Abstract
Förster resonance energy transfer (FRET)-based reporters are important tools to study the spatiotemporal compartmentalization of cyclic adenosine monophosphate (cAMP) in living cells. To increase the spatial resolution of cAMP detection, new reporters with specific intracellular targeting have been developed. Therefore it has become critical to be able to appropriately compare the signals revealed by the different sensors. Here we illustrate a protocol to calibrate the response detected by different targeted FRET reporters involving the generation of a dose–response curve to the cAMP raising agent forskolin. This method represents a general tool for the accurate analysis and interpretation of intracellular cAMP changes detected at the level of different subcellular compartments.
Alessandra Stangherlin and Andreas Koschinski contributed equally to this work.
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Acknowledgments
We thank Alsbetha Hulikova and Thomas Fritz for valuable discussions. The work described in this paper was supported by the British Heart Foundation (PG/07/091/23698) and the NSF–NIH CRCNS program (NIH R01 AA18060).
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Stangherlin, A. et al. (2014). Analysis of Compartmentalized cAMP: A Method to Compare Signals from Differently Targeted FRET Reporters. In: Zhang, J., Ni, Q., Newman, R. (eds) Fluorescent Protein-Based Biosensors. Methods in Molecular Biology, vol 1071. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-622-1_5
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DOI: https://doi.org/10.1007/978-1-62703-622-1_5
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