Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status

Detalhes bibliográficos
Autor(a) principal: Vieira-da-Silva, Beatriz
Data de Publicação: 2023
Outros Autores: Castanho, Miguel A. R. B.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/57604
Resumo: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
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spelling Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic statusResazurinCell viabilityMetabolic activityFluorometric assayUV–Vis assayCytotoxicity© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV–Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical “black box” use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.The project leading to these results has received funding from “la Caixa” Foundation and FCT, I.P. under the project code [LCF/PR/HR21/00605], BREAST-BRAIN-N-BBB.MDPIRepositório da Universidade de LisboaVieira-da-Silva, BeatrizCastanho, Miguel A. R. B.2023-05-25T10:49:16Z20232023-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10451/57604engMolecules 2023, 28(5), 22831439-422710.3390/molecules28052283info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T17:06:12Zoai:repositorio.ul.pt:10451/57604Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T22:08:05.637832Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
title Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
spellingShingle Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
Vieira-da-Silva, Beatriz
Resazurin
Cell viability
Metabolic activity
Fluorometric assay
UV–Vis assay
Cytotoxicity
title_short Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
title_full Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
title_fullStr Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
title_full_unstemmed Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
title_sort Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status
author Vieira-da-Silva, Beatriz
author_facet Vieira-da-Silva, Beatriz
Castanho, Miguel A. R. B.
author_role author
author2 Castanho, Miguel A. R. B.
author2_role author
dc.contributor.none.fl_str_mv Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Vieira-da-Silva, Beatriz
Castanho, Miguel A. R. B.
dc.subject.por.fl_str_mv Resazurin
Cell viability
Metabolic activity
Fluorometric assay
UV–Vis assay
Cytotoxicity
topic Resazurin
Cell viability
Metabolic activity
Fluorometric assay
UV–Vis assay
Cytotoxicity
description © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
publishDate 2023
dc.date.none.fl_str_mv 2023-05-25T10:49:16Z
2023
2023-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/57604
url http://hdl.handle.net/10451/57604
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Molecules 2023, 28(5), 2283
1439-4227
10.3390/molecules28052283
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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instacron_str RCAAP
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collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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