Abstract
Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons.
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Acknowledgements
This work was supported by a Monash Fellowship to M. Canals, NHMRC RD Wright Career Development Fellowship to M.L. Halls (1061687), NHMRC Project Grants (1047633, 1047730, 1062230) to M. Canals, M.L. Halls and A.M. Ellisdon, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences Large Grant Support Scheme grants to M. Canals and M.L. Halls, ARC Centre of Excellence in Advanced Molecular Imaging and Faculty of Medicine Nursing and Health Sciences Early Career Development Strategic Grant to A.M. Ellisdon.
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Halls, M.L., Poole, D.P., Ellisdon, A.M., Nowell, C.J., Canals, M. (2015). Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging. In: Filizola, M. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 1335. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2914-6_10
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DOI: https://doi.org/10.1007/978-1-4939-2914-6_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2913-9
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