Abstract
Nuclear magnetic resonance (NMR) is one of the key analytical platforms used in the analysis of intracellular and extracellular metabolites. Despite the technological advances that allow for the production of high-quality data, the sampling procedures of cultured cells are less well standardized. Different cell lines and culture media composition require adjustments of the protocols to result meaningful quantitative information. Here we provide the workflow for obtaining quantitative metabolic data from adherent mammalian cells using NMR spectroscopy. The robustness of NMR allows for the implementation of the here described protocol to other cell types with only minor adjustments.
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References
Liu W, Deng Y, Liu Y et al (2013) Stem cell models for drug discovery and toxicology studies. J Biochem Mol Toxicol 27:17–27
Benjamin DI, Cravatt BF, Nomura DK (2012) Global profiling strategies for mapping Dysregulated metabolic pathways in cancer. Cell Metab 16:565–577
Schlegel M, Köhler D, Körner A et al (2016) The neuroimmune guidance cue netrin-1 controls resolution programs and promotes liver regeneration. Hepatology 63:1689–1705
McNamara LE, Sjostrom T, Meek RMD et al (2012) Metabolomics: a valuable tool for stem cell monitoring in regenerative medicine. J R Soc Interface 9:1713–1724
Kelly B, O’Neill LA (2015) Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res 25:771–784
Johnson CH, Ivanisevic J, Siuzdak G (2016) Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol 17:451–459
Bennett BD, Yuan J, Kimball EH et al (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3:1299–1311
Paglia G, Hrafnsdóttir S, Magnúsdóttir M et al (2012) Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole–time of flight mass spectrometry (UPLC–Q–ToF-MS). Anal Bioanal Chem 402:1183–1198
Nagana Gowda GA, Abell L, Lee CF et al (2016) Simultaneous analysis of major coenzymes of cellular redox reactions and energy using ex vivo 1 H NMR spectroscopy. Anal Chem 88:4817–4824
Goldoni L, Beringhelli T, Rocchia W et al (2016) A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance. Anal Biochem 501:26–34
León Z, García-Cañaveras JC, Donato MT et al (2013) Mammalian cell metabolomics: experimental design and sample preparation. Electrophoresis 34:2762–2775
Dietmair S, Timmins NE, Gray PP et al (2010) Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. Anal Biochem 404:155–164
Sellick CA, Hansen R, Stephens GM et al (2011) Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nat Protoc 6:1241–1249
Lorenz MA, Burant CF, Kennedy RT (2011) Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics. Anal Chem 83:3406–3414
Dettmer K, Nürnberger N, Kaspar H et al (2011) Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols. Anal Bioanal Chem 399:1127–1139
Bi H, Krausz KW, Manna SK et al (2013) Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells. Anal Bioanal Chem 405:5279–5289
Ser Z, Liu X, Tang NN et al (2015) Extraction parameters for metabolomics from cultured cells. Anal Biochem 475:22–28
Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Findeisen M, Brand T, Berger S (2007) A 1H-NMR thermometer suitable for cryoprobes. Magn Reson Chem 45:175–178
Wu PSC, Otting G (2005) Rapid pulse length determination in high-resolution NMR. J Magn Reson 176:115–119
Wishart DS, Jewison T, Guo AC et al (2013) HMDB 3.0–the human Metabolome database in 2013. Nucleic Acids Res 41:D801–D807
Ulrich EL, Akutsu H, Doreleijers JF et al (2008) BioMagResBank. Nucleic Acids Res 36:D402–D408
Ludwig C, Easton JM, Lodi A et al (2012) Birmingham metabolite library: a publicly accessible database of 1-D 1H and 2-D 1H J-resolved NMR spectra of authentic metabolite standards (BML-NMR). Metabolomics 8:8–18
Hao J, Liebeke M, Astle W et al (2014) Bayesian deconvolution and quantification of metabolites in complex 1D NMR spectra using BATMAN. Nat Protoc 9:1416–1427
Bharti SK, Roy R (2012) Quantitative 1H NMR spectroscopy. TrAC Trends Anal Chem 35:5–26
Sokolenko S, Blondeel EJMM, Azlah N et al (2014) Profiling convoluted single-dimension proton NMR spectra: a Plackett–Burman approach for assessing quantification error of metabolites in complex mixtures with application to cell culture. Anal Chem 86:3330–3337
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Kostidis, S. (2018). Quantitative Analysis of Central Energy Metabolism in Cell Culture Samples. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_25
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DOI: https://doi.org/10.1007/978-1-4939-7592-1_25
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7591-4
Online ISBN: 978-1-4939-7592-1
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