title: Removing the bottlenecks of cell culture metabolomics: fast normalization procedure, correlation of metabolites to cell number, and impact of the cell harvesting method.
abstract: BACKGROUND
  Although cultured cells are nowadays regularly analyzed by metabolomics technologies, some issues in study setup and data processing are still not resolved to complete satisfaction: a suitable harvesting method for adherent cells, a fast and robust method for data normalization, and the proof that metabolite levels can be normalized to cell number.
  
  
  OBJECTIVE
  We intended to develop a fast method for normalization of cell culture metabolomics samples, to analyze how metabolite levels correlate with cell numbers, and to elucidate the impact of the kind of harvesting on measured metabolite profiles.
  
  
  METHODS
  We cultured four different human cell lines and used them to develop a fluorescence-based method for DNA quantification. Further, we assessed the correlation between metabolite levels and cell numbers and focused on the impact of the harvesting method (scraping or trypsinization) on the metabolite profile.
  
  
  RESULTS
  We developed a fast, sensitive and robust fluorescence-based method for DNA quantification showing excellent linear correlation between fluorescence intensities and cell numbers for all cell lines. Furthermore, 82-97 % of the measured intracellular metabolites displayed linear correlation between metabolite concentrations and cell numbers. We observed differences in amino acids, biogenic amines, and lipid levels between trypsinized and scraped cells.
  
  
  CONCLUSIONS
  We offer a fast, robust, and validated normalization method for cell culture metabolomics samples and demonstrate the eligibility of the normalization of metabolomics data to the cell number. We show a cell line and metabolite-specific impact of the harvesting method on metabolite concentrations.
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Muschet
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Möller
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Prehn
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany ; Lehrstuhl für Experimentelle Genetik, Technische Universität München, 85350 Freising-Weihenstephan, Germany ; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany.
authors.forename: Martin Hrabě
authors.initials: MH
authors.lastname: de Angelis
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany ; Lehrstuhl für Experimentelle Genetik, Technische Universität München, 85350 Freising-Weihenstephan, Germany ; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Adamski
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
authors.forename: Janina
authors.identifier: 0000-0003-4344-4149
authors.initials: J
authors.lastname: Tokarz
keywords: Cell culture metabolomics; Harvesting; Metabolite–cell number correlation; Normalization method