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

Giampaolo Morciano, Alba Clara Sarti, Saverio Marchi, Sonia Missiroli, Simonetta Falzoni, Lizzia Raffaghello, Vito Pistoia, Carlotta Giorgi, Francesco Di Virgilio, Paolo Pinton

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1542-1562
Number of pages21
JournalNature Protocols
Volume12
Issue number8
DOIs
Publication statusPublished - Aug 1 2017

Fingerprint

Luciferases
Animals
Adenosine Triphosphate
Cells
Phosphorylation
Active Biological Transport
Light emission
Cell membranes
Muscle Contraction
Luminescence
Cytosol
Magnesium
Muscle
Cell Membrane
Oxygen
Inflammation
Light
Oxidation
Molecules
Monitoring

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Morciano, G., Sarti, A. C., Marchi, S., Missiroli, S., Falzoni, S., Raffaghello, L., ... Pinton, P. (2017). Use of luciferase probes to measure ATP in living cells and animals. Nature Protocols, 12(8), 1542-1562. https://doi.org/10.1038/nprot.2017.052

Use of luciferase probes to measure ATP in living cells and animals. / Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo.

In: Nature Protocols, Vol. 12, No. 8, 01.08.2017, p. 1542-1562.

Research output: Contribution to journalArticle

Morciano, G, Sarti, AC, Marchi, S, Missiroli, S, Falzoni, S, Raffaghello, L, Pistoia, V, Giorgi, C, Di Virgilio, F & Pinton, P 2017, 'Use of luciferase probes to measure ATP in living cells and animals', Nature Protocols, vol. 12, no. 8, pp. 1542-1562. https://doi.org/10.1038/nprot.2017.052
Morciano G, Sarti AC, Marchi S, Missiroli S, Falzoni S, Raffaghello L et al. Use of luciferase probes to measure ATP in living cells and animals. Nature Protocols. 2017 Aug 1;12(8):1542-1562. https://doi.org/10.1038/nprot.2017.052
Morciano, Giampaolo ; Sarti, Alba Clara ; Marchi, Saverio ; Missiroli, Sonia ; Falzoni, Simonetta ; Raffaghello, Lizzia ; Pistoia, Vito ; Giorgi, Carlotta ; Di Virgilio, Francesco ; Pinton, Paolo. / Use of luciferase probes to measure ATP in living cells and animals. In: Nature Protocols. 2017 ; Vol. 12, No. 8. pp. 1542-1562.
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