A direct cell quenching method for cell-culture based metabolomics
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.
KeywordsCellular metabolite Cell-culture based metabolomics Direct cell quenching Metabolite profiling of breast cancer cells
This work was funded in part through the Computational Toxicology Program of the U.S. EPA Office of Research and Development and the U.S. EPA Office of Science Council Policy. It has been subjected to review by the National Exposure Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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