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

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