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A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity

PURPOSE: Cancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metaboli...

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Autores principales: Herst, Patries M., Grasso, Carole, Fabre, Marie-Sophie, Boukalova, Stepana, Ezrova, Zuzana, Neuzil, Jiri, Berridge, Michael V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691152/
https://www.ncbi.nlm.nih.gov/pubmed/33294636
http://dx.doi.org/10.1016/j.bbrep.2020.100858
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author Herst, Patries M.
Grasso, Carole
Fabre, Marie-Sophie
Boukalova, Stepana
Ezrova, Zuzana
Neuzil, Jiri
Berridge, Michael V.
author_facet Herst, Patries M.
Grasso, Carole
Fabre, Marie-Sophie
Boukalova, Stepana
Ezrova, Zuzana
Neuzil, Jiri
Berridge, Michael V.
author_sort Herst, Patries M.
collection PubMed
description PURPOSE: Cancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET. METHODS: We measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines. RESULTS: We found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype. CONCLUSION: Indirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.
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spelling pubmed-76911522020-12-07 A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity Herst, Patries M. Grasso, Carole Fabre, Marie-Sophie Boukalova, Stepana Ezrova, Zuzana Neuzil, Jiri Berridge, Michael V. Biochem Biophys Rep Research Article PURPOSE: Cancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET. METHODS: We measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines. RESULTS: We found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype. CONCLUSION: Indirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells. Elsevier 2020-11-20 /pmc/articles/PMC7691152/ /pubmed/33294636 http://dx.doi.org/10.1016/j.bbrep.2020.100858 Text en © 2020 Published by Elsevier B.V. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
Herst, Patries M.
Grasso, Carole
Fabre, Marie-Sophie
Boukalova, Stepana
Ezrova, Zuzana
Neuzil, Jiri
Berridge, Michael V.
A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title_full A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title_fullStr A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title_full_unstemmed A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title_short A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
title_sort simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691152/
https://www.ncbi.nlm.nih.gov/pubmed/33294636
http://dx.doi.org/10.1016/j.bbrep.2020.100858
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