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Fluorescence resonance energy transfer (FRET)-based reporters are invaluable tools to study spatiotemporal aspects of cyclic adenosine monophosphate (cAMP) signaling and compartmentalization in living cells. These sensors allow estimation of relative changes of cAMP levels in real-time and intact cells. However, one of their major shortcomings is that they do not easily allow direct measurement of cAMP concentrations. This is mainly due to the fact that the methods to calibrate these sensors in their physiological microenvironment are not generally available. All published approaches to calibrate FRET-based reporters rely at least in part on data derived under nonphysiological conditions. Here, we present a protocol to calibrate FRET reporters completely "in cell." We introduce a combination of FRET imaging of cAMP and the whole-cell patch-clamp techniques to microinfuse or dilute intracellular cAMP to known concentrations. This method represents a general tool to accurately estimate intracellular cAMP concentrations by allocating concentration values to FRET ratio changes.

Original publication

DOI

10.1007/978-1-4939-2537-7_3

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2015

Volume

1294

Pages

25 - 40

Keywords

Animals, Biosensing Techniques, CHO Cells, Calibration, Cellular Microenvironment, Cricetulus, Cyclic AMP, Fluorescence Resonance Energy Transfer, Patch-Clamp Techniques