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Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes

  • Gaia Gherardi
  • Cristina MammucariEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)

Abstract

We report a method for ex vivo measurements of Ca2+ transients in skeletal muscle fibers, both in the sarcoplasma and into the mitochondria. These measurements are based on the use of genetically encoded probes. Addition of targeting DNA sequences, in frame with the probe encoding sequence, ensures protein expression in specific compartments. The use of probes with different excitation spectra allows the simultaneous determination of cytosolic and mitochondrial Ca2+ transients in the same fiber. Probe encoding plasmids are expressed in flexor digitorum brevis (FDB) muscles by means of the in vivo electroporation technique. Measurements are then performed ex vivo in isolated single myofibers.

Key words

Skeletal muscle fibers Ca2+ measurements Cytosol Mitochondria Genetically encoded probes 

Notes

Acknowledgments

Research is supported by fundings from the Italian Ministry of Education, University, and Research (PRIN 2015W2N883_003) and the French Muscular Dystrophy Association AFM-Téléthon (18857).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of PaduaPaduaItaly

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