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A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlying cAMP change. In this chapter, we provide examples of the problems that may arise when using normalized FRET data and present a method based on the conversion of FRET ratio changes into actual cAMP concentrations that mitigates these issues.

Original publication

DOI

10.1007/978-1-4939-9121-1_12

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2019

Volume

1947

Pages

217 - 237

Keywords

Biosensors, FRET, Fluorescence resonance energy transfer, Intracellular signaling, Protein kinase A, Real time imaging, cAMP