Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.22/10261 |
Resumo: | The green alga Pseudokirchneriella subcapitata has been extensively used for the assessment of adverse impacts of pollutants. Glutathione is involved in antioxidant defence and drug detoxification. Intracellular reduced glutathione (GSH) concentration can be used as an indicator of the health of cells. This work describes a simple and fast fluorescent cell-based assay for the evaluation of intracellular GSH in the alga P. subcapitata, using monochlorobimane (mBCl). Metabolically active algal cells incubated with 50 μmol L−1 mBCl form fluorescent bimane–glutathione (B-SG) adducts that can be measured fluorometrically. The distribution of GSH (B-SG adducts) in whole cells can be observed by epifluorescence microscopy, in the form of blue fluorescent spots. Depletion of cellular GSH with iodoacetamide, inhibition of glutathione S-transferase with ethacrynic acid or heat-induced death of the cells inhibited the formation of fluorescent adducts in the presence of mBCl. The fluorometric assay, using the 96-well microplate format, was able to detect GSH depletion in algal cells. This cell-based assay can be used to evaluate decreases in GSH content due to exposure to toxicants. This assay is amenable to automation and may be useful in high-throughput toxicity screening using the alga P. subcapitata. |
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Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimaneFluorescence microscopyGlutathioneMicroplate assayMonochlorobimaneSelenastrum capricornutumToxicity assayThe green alga Pseudokirchneriella subcapitata has been extensively used for the assessment of adverse impacts of pollutants. Glutathione is involved in antioxidant defence and drug detoxification. Intracellular reduced glutathione (GSH) concentration can be used as an indicator of the health of cells. This work describes a simple and fast fluorescent cell-based assay for the evaluation of intracellular GSH in the alga P. subcapitata, using monochlorobimane (mBCl). Metabolically active algal cells incubated with 50 μmol L−1 mBCl form fluorescent bimane–glutathione (B-SG) adducts that can be measured fluorometrically. The distribution of GSH (B-SG adducts) in whole cells can be observed by epifluorescence microscopy, in the form of blue fluorescent spots. Depletion of cellular GSH with iodoacetamide, inhibition of glutathione S-transferase with ethacrynic acid or heat-induced death of the cells inhibited the formation of fluorescent adducts in the presence of mBCl. The fluorometric assay, using the 96-well microplate format, was able to detect GSH depletion in algal cells. This cell-based assay can be used to evaluate decreases in GSH content due to exposure to toxicants. This assay is amenable to automation and may be useful in high-throughput toxicity screening using the alga P. subcapitata.Springer VerlagRepositório Científico do Instituto Politécnico do PortoMachado, Manuela D.Soares, Eduardo V.2017-09-14T13:29:41Z20122012-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.22/10261eng10.1007/s10811-012-9811-7info: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:RCAAP2023-03-13T12:51:43Zoai:recipp.ipp.pt:10400.22/10261Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T17:30:39.616552Repositório 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 |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
title |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
spellingShingle |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane Machado, Manuela D. Fluorescence microscopy Glutathione Microplate assay Monochlorobimane Selenastrum capricornutum Toxicity assay |
title_short |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
title_full |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
title_fullStr |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
title_full_unstemmed |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
title_sort |
Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane |
author |
Machado, Manuela D. |
author_facet |
Machado, Manuela D. Soares, Eduardo V. |
author_role |
author |
author2 |
Soares, Eduardo V. |
author2_role |
author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Politécnico do Porto |
dc.contributor.author.fl_str_mv |
Machado, Manuela D. Soares, Eduardo V. |
dc.subject.por.fl_str_mv |
Fluorescence microscopy Glutathione Microplate assay Monochlorobimane Selenastrum capricornutum Toxicity assay |
topic |
Fluorescence microscopy Glutathione Microplate assay Monochlorobimane Selenastrum capricornutum Toxicity assay |
description |
The green alga Pseudokirchneriella subcapitata has been extensively used for the assessment of adverse impacts of pollutants. Glutathione is involved in antioxidant defence and drug detoxification. Intracellular reduced glutathione (GSH) concentration can be used as an indicator of the health of cells. This work describes a simple and fast fluorescent cell-based assay for the evaluation of intracellular GSH in the alga P. subcapitata, using monochlorobimane (mBCl). Metabolically active algal cells incubated with 50 μmol L−1 mBCl form fluorescent bimane–glutathione (B-SG) adducts that can be measured fluorometrically. The distribution of GSH (B-SG adducts) in whole cells can be observed by epifluorescence microscopy, in the form of blue fluorescent spots. Depletion of cellular GSH with iodoacetamide, inhibition of glutathione S-transferase with ethacrynic acid or heat-induced death of the cells inhibited the formation of fluorescent adducts in the presence of mBCl. The fluorometric assay, using the 96-well microplate format, was able to detect GSH depletion in algal cells. This cell-based assay can be used to evaluate decreases in GSH content due to exposure to toxicants. This assay is amenable to automation and may be useful in high-throughput toxicity screening using the alga P. subcapitata. |
publishDate |
2012 |
dc.date.none.fl_str_mv |
2012 2012-01-01T00:00:00Z 2017-09-14T13:29:41Z |
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/10400.22/10261 |
url |
http://hdl.handle.net/10400.22/10261 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1007/s10811-012-9811-7 |
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 |
Springer Verlag |
publisher.none.fl_str_mv |
Springer Verlag |
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 instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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 |
repository.mail.fl_str_mv |
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1799131402509221888 |