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Use of luciferase probes to measure ATP in living cells and animals

Nature Protocols volume 12, pages 1542–1562 (2017)Cite this article

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Abstract

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

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Figure 1: Schematic rendition of pmeLUC, plasma membrane localization and pathways for ATP release.
Figure 2: Schematics of the in vitro protocol.
Figure 3: Schematics of the in vivo protocol.
Figure 4: ATP kinetics in mitochondrial and cytosolic compartments.
Figure 5: Calculation of basal ATP content and mitochondrial ATP production.
Figure 6: The luminometer.
Figure 7: OXPHOS contribution in ATP synthesis.
Figure 8: The in vivo imaging system.

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Acknowledgements

P.P. is grateful to C. degli Scrovegni for continuous support. P.P. is supported by the Italian Ministry of Education, University and Research (COFIN no. 20129JLHSY_002, FIRB no. RBAP11FXBC_002 and Futuro in Ricerca no. RBFR10EGVP_001), the Italian Cystic Fibrosis Research Foundation (no. 19/2014) and the Telethon Foundation (no. GGP15219/B). P.P. and C.G. are supported by local funds from the University of Ferrara and the Italian Ministry of Health, as well as by the Italian Association for Cancer Research (AIRC nos. IG-18624 and MFAG-13521). F.D.V. is supported by grants from the Italian Association for Cancer Research (no. IG 2016 Id.18581), the Ministry of Health of Italy (no. RF-2011–02348435) and EU-COST Action (no. BM 1406). L.R. is funded by 'Cinque per mille dell'IRPEF–Finanziamento della ricerca sanitaria'.

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  1. Giampaolo Morciano and Alba Clara Sarti: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Morphology, Section of Pathology, Oncology and Experimental Biology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy

    Giampaolo Morciano, Alba Clara Sarti, Saverio Marchi, Sonia Missiroli, Simonetta Falzoni, Carlotta Giorgi, Francesco Di Virgilio & Paolo Pinton

  2. Laboratorio di Oncologia, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) G. Gaslini, Genoa, Italy

    Lizzia Raffaghello

  3. Immunology Area, Ospedale Pediatrico Bambino Gesù, Rome, Italy

    Vito Pistoia

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  1. Giampaolo Morciano
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Contributions

G.M., A.C.S., S. Marchi, S. Missiroli, S.F., L.R., V.P., C.G., F.D.V. and P.P. contributed extensively to the writing of this paper. G.M. and A.C.S. performed the experiments, analyzed the data and generated the figures.

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Correspondence to Francesco Di Virgilio or Paolo Pinton.

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Competing interests

F.D.V. serves as a member of the Scientific Advisory Board of Biosceptre International, a UK-based R&D company developing therapeutic applications of P2X7-receptor-targeting Abs.

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Supplementary Data. Nucleotide sequences of luciferases. (PDF 273 kb)

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Morciano, G., Sarti, A., Marchi, S. et al. Use of luciferase probes to measure ATP in living cells and animals. Nat Protoc 12, 1542–1562 (2017). https://doi.org/10.1038/nprot.2017.052

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