Effective quenching processes for physiologically valid metabolite profiling of suspension cultured Mammalian cells
| Autor: | Gillian M. Stephens, Warwick B. Dunn, Alan J. Dickson, Rasmus Hansen, Arfa Maqsood, Christopher A. Sellick, Royston Goodacre |
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| Rok vydání: | 2008 |
| Předmět: |
Cell
Glucose-6-Phosphate CHO Cells Gas Chromatography-Mass Spectrometry Analytical Chemistry law.invention chemistry.chemical_compound Metabolomics Cricetulus law Cricetinae medicine Animals Cells Cultured Quenching (fluorescence) biology Chemistry Adenine Nucleotides Chinese hamster ovary cell Methanol biology.organism_classification NAD Rats Bicarbonates Ammonium bicarbonate medicine.anatomical_structure Biochemistry Metabolite profiling Recombinant DNA |
| Zdroj: | Analytical chemistry. 81(1) |
| ISSN: | 1520-6882 |
| Popis: | Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. This type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures. |
| Databáze: | OpenAIRE |
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