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Rational cell culture optimization enhances experimental reproducibility in cancer cells

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data. B...

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Autores principales: Wright Muelas, Marina, Ortega, Fernando, Breitling, Rainer, Bendtsen, Claus, Westerhoff, Hans V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813001/
https://www.ncbi.nlm.nih.gov/pubmed/29445172
http://dx.doi.org/10.1038/s41598-018-21050-4
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author Wright Muelas, Marina
Ortega, Fernando
Breitling, Rainer
Bendtsen, Claus
Westerhoff, Hans V.
author_facet Wright Muelas, Marina
Ortega, Fernando
Breitling, Rainer
Bendtsen, Claus
Westerhoff, Hans V.
author_sort Wright Muelas, Marina
collection PubMed
description Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data. Baseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce. We discuss the implications of the cells’ metabolic environment when studying the effects of perturbations to cell function by any type of inhibitor. We then devised ‘metabolically rationalized standard’ assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.
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spelling pubmed-58130012018-02-21 Rational cell culture optimization enhances experimental reproducibility in cancer cells Wright Muelas, Marina Ortega, Fernando Breitling, Rainer Bendtsen, Claus Westerhoff, Hans V. Sci Rep Article Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data. Baseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce. We discuss the implications of the cells’ metabolic environment when studying the effects of perturbations to cell function by any type of inhibitor. We then devised ‘metabolically rationalized standard’ assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays. Nature Publishing Group UK 2018-02-14 /pmc/articles/PMC5813001/ /pubmed/29445172 http://dx.doi.org/10.1038/s41598-018-21050-4 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Wright Muelas, Marina
Ortega, Fernando
Breitling, Rainer
Bendtsen, Claus
Westerhoff, Hans V.
Rational cell culture optimization enhances experimental reproducibility in cancer cells
title Rational cell culture optimization enhances experimental reproducibility in cancer cells
title_full Rational cell culture optimization enhances experimental reproducibility in cancer cells
title_fullStr Rational cell culture optimization enhances experimental reproducibility in cancer cells
title_full_unstemmed Rational cell culture optimization enhances experimental reproducibility in cancer cells
title_short Rational cell culture optimization enhances experimental reproducibility in cancer cells
title_sort rational cell culture optimization enhances experimental reproducibility in cancer cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813001/
https://www.ncbi.nlm.nih.gov/pubmed/29445172
http://dx.doi.org/10.1038/s41598-018-21050-4
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