Kapoore, R.V. orcid.org/0000-0002-2287-0619, Coyle, R. orcid.org/0000-0003-4137-1230, Staton, C.A. et al. (2 more authors) (2017) Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: A case study with the metastatic breast cancer cell line MDA-MB-231. Analyst, 11. 142. pp. 2038-2049. ISSN 0003-2654
Abstract
Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Royal Society of Chemistry 2017. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > The Medical School (Sheffield) > Division of Genomic Medicine (Sheffield) > Department of Oncology and Metabolism (Sheffield) The University of Sheffield > Sheffield Teaching Hospitals |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 15 Jun 2017 10:06 |
Last Modified: | 15 Jun 2017 10:06 |
Published Version: | https://doi.org/10.1039/C7AN00207F |
Status: | Published |
Publisher: | Royal Society of Chemistry |
Refereed: | Yes |
Identification Number: | 10.1039/C7AN00207F |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:117744 |