Abstract
Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.
MeSH terms
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Angiogenesis Inhibitors / pharmacology*
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Benzimidazoles / pharmacology*
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Cell Culture Techniques
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Cells, Cultured
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Coculture Techniques
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Green Fluorescent Proteins / biosynthesis
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Green Fluorescent Proteins / genetics
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High-Throughput Screening Assays / methods*
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Human Umbilical Vein Endothelial Cells / drug effects
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Humans
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Image Processing, Computer-Assisted*
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Microscopy, Fluorescence
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Myocytes, Smooth Muscle / drug effects
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Phthalazines / pharmacology*
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Pulmonary Artery / cytology
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Pyridines / pharmacology*
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Quinolones / pharmacology*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / genetics
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Staining and Labeling
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Transduction, Genetic
Substances
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4-amino-5-fluoro-3-(5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl)quinolin-2(1H)-one
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Angiogenesis Inhibitors
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Benzimidazoles
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Phthalazines
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Pyridines
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Quinolones
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Recombinant Proteins
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Green Fluorescent Proteins
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vatalanib