{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T18:38:35Z","timestamp":1775155115116,"version":"3.50.1"},"reference-count":33,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2019,12,18]],"date-time":"2019-12-18T00:00:00Z","timestamp":1576627200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/metabo10010002","type":"journal-article","created":{"date-parts":[[2019,12,23]],"date-time":"2019-12-23T03:23:12Z","timestamp":1577071392000},"page":"2","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["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"],"prefix":"10.3390","volume":"10","author":[{"given":"Raphaela","family":"Fritsche-Guenther","sequence":"first","affiliation":[{"name":"Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 10178 Berlin, Germany"},{"name":"Max-Delbr\u00fcck-Center for Molecular Medicine (MDC) in the Helmholtz Association, 13125 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Bauer","sequence":"additional","affiliation":[{"name":"Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 10178 Berlin, Germany"},{"name":"Max-Delbr\u00fcck-Center for Molecular Medicine (MDC) in the Helmholtz Association, 13125 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0493-8592","authenticated-orcid":false,"given":"Yoann","family":"Gloaguen","sequence":"additional","affiliation":[{"name":"Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 10178 Berlin, Germany"},{"name":"Max-Delbr\u00fcck-Center for Molecular Medicine (MDC) in the Helmholtz Association, 13125 Berlin, Germany"},{"name":"Core Unit Bioinformatics, Berlin Institute of Health (BIH), 10178 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5267-4101","authenticated-orcid":false,"given":"Mario","family":"Lorenz","sequence":"additional","affiliation":[{"name":"German Centre for Cardiovascular Research (DZHK), partner site Berlin, 13353 Berlin, Germany"},{"name":"Charit\u00e9-Universit\u00e4tsmedizin Berlin, corporate member of Freie Universit\u00e4t Berlin, Humboldt-Universit\u00e4t zu Berlin, and Berlin Institute of Health, Medizinische Klinik f\u00fcr Kardiologie und Angiologie, Campus Mitte, 10117 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5423-1651","authenticated-orcid":false,"given":"Jennifer A.","family":"Kirwan","sequence":"additional","affiliation":[{"name":"Berlin Institute of Health Metabolomics Platform, Berlin Institute of Health (BIH), 10178 Berlin, Germany"},{"name":"Max-Delbr\u00fcck-Center for Molecular Medicine (MDC) in the Helmholtz Association, 13125 Berlin, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kapoore, R.V., and Vaidyanathan, S. 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