BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem
Gewählte Publikation:
Bort, JAH; Shanmukam, V; Pabst, M; Windwarder, M; Neumann, L; Alchalabi, A; Krebiehl, G; Koellensperger, G; Hann, S; Sonntag, D; Altmann, F; Heel, C; Borth, N.
(2014):
Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction
J BIOTECHNOL. 2014; 182: 97-103.
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- Abstract:
- In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15 s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
- Autor*innen der BOKU Wien:
- Altmann Friedrich
- Borth Nicole
- Hann Stephan
- Köllensperger Gunda
- Neumann Laura
- Pabst Martin
- Windwarder Markus
- Find related publications in this database (using NML MeSH Indexing)
-
Animals
Arginine/analysis
CHO Cells
Cricetinae
Cricetulus
Filtration/instrumentation
Filtration/methods*
Intracellular Space/chemistry
Metabolomics/instrumentation
Metabolomics/methods*
Syringes*
Tryptophan/analysis
- Find related publications in this database (Keywords)
- Metabolomics
- Quenching
- Metabolite extraction
- Fast filtration
- CHO cells
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