Titlebook: cAMP Signaling; Methods and Protocol Manuela Zaccolo Book 2022Latest edition The Editor(s) (if applicable) and The Author(s), under exclusi

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Real-Time Measurements of Intracellular cAMP Gradients Using FRET-Based cAMP Nanorulers,eptors (GPCRs). In a single cell, cAMP can exert innumerous specific cell functions in response to more than one hundred different GPCRs. Cells achieve this extraordinary functional specificity of cAMP signaling by limiting the spread of these signals in space and time. To do so, cells establish nan

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Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells,lly performed in vitro using radio-labeled ATP. For in vivo studies, genetically encoded FRET-based sensors have become popular for monitoring PKA activity. Here, we show that it is also possible to apply such reporters in vitro. We describe how to express and purify milligram quantities of a FRET-b

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MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET,n. Here we describe a detailed protocol for setup and use of this software for any purpose requiring instant feedback during fluorescence measurement experiments. We further describe its non-primary features including beam splitter misalignment correction, custom calculations through input of simple

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Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity,genetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of ne

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Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors,sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-ef

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Micro-2D Cell Culture for cAMP Measurements Using FRET Reporters in Human iPSC-Derived Cardiomyocytiomyocytes. However, as the differentiation process is lengthy and commercially available cells are expensive, the cell number is limited. Here we provide detailed information on how to scale down 2D cell cultures of hIPS-CMs for the purpose of cAMP FRET measurements, thereby extending the number of

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Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification,ch to measure localized cAMP signals. However, given the low signal-to-noise ratio of most FRET probes and the dynamic nature of the intracellular environment, there have been marked limitations in the ability to use FRET probes to study localized signaling events within the same cell. Here, we outl