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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References
Beis I, Newsholme EA (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem J 152:23–32
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795
Hynes J, Natoli E, Will Y (2009) Fluorescent pH and oxygen probes of the assessment of mitochondrial toxicity in isolated mitochondria and whole cells. Curr Protoc Toxicol 2.16. 11–12.16. 22
Ferrick DA, Neilson A, Beeson C (2008) Advances in measuring cellular bioenergetics using extracellular flux. Drug Discov Today 13:268–274
Zhdanov AV, Favre C, O’Flaherty L, Adam J, O’Connor R, Pollard PJ, Papkovsky DB (2011) Comparative bioenergetic assessment of transformed cells using a cell energy budget platform. Integr Biol (Camb) 3:1135–1142
Zhdanov AV, Waters AH, Golubeva AV, Dmitriev RI, Papkovsky DB (2014) Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling. Biochim Biophys Acta 1837:51–62
Ballew RM, Demas J (1989) An error analysis of the rapid lifetime determination method for the evaluation of single exponential decays. Anal Chem 61:30–33
O’Flaherty L, Adam J, Heather LC, Zhdanov AV, Chung YL, Miranda MX, Croft J, Olpin S, Clarke K, Pugh CW, Griffiths J, Papkovsky D, Ashrafian H, Ratcliffe PJ, Pollard PJ (2010) Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism. Hum Mol Genet 19:3844–3851
Favre C, Zhdanov A, Leahy M, Papkovsky D, O’Connor R (2010) Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells. Oncogene 29:3964–3976
Zhdanov AV, Dmitriev RI, Papkovsky DB (2012) Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling. Biosci Rep 32:587–595
Zhdanov AV, Dmitriev RI, Papkovsky DB (2011) Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria. Cell Mol Life Sci 68:903–917
Hynes J, O’Riordan TC, Zhdanov AV, Uray G, Will Y, Papkovsky DB (2009) In vitro analysis of cell metabolism using a long-decay pH-sensitive lanthanide probe and extracellular acidification assay. Anal Biochem 390:21–28
Hynes J, Floyd S, Soini AE, O’Connor R, Papkovsky DB (2003) Fluorescence-based cell viability screening assays using water-soluble oxygen probes. J Biomol Screen 8:264–272
Zhdanov AV, Ogurtsov VI, Taylor CT, Papkovsky DB (2010) Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique. Integr Biol (Camb) 2:443–451
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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