Next Article in Journal
Glutathione Injection Alleviates the Fluctuation of Metabolic Response under Thermal Stress in Olive Flounder, Paralichthys olivaceus
Next Article in Special Issue
Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma
Previous Article in Journal
Metatranscriptomic Analysis of the Mouse Gut Microbiome Response to the Persistent Organic Pollutant 2,3,7,8-Tetrachlorodibenzofuran
Previous Article in Special Issue
Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids
Open AccessArticle

Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions

1
Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 10178 Berlin, Germany
2
Max-Delbrück-Center for Molecular Medicine (MDC) in the Helmholtz Association, 13125 Berlin, Germany
3
Core Unit Bioinformatics, Berlin Institute of Health (BIH), 10178 Berlin, Germany
4
German Centre for Cardiovascular Research (DZHK), partner site Berlin, 13353 Berlin, Germany
5
Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Medizinische Klinik für Kardiologie und Angiologie, Campus Mitte, 10117 Berlin, Germany
*
Author to whom correspondence should be addressed.
Metabolites 2020, 10(1), 2; https://doi.org/10.3390/metabo10010002
Received: 13 November 2019 / Revised: 9 December 2019 / Accepted: 11 December 2019 / Published: 18 December 2019
(This article belongs to the Special Issue Sample Preparation in Metabolomics)
A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials. View Full-Text
Keywords: GC-MS; 20% FCS; harvesting; extraction; metabolites; normalization GC-MS; 20% FCS; harvesting; extraction; metabolites; normalization
Show Figures

Figure 1

MDPI and ACS Style

Fritsche-Guenther, R.; Bauer, A.; Gloaguen, Y.; Lorenz, M.; Kirwan, J.A. Modified Protocol of Harvesting, Extraction, and Normalization Approaches for Gas Chromatography Mass Spectrometry-Based Metabolomics Analysis of Adherent Cells Grown Under High Fetal Calf Serum Conditions. Metabolites 2020, 10, 2.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop