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Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase
Division of Bio-Imaging, Chuncheon Center, Korea Basic Science Institute, Chuncheon 200-701, Korea
Acupuncture & Meridian Science Research Center, College of Oriental Medicine, Kyung Hee University, Seoul 130-701, Korea
* Author to whom correspondence should be addressed.
Received: 27 September 2012; in revised form: 8 November 2012 / Accepted: 8 November 2012 / Published: 12 November 2012
Abstract: Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10–9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.
Keywords: intracellular ATP; PTD-Conjugated luciferase; live cell-based assay; 2-deoxy- glucose; iodoacetic acid; potassium cyanide
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Lee, M.-S.; Park, W.-S.; Kim, Y.H.; Ahn, W.G.; Kwon, S.-H.; Her, S. Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase. Sensors 2012, 12, 15628-15637.
Lee M-S, Park W-S, Kim YH, Ahn WG, Kwon S-H, Her S. Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase. Sensors. 2012; 12(11):15628-15637.
Lee, Mi-Sook; Park, Wan-Soon; Kim, Young H.; Ahn, Won G.; Kwon, Seung-Hae; Her, Song. 2012. "Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase." Sensors 12, no. 11: 15628-15637.