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Int. J. Mol. Sci. 2018, 19(9), 2670; https://doi.org/10.3390/ijms19092670

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

1
Department of Laboratory Medicine, Medical School, University of Pécs, Ifjúság u. 13, H-7624 Pécs, Hungary
2
János Szentágothai Research Center, Ifjúság u. 20, H-7624 Pécs, Hungary
3
Department of Pharmacology, Faculty of Pharmacy, University of Pécs, Szigeti u. 12, H-7624 Pécs, Hungary
4
Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UA, UK
*
Author to whom correspondence should be addressed.
Received: 25 August 2018 / Revised: 31 August 2018 / Accepted: 31 August 2018 / Published: 8 September 2018
(This article belongs to the Section Biochemistry)
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Abstract

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds. View Full-Text
Keywords: Intracellular glucose assay; luminescence; validation; ATP; metabolic inhibitors; flow cytometry Intracellular glucose assay; luminescence; validation; ATP; metabolic inhibitors; flow cytometry
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Csepregi, R.; Temesfői, V.; Sali, N.; Poór, M.; W. Needs, P.; A. Kroon, P.; Kőszegi, T. A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells. Int. J. Mol. Sci. 2018, 19, 2670.

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