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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.

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

Publication Date Jan 1, 2015
Deposit Date Jan 17, 2020
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
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; animal cell; animal tissue; Article; biosensor; cell line; controlled study; enzyme activity; fluorescence resonance energy transfer; human; human cell; image analysis; intracellular signaling; molecular imaging; mouse; nonhuman; priority journal; second messenger; spinal ganglion; cell culture technique; cytology; female; fluorescence resonance energy transfer; genetic procedures; genetic transfection; HEK293 cell line; image processing; intracellular space; male; MCF 7 cell line; metabolism; molecular imaging; nerve cell; procedures; signal transduction; Biosensing Techniques; Cell Culture Techniques; Female; Fluorescence Resonance Energy Transfer; Ganglia, Spinal; HEK293 Cells; Humans; Image Processing, Computer-Assisted; Intracellular Space; Male; MCF-7 Cells; Molecular Imaging; Neurons; Signal Transduction; Transfection
Public URL https://nottingham-repository.worktribe.com/output/1312043
Publisher URL https://www.scopus.com/inward/record.uri?eid=2-s2.0-84938901798&doi=10.1007%2f978-1-4939-2914-6_10&partnerID=40&md5=92796c0d2ff5ac7e38f53eab79a7fc52