| Dokumentenart: | Artikel | ||||||||||||||||||||||||||||||||
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| Titel eines Journals oder einer Zeitschrift: | Analytical and bioanalytical chemistry | ||||||||||||||||||||||||||||||||
| Verlag: | Springer | ||||||||||||||||||||||||||||||||
| Band: | 399 | ||||||||||||||||||||||||||||||||
| Nummer des Zeitschriftenheftes oder des Kapitels: | 3 | ||||||||||||||||||||||||||||||||
| Seitenbereich: | S. 1127-1139 | ||||||||||||||||||||||||||||||||
| Datum: | Januar 2011 | ||||||||||||||||||||||||||||||||
| Institutionen: | Medizin > Lehrstuhl für Anästhesiologie Medizin > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) | ||||||||||||||||||||||||||||||||
| Identifikationsnummer: |
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| Stichwörter / Keywords: | Metabolomics GC-MS Adherent cells Detachment Extraction Amino acids | ||||||||||||||||||||||||||||||||
| Dewey-Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin | ||||||||||||||||||||||||||||||||
| Status: | Veröffentlicht | ||||||||||||||||||||||||||||||||
| Begutachtet: | Ja, diese Version wurde begutachtet | ||||||||||||||||||||||||||||||||
| An der Universität Regensburg entstanden: | Ja | ||||||||||||||||||||||||||||||||
| Dokumenten-ID: | 30623 |
Zusammenfassung
Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, ...
Zusammenfassung
Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid-base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid-base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.
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