HyPer biosensor to monitor intracellular hydrogen peroxide in skeletal muscle cells
FREE RADICAL BIOLOGY AND MEDICINE(2017)
摘要
Hydrogen peroxide (H 2 O 2 ) is one of the reactive oxygen species (ROS) that seems to play an essential role in cellular signalling pathways coupled to frequent pathophysiological processes. However, using traditional methodology it is virtually impossible to identify and quantify H 2 O 2 flux in cells. We have developed methodological approaches based on the use of a hydrogen peroxide biosensor, HyPer, in skeletal muscle cells: myoblasts and myotubes C2C12, and individual matured muscle fibres isolated from the mouse muscle. Using transfection techniques with chemical agents and microinjection/electroporation techniques, we have achieved the expression of HyPer biosensor in those cells. In combination with live cell fluorescence microscopy image analysis we have monitored the intracellular flow of H 2 O 2 in situ and in real time in those skeletal muscle cells that expressed HyPer. The HyPer fluorescence emitted by cells was registered during a period in which cells were exposed either to extracellular hydrogen peroxide or to a reducing agent, dithiothreitol, in the medium. Conclusion: i) it is possible the expression of hydrogen peroxide biosensor HyPer in myoblasts and myotubes C2C12 and in single isolated skeletal muscle fibres, and ii) HyPer biosensor is functional and detects changes in the intracellular concentration of hydrogen peroxide in situ and in real time in skeletal muscle cells.
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关键词
HyPer,hydrogen peroxide,myoblast,myotubes,skeletal muscle fibres
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