Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis
| Autor(a) principal: | |
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| Data de Publicação: | 2017 |
| 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/10451/28768 |
Resumo: | Copyright: Pereira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
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Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysisCell cycleEdUS phaseDNA replicationCopyright: Pereira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.João A. Ferreira received support from a Calouste Gulbenkian Foundation grant (96526) and Pedro Pereira is an FCT fellow (SFRH/BD/45502/2008). Evguenia Bekman is the recipient of an IMM-Lisbon fellowship (iMM/BPD/60-2016; project PTDC/BEXBCM/5899/2014).Impact JournalsRepositório da Universidade de LisboaPereira, Pedro D.Caetano, Ana SerraCabrita, MarisaBekman, EvgueniaBraga, JoséRino, JoséSantus, RenèFilipe, Paulo L.Sousa, Ana E.Ferreira, João A.2017-08-24T13:18:36Z20172017-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10451/28768engOncotarget, 2017, Vol. 8, (No. 25), pp: 40514-405321949-255310.18632/oncotarget.17121info: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:RCAAP2023-11-08T16:20:49Zoai:repositorio.ul.pt:10451/28768Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:44:55.988219Repositório 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 |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| title |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| spellingShingle |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis Pereira, Pedro D. Cell cycle EdU S phase DNA replication |
| title_short |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| title_full |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| title_fullStr |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| title_full_unstemmed |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| title_sort |
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis |
| author |
Pereira, Pedro D. |
| author_facet |
Pereira, Pedro D. Caetano, Ana Serra Cabrita, Marisa Bekman, Evguenia Braga, José Rino, José Santus, Renè Filipe, Paulo L. Sousa, Ana E. Ferreira, João A. |
| author_role |
author |
| author2 |
Caetano, Ana Serra Cabrita, Marisa Bekman, Evguenia Braga, José Rino, José Santus, Renè Filipe, Paulo L. Sousa, Ana E. Ferreira, João A. |
| author2_role |
author author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Repositório da Universidade de Lisboa |
| dc.contributor.author.fl_str_mv |
Pereira, Pedro D. Caetano, Ana Serra Cabrita, Marisa Bekman, Evguenia Braga, José Rino, José Santus, Renè Filipe, Paulo L. Sousa, Ana E. Ferreira, João A. |
| dc.subject.por.fl_str_mv |
Cell cycle EdU S phase DNA replication |
| topic |
Cell cycle EdU S phase DNA replication |
| description |
Copyright: Pereira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
| publishDate |
2017 |
| dc.date.none.fl_str_mv |
2017-08-24T13:18:36Z 2017 2017-01-01T00:00:00Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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publishedVersion |
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http://hdl.handle.net/10451/28768 |
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http://hdl.handle.net/10451/28768 |
| dc.language.iso.fl_str_mv |
eng |
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eng |
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Oncotarget, 2017, Vol. 8, (No. 25), pp: 40514-40532 1949-2553 10.18632/oncotarget.17121 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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Impact Journals |
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Impact Journals |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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