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Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes

Nature Protocols volume 8, pages 2105–2118 (2013)Cite this article

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Abstract

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca2+) signaling. Aequorin reacts with Ca2+ via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca2+ probe and how to measure Ca2+ in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

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Figure 1: Setup of an aequorin experiment.
Figure 2: Setup of the aequorin plate-reader assay.
Figure 3: Aequorin calibration into [Ca2+] values.
Figure 4: Measurements of Ca2+ in different cellular compartments.

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Acknowledgements

This research was supported by the Italian Association for Cancer Research (AIRC), Telethon (GGP11139B), local funds from the University of Ferrara, the Italian Ministry of Education, University and Research (COFIN, FIRB and Futuro in Ricerca) and Italian Ministry of Health to P.P.; by Italian Ministry of Health to A.R.; by AIRC to C.G.; by grants from the Italian Ministries of Health and of Education, University and Research, the European Union (ERC mitoCalcium, no. 294777 and FP7 'MyoAGE', no. 223576), US National Institutes of Health (grant no. 2P01AG025532-06A1), Cariparo Foundation (Padua), Cariplo (no. 2012-06-46), AIRC and Telethon-Italy (GPP1005A, GGP11082) to R.R. S.M. was supported by a FIRC fellowship. S.P. was supported by a research fellowship FISM (Cod. 2012/B/11).

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Author notes

  1. Massimo Bonora and Carlotta Giorgi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Interdisciplinary Center for the Study of Inflammation (ICSI), Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy

    Massimo Bonora, Carlotta Giorgi, Angela Bononi, Saverio Marchi, Simone Patergnani, Alessandro Rimessi & Paolo Pinton

  2. Department of Biomedical Sciences, University of Padua and Institute of Neuroscience, Consiglio Nazionale delle Recherche, Padua, Italy

    Rosario Rizzuto

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Contributions

All authors contributed extensively to the work presented in this paper. M.B., C.G., A.B., S.M. and A.R. performed experiments; M.B., C.G., A.B., S.M., A.R., R.R. and P.P. analyzed data; and M.B., C.G. and P.P. wrote the paper.

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Correspondence to Paolo Pinton.

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Bonora, M., Giorgi, C., Bononi, A. et al. Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat Protoc 8, 2105–2118 (2013). https://doi.org/10.1038/nprot.2013.127

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