Abstract
Changes in bioenergetic parameters report on metabolic rearrangement, dysfunction of major pathways, and regulatory processes within the cell, adaptation to energy stress, or new physiological condition. A combined measurement of oxidative phosphorylation, glycolytic flux, the Krebs cycle activity, ATP levels, and total biomass allows detailed metabolic assessment. We describe a simple methodology for high-throughput multiparametric assessment of cell bioenergetics, called cell energy budget (CEB) platform, and demonstrate its practical use with cell models. The CEB relies on a standard multi-label reader with time-resolved fluorescence capabilities, the lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA) extrusion, the phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), the bioluminescent total ATP assay, and absorbance-based total protein assay. This approach can be further extended with the measurement of other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, and redox state, using the corresponding fluorescent or luminescent probes.
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Acknowledgments
Support of this work by the Science Foundation Ireland, grant No. 07/IN.1/B1804, is gratefully acknowledged.
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Papkovsky, D.B., Zhdanov, A.V. (2015). Cell Energy Budget Platform for Assessment of Cell Metabolism. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1265. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2288-8_23
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DOI: https://doi.org/10.1007/978-1-4939-2288-8_23
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2287-1
Online ISBN: 978-1-4939-2288-8
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