A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis
Autor(a) principal: | |
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Data de Publicação: | 2002 |
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/1822/1505 |
Resumo: | A novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa. |
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A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformisCalcein AMCiliated protozoaCytotoxicity assayEthD-1Science & TechnologyA novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa.Instituto de Biotecnologia e Química Fina (IBQF).Fundação para a Ciência e a Tecnologia - PRAXIS XXI/BD/20328/99.ElsevierUniversidade do MinhoDias, NicolinaLima, Nelson20022002-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/1505eng"Research microbiology". ISSN 0923-2508. 153 (2002) 313-322.0923-250810.1016/S0923-2508(02)01326-812160323info: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:RCAAP2023-07-21T12:32:54Zoai:repositorium.sdum.uminho.pt:1822/1505Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:28:19.425023Repositório 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 |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
title |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
spellingShingle |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis Dias, Nicolina Calcein AM Ciliated protozoa Cytotoxicity assay EthD-1 Science & Technology |
title_short |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
title_full |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
title_fullStr |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
title_full_unstemmed |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
title_sort |
A comparative study using a fluorescence-based and a direct-count assay to determine cytotoxicity in Tetrahymena pyriformis |
author |
Dias, Nicolina |
author_facet |
Dias, Nicolina Lima, Nelson |
author_role |
author |
author2 |
Lima, Nelson |
author2_role |
author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Dias, Nicolina Lima, Nelson |
dc.subject.por.fl_str_mv |
Calcein AM Ciliated protozoa Cytotoxicity assay EthD-1 Science & Technology |
topic |
Calcein AM Ciliated protozoa Cytotoxicity assay EthD-1 Science & Technology |
description |
A novel cellular cytotoxicity assay using two fluorescent dyes was developed as an alternative method to the standard direct count of viable protozoa under light microscopy. The compound calcein AM is a non-fluorescent substance that diffuses passively across intact cell membranes and is converted by intracellular esterases to the green fluorescent calcein, which is retained in viable cells. The addition of EthD-1 that binds to DNA stained nuclei of dead cells red. The experiments were carried out in order to assess viability in the freshwater ciliate Tetrahymena pyriformis after exposure to eight surfactants, two of each representing one of four ionic class (non-ionic, anionic, cationic and amphoteric), and two heavy metals, copper and zinc, at several concentrations. In earlier time exposure, less than one hour of contact with surfactants at sublethal concentrations, the fluorescent method is more sensitive and provides more accurate results than direct counting under light microscopy. In contrast, with increasing time exposure, the results obtained by the two methods were similar. Calcein was shown to be a poor viability marker in the presence of zinc and copper since the fluorescence intensity was affected by the metal presence. However, the fluorescent method offers new opportunities to use advanced techniques, such as flow cytometry, to assess cytotoxicity in protozoa. |
publishDate |
2002 |
dc.date.none.fl_str_mv |
2002 2002-01-01T00:00:00Z |
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/1822/1505 |
url |
http://hdl.handle.net/1822/1505 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
"Research microbiology". ISSN 0923-2508. 153 (2002) 313-322. 0923-2508 10.1016/S0923-2508(02)01326-8 12160323 |
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 |
Elsevier |
publisher.none.fl_str_mv |
Elsevier |
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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1799132777814163456 |