Section of Pathology, Oncology and Experimental Biology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.
Laboratorio di Oncologia, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) G. Gaslini, Genoa, Italy.
Nat Protoc. 2017 Aug;12(8):1542-1562. doi: 10.1038/nprot.2017.052. Epub 2017 Jul 6.
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.
ATP 是连接合成代谢和分解代谢的能量交换因子，是发生在活细胞中的主要反应和过程所必需的，如肌肉收缩、磷酸化和主动运输。ATP 也是细胞外嘌呤能信号机制的关键分子，在炎症和几种其他疾病状态中具有既定的关键作用。在这里，我们描述了使用基于靶向荧光素酶探针的发光技术来测量分离的活细胞和动物中 ATP 浓度的详细方案。在镁、氧气和 ATP 的存在下，蛋白质荧光素酶催化底物荧光素的氧化，这与发光有关。重组表达的野生型荧光素酶仅存在于细胞质中；然而，添加特定的靶向序列可以改变其细胞定位。使用这种策略，我们构建了靶向线粒体基质和质膜外表面的荧光素酶嵌合体。在这里，我们描述了通过平均需要 3 天的总体程序，优化了监测活细胞中细胞质、线粒体基质和细胞周空间中 ATP 浓度的方案。此外，我们还介绍了一种使用转染了荧光素酶的报告细胞在小鼠体内检测细胞外 ATP 的详细方案。该过程可能需要长达 25 天才能完成。
