from rings using the Nucleospin kit. P.R.B. developed the TRI2 program and wrote the automated microvessel area quantification protocol. B.T. optimized the fibrin protocol and provided confocal images and data. G.D. provided Rac1 knockdown data and D.T.J. provided Flk1 knockdown data. B.V. oversaw the design and application of the automated vessel-counting system. K.H.-D. contributed to the development of the methods and oversaw the writing of the paper.
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Supplementary information
Supplementary Figure 1
Automatic quantification of microvessel area in TRI2. An image of an aortic ring embedded in collagen, stained with BS1 lectin-FITC and imaged on an epifluorescence microscope after 9 days has been loaded into the TRI2 program (Original). The final binary image is shown (Segmented) in addition to being used as an overlay over the original data (Masked). The resulting statistics for total and average intensity with standard deviation plus the total vessel area in pixels are highlighted in the red box. See BOX 2. (TIFF 4489 kb)
Supplementary Video 1
Dissection of the murine thoracic aorta. After cleaning the fur with 70% ethanol, a single cut and blunt dissection are used to expose the rib cage. The thoracic cavity is opened and the heart and lungs removed. The aorta is indicated with white arrowheads in the still image. The aorta is carefully dissected as described in Figure 2 and the text and transferred to Opti-MEM®. Images of an aorta before and after cleaning are shown with the fatty layer (arrowheads) and aortic vessel (arrow) indicated. The approximate size of rings to be cut is indicated alongside the cleaned aorta with a scale bar. (MOV 16258 kb)
Supplementary Video 2
Embedding aortic rings in a collagen matrix. Materials required for embedding are shown: the 96-well plate and sterile forceps. The unpolymerised collagen mixture consisting of 10x DMEM, sterile distilled water and type 1 rat tail collagen is visible as a yellow liquid. After adding a small drop of 5N NaOH to the mixture and mixing well, the pH increases and the mixture turns pink. 50 µl aliquots of this mixture are transferred to the 96-well plate and aortic rings carefully embedded in them, with care taken to remove as much Opti-MEM® from the ring as possible so as not to dilute the collagen matrix excessively (as this would impair polymerization). (MOV 13427 kb)
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Baker, M., Robinson, S., Lechertier, T. et al. Use of the mouse aortic ring assay to study angiogenesis. Nat Protoc 7, 89–104 (2012). https://doi.org/10.1038/nprot.2011.435
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DOI: https://doi.org/10.1038/nprot.2011.435
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