Abstract
This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatography–mass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t1/2≤5 min for central metabolites in Escherichia coli), variations on this approach can effectively probe dynamically changing metabolic fluxes. This protocol is exemplified using E. coli and nitrogen labeling, for which quantitative flux data for ∼15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.
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Acknowledgements
This research was supported by the Beckman Foundation, NSF DDDAS grant CNS-0540181, American Heart Association grant 0635188N, NSF CAREER Award MCB-0643859, NIH grant AI078063, and NIH grant GM071508 for Center of Quantitative Biology at Princeton University. We thank Wenyun Lu, Elizabeth Kimball, Sunil Bajad and Joshua Munger for their contributions to the development of the protocols presented here, and David Botstein for suggesting the filter culture approach which played a pivotal role in the development of KFP.
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Yuan, J., Bennett, B. & Rabinowitz, J. Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nat Protoc 3, 1328–1340 (2008). https://doi.org/10.1038/nprot.2008.131
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DOI: https://doi.org/10.1038/nprot.2008.131