A direct cell quenching method for cell-culture based metabolomics |
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Authors: | Quincy Teng Wenlin Huang Timothy W Collette Drew R Ekman Chalet Tan |
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Institution: | (1) National Exposure Research Laboratory, U.S. EPA, 960 College Station Road, 30605 Athens, GA, USA;(2) Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Mercer University, Atlanta, GA 30341, USA |
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Abstract: | A crucial step in metabolomic analysis of cellular extracts is the cell quenching process. The conventional method first uses
trypsin to detach cells from their growth surface. This inevitably changes the profile of cellular metabolites since the detachment
of cells from the extracellular matrix alters their physiology. This conventional method also includes time consuming wash/centrifuge
steps after trypsinization, but prior to quenching cell activity. During this time, a considerable portion of intracellular
metabolites are lost, rendering the conventional method less than ideal for application to metabolomics. We report here a
novel sample preparation method for metabolomics applications using adherent mammalian cells, which eliminates the time consumption
and physiological stress of the trypsinization and wash/centrifuge steps. This new method was evaluated in the study of metabolic
changes caused by 17α-ethynylestradiol (EE2) in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast
cancer cell lines using NMR spectroscopy. The results demonstrated that our direct cell quenching method is rapid, effective,
and exhibits greater metabolite retention, providing an increase of approximately a factor of 50 compared to the conventional
method. |
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Keywords: | Cellular metabolite Cell-culture based metabolomics Direct cell quenching Metabolite profiling of breast cancer cells |
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