Journal Article

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

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

2017

Overview

This protocol describes how to construct luciferase probes that are targeted to the mitochondrial matrix or the outer surface of the plasma membrane. These probes can be used to measure ATP concentrations in different cellular compartments. 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/1888 / 631/1647/245/2222 / 631/1647/767 / 631/443/319/333 / Active transport / Adenosine Triphosphate - analysis / Analysis / Analytical Chemistry / Animals / Apoptosis / ATP (Adenosine triphosphate) / Biological Techniques / Bioluminescence / Biosensing Techniques - methods / Catabolism / Cell growth / Cell Line / Cells (biology) / Chimeras / Computational Biology/Bioinformatics / Cytological Techniques - methods / Cytosol / Enzymes / Fluorescent indicators / Humans / Inflammation / Life Sciences / Light emission / Localization / Luciferase / Luciferases - metabolism / Luciferin / Luminescence / Luminescent Agents - metabolism / Magnesium / Measurement / Membranes / Metabolism / Mice / Microarrays / Mitochondria / Muscle contraction / Muscles / Muscular function / Organic Chemistry / Oxidation / Phosphorylation / Plasma / Probes / Proteins / Protocol / Risk factors / Staining and Labeling - methods / Substrates