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/CO 2 (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, Ca 2+, mitochondrial pH, membrane potential, and redox state, using the corresponding fluorescent or luminescent probes.
CITATION STYLE
Papkovsky, D. B., & Zhdanov, A. V. (2015). Cell energy budget platform for assessment of cell metabolism. Methods in Molecular Biology, 1265, 333–348. https://doi.org/10.1007/978-1-4939-2288-8_23
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