Imaging Intracellular H2O2 with the Genetically Encoded PerFRET and OxyFRET Probes
Hydrogen peroxide (H2O2) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of H2O2 signals is poorly understood. Introducing novel techniques for the intracellular detection of H2O2 would be essential for a more complete understanding of its role in cellular signaling. We previously reported the development of two novel fluorescence resonance energy transfer (FRET)-based protein sensors that showed opposite emission ratio changes upon reaction with H2O2. In this chapter, we detail the methods for using OxyFRET and PerFRET for the assessment of changes in subcellular H2O2 levels.
Key wordsHydrogen peroxide PerFRET OxyFRET Genetically encoded sensor Fluorescent biosensor imaging
This work was supported by grants from the Hungarian Research Fund (OTKA NF72669) and from the National Office for Research and Technology (OMFB-01680/2009). The work was also financed by the Higher Education Institutional Excellence Programme (FIKP) of the Ministry of Human Capacities in Hungary, within the framework of the Molecular Biology thematic programme of the Semmelweis University and by “Lendület” grants from the Hungarian Academy of Sciences to both MG (LP2011-001/2011) and BE (LP2018-13/2018). EB is also grateful for the support of the Premium postdoctoral research program (PPD-016/2017) of the Hungarian Academy of Sciences.
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