Abstract B49: Bioluminescent assays for measuring glycolytic rate and glucose homeostasis
| Autor: | James J. Cali, Jolanta Vidugiriene, Donna Leippe, Mike Valley, Sarah Duellman, Mary Sobol, Natasha Karassina |
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| Rok vydání: | 2016 |
| Předmět: | |
| Zdroj: | Molecular Cancer Research. 14:B49-B49 |
| ISSN: | 1557-3125 1541-7786 |
| DOI: | 10.1158/1557-3125.metca15-b49 |
| Popis: | We describe a set of bioluminescence assays for systematic evaluation of metabolic alterations in response to different growth conditions or treatments. These newly developed cell-based bioluminescence assays offer new methods to detect different metabolites such as NAD(P)/NAD(P)H, lactate, glutamate, glucose-6-phosphate, glucose and 2-deoxyglucose-6-phosphate (for glucose uptake). These assays address the need for more rapid, sensitive and easier to use approaches to quantify key metabolites in cell lysates and tissues. Using the glucose uptake assay, compounds altering translocation of glucose transporters can be screened and characterized rapidly. The lactate detection assay offers a sensitive and rapid approach for glycolytic rate measurements. The glucose detection assay provides information on glucose consumption rates. When these glucose consumption rates are correlated with lactate secretion, the results serve as an indicator of the metabolic status of the cells (e.g. a shift from oxidative phosphorylation to glycolysis). The glucose assay also provides a convenient and robust approach for studying activation or inhibition of gluconeogenesis or glycogenolysis. These bioluminescent assays are robust and sensitive with assay windows significantly larger than analogous fluorescent or colorimetric methods. The sensitivity (1-5pmol/sample) and wide dynamic range (maximum signal-to-background > 100 fold) of the assays allow simultaneous detection of multiple metabolites from the same set of samples. Most importantly, the assays incorporate rapid inactivation of endogenous enzymes, which overcomes the limitations of the deprotonization step required by other methods. The improved workflow in combination with the high sensitivity of luminescence enables measurement of intracellular metabolite levels by simply adding detection reagents directly to treated cells. Alternatively, small samples (2-5ul) can be removed from the media and used for multiple metabolite (lactate, glucose, glutamate) measurements. By consolidating dose- and time-dependent measurements, these assays conserve cells and reagents, streamline workflow, and produce internally controlled data sets. The assays can be used in various formats (e.g. 96- and 384-well plates), applied to various sample types (e.g. mammalian cells, tissues, and 3D microtissues), and multiplexed with other assays, including our new real time cell viability assay. The bioluminescent real time cell viability assay is a homogeneous, non-lytic method to measure cell viability in real time. It overcomes the limitations of standard end-point lytic assays by providing a comprehensive representation of changes in cell viability occurring in live cells over the entire length of an experiment. Changes in cell viability can be conveniently correlated with changes in metabolite levels. Citation Format: Jolanta Vidugiriene, Donna Leippe, Mary Sobol, Mike Valley, Natasha Karassina, Sarah Duellman, James Cali. Bioluminescent assays for measuring glycolytic rate and glucose homeostasis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B49. |
| Databáze: | OpenAIRE |
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