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Detection and quantifi cation of intracellular signaling using fret-based biosensors and high content imaging

Halls, Michelle L.; Poole, Daniel P.; Ellisdon, Andrew M.; Nowell, Cameron J.; Canals, Meritxell

Authors

Michelle L. Halls

Daniel P. Poole

Andrew M. Ellisdon

Cameron J. Nowell



Abstract

© Springer Science+Business Media New York 2015. 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.

Publication Date Jan 1, 2015
Publisher Humana Press
Pages 131-161
Series Title Methods in Molecular Biology book series
Series Number Volume 1335
Series ISSN 1940-6029
Edition 2nd
Book Title G Protein-Coupled Receptors in Drug Discovery
Chapter Number 10
ISBN 9781493929139
APA6 Citation Halls, M. L., Poole, D. P., Ellisdon, A. M., Nowell, C. J., & Canals, M. (2015). Detection and quantifi cation of intracellular signaling using fret-based biosensors and high content imaging. In G Protein-Coupled Receptors in Drug Discovery, (131-161). (2nd). Humana Press. https://doi.org/10.1007/978-1-4939-2914-6_10
DOI https://doi.org/10.1007/978-1-4939-2914-6_10
Keywords Biosensors; Cell signaling; Cyclic AMP; Extracellular-regulated kinase; Förster resonance energy transfer; High content imaging; Protein kinase C; Rac1; cyclic AMP; guanosine triphosphatase; mitogen activated protein kinase; protein kinase C; adult; anima
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