Cell Energy Budget Platform for Assessment of Cell Metabolism

Part of the Methods in Molecular Biology book series (MIMB, volume 1265)

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.

Key words

Cell metabolism Bioenergetics Oxidative phosphorylation Glycolysis Extracellular acidification Oxygen consumption rate Cell energy budget O2- and pH-sensitive probes Time-resolved fluorescence ATP assay 

Notes

Acknowledgments

Support of this work by the Science Foundation Ireland, grant No. 07/IN.1/B1804, is gratefully acknowledged.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of Biochemistry and Cell BiologyUniversity College CorkCorkIreland

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