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.
Highlights
Assessing metabolic function via the cellular ATP content is a widely used method of measuring cellular toxicity in assays that are essential for screening drugs and testing toxicological safety
ATP powers the luciferase-mediated conversion of luciferin into oxyluciferin, which produces a chemiluminescent signal that is proportional to the amount of ATP, according to the following equation: Luciferase
HeLa cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin under 5% CO2 at 37 °C, as recommended by the supplier
Summary
Assessing metabolic function via the cellular ATP content is a widely used method of measuring cellular toxicity in assays that are essential for screening drugs and testing toxicological safety. The current bioluminescent ATP assays are necessary to release the intracellular ATP by the cell extraction processes including cell harvesting, lysis, and separation steps. Reduced membrane integrity caused by the extraction process results in the rapid loss of cytoplasmic ATP, leading to inaccurate measurement of the intracellular ATP content and limiting the application of the method to homogenous high-throughput screening (HTS). To reduce the problems caused by cell extraction processes, methods have been developed that use modified cell lysis solutions, such as the single-step homogenous method [2,6]. This method shows high sensitivity, excellent linearity, simplicity, and rapidity, and requires no cell harvesting or separation steps. The greatest advantage of PTD-Luc is that it allows the rapid, direct measurement of the intracellular ATP content in live cells without requiring cell extraction processes
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