| Item type: | Article | ||||||||||||||||||||||||||||||||
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| Journal or Publication Title: | Analytical and bioanalytical chemistry | ||||||||||||||||||||||||||||||||
| Publisher: | Springer | ||||||||||||||||||||||||||||||||
| Volume: | 399 | ||||||||||||||||||||||||||||||||
| Number of Issue or Book Chapter: | 3 | ||||||||||||||||||||||||||||||||
| Page Range: | pp. 1127-1139 | ||||||||||||||||||||||||||||||||
| Date: | January 2011 | ||||||||||||||||||||||||||||||||
| Institutions: | Medicine > Lehrstuhl für Anästhesiologie Medicine > Institut für Funktionelle Genomik > Lehrstuhl für Funktionelle Genomik (Prof. Oefner) | ||||||||||||||||||||||||||||||||
| Identification Number: |
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| Classification: |
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| Keywords: | Metabolomics GC-MS Adherent cells Detachment Extraction Amino acids | ||||||||||||||||||||||||||||||||
| Dewey Decimal Classification: | 500 Science > 500 Natural sciences & mathematics 500 Science > 570 Life sciences 600 Technology > 610 Medical sciences Medicine | ||||||||||||||||||||||||||||||||
| Status: | Published | ||||||||||||||||||||||||||||||||
| Refereed: | Yes, this version has been refereed | ||||||||||||||||||||||||||||||||
| Created at the University of Regensburg: | Yes | ||||||||||||||||||||||||||||||||
| Item ID: | 30623 |
Abstract
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, ...
Abstract
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.
Metadata last modified: 29 Sep 2021 07:40
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