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High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels

  • Simona Feno
  • Giulia Di Marco
  • Agnese De Mario
  • Halenya Monticelli
  • Denis Vecellio ReaneEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)

Abstract

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.

Key words

Calcium probes Aequorin Calcium Cytosol Mitochondria ER High-throughput screening 

Notes

Acknowledgments

We thank Anna Raffaello for the critical reading of the manuscript. This work was supported by the Italian Telethon Foundation (GGP16026) and the French Muscular Dystrophy Association (AFM-Téléthon) (19471).

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Copyright information

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

Authors and Affiliations

  • Simona Feno
    • 1
  • Giulia Di Marco
    • 1
  • Agnese De Mario
    • 1
  • Halenya Monticelli
    • 2
  • Denis Vecellio Reane
    • 1
    Email author
  1. 1.Department of Biomedical SciencesUniversity of PaduaPaduaItaly
  2. 2.Department of Surgery, Oncology and GastroenterologyUniversity of PaduaPaduaItaly

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