Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells

Abstract Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex...

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Autores principales:	Joëlle J. E. Janssen, Bart Lagerwaard, Annelies Bunschoten, Huub F. J. Savelkoul, R. J. Joost van Neerven, Jaap Keijer, Vincent C. J. de Boer
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9a7cd53fcbaa4e87b59307e0047c718b
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spelling	oai:doaj.org-article:9a7cd53fcbaa4e87b59307e0047c718b2021-12-02T13:48:41ZNovel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells10.1038/s41598-021-81217-42045-2322https://doaj.org/article/9a7cd53fcbaa4e87b59307e0047c718b2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81217-4https://doaj.org/toc/2045-2322Abstract Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.Joëlle J. E. JanssenBart LagerwaardAnnelies BunschotenHuub F. J. SavelkoulR. J. Joost van NeervenJaap KeijerVincent C. J. de BoerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Joëlle J. E. Janssen Bart Lagerwaard Annelies Bunschoten Huub F. J. Savelkoul R. J. Joost van Neerven Jaap Keijer Vincent C. J. de Boer Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
description	Abstract Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.
format	article
author	Joëlle J. E. Janssen Bart Lagerwaard Annelies Bunschoten Huub F. J. Savelkoul R. J. Joost van Neerven Jaap Keijer Vincent C. J. de Boer
author_facet	Joëlle J. E. Janssen Bart Lagerwaard Annelies Bunschoten Huub F. J. Savelkoul R. J. Joost van Neerven Jaap Keijer Vincent C. J. de Boer
author_sort	Joëlle J. E. Janssen
title	Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
title_short	Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
title_full	Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
title_fullStr	Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
title_full_unstemmed	Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
title_sort	novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/9a7cd53fcbaa4e87b59307e0047c718b
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_version_	1718392453634981888

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells

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