A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells

Detalhes bibliográficos
Autor(a) principal: Chometon, Thaize Quiroga
Data de Publicação: 2020
Outros Autores: Siqueira, Mariana da Silva, Sant`Anna, Julie Carmo, Almeida, Matheus Rogério, Gandini, Mariana, Nogueira, Ana Cristina Martins de Almeida, Antas, Paulo Renato Zuquim
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/40931
Resumo: Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Programa de Pós-Graduação em Vigilância Sanitária. Rio de Janeiro, RJ, Brasil.
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spelling Chometon, Thaize QuirogaSiqueira, Mariana da SilvaSant`Anna, Julie CarmoAlmeida, Matheus RogérioGandini, MarianaNogueira, Ana Cristina Martins de AlmeidaAntas, Paulo Renato Zuquim2020-04-23T13:28:54Z2020-04-23T13:28:54Z2020CHOMETON, Thaize Quiroga et al. A protocol for rapid monocyte isolation and generation of singular human monocytederived dendritic cells. PLoS ONE, v. 15, n. 4, p. 1-16, 9 Apr. 2020.1932-6203https://www.arca.fiocruz.br/handle/icict/4093110.1371/journal.pone.0231132engPublic Library of ScienceProtocoloIsolamenteMonocitos humanos singularesCélulas dendríticasSingular human monocyteProtocolIsolationDendritic cellsA protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cellsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Programa de Pós-Graduação em Vigilância Sanitária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Programa de Pós-Graduação em Vigilância Sanitária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil.The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/40931/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALPauloRZuquim_Antas_etal_IOC_2020.pdfPauloRZuquim_Antas_etal_IOC_2020.pdfapplication/pdf3327169https://www.arca.fiocruz.br/bitstream/icict/40931/2/PauloRZuquim_Antas_etal_IOC_2020.pdfe1f4849fbb99e090d25279d5ca0c96bbMD52TEXTPauloRZuquim_Antas_etal_IOC_2020.pdf.txtPauloRZuquim_Antas_etal_IOC_2020.pdf.txtExtracted 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dc.title.en.fl_str_mv A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
title A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
spellingShingle A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
Chometon, Thaize Quiroga
Protocolo
Isolamente
Monocitos humanos singulares
Células dendríticas
Singular human monocyte
Protocol
Isolation
Dendritic cells
title_short A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
title_full A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
title_fullStr A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
title_full_unstemmed A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
title_sort A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells
author Chometon, Thaize Quiroga
author_facet Chometon, Thaize Quiroga
Siqueira, Mariana da Silva
Sant`Anna, Julie Carmo
Almeida, Matheus Rogério
Gandini, Mariana
Nogueira, Ana Cristina Martins de Almeida
Antas, Paulo Renato Zuquim
author_role author
author2 Siqueira, Mariana da Silva
Sant`Anna, Julie Carmo
Almeida, Matheus Rogério
Gandini, Mariana
Nogueira, Ana Cristina Martins de Almeida
Antas, Paulo Renato Zuquim
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Chometon, Thaize Quiroga
Siqueira, Mariana da Silva
Sant`Anna, Julie Carmo
Almeida, Matheus Rogério
Gandini, Mariana
Nogueira, Ana Cristina Martins de Almeida
Antas, Paulo Renato Zuquim
dc.subject.other.pt_BR.fl_str_mv Protocolo
Isolamente
Monocitos humanos singulares
Células dendríticas
topic Protocolo
Isolamente
Monocitos humanos singulares
Células dendríticas
Singular human monocyte
Protocol
Isolation
Dendritic cells
dc.subject.en.en.fl_str_mv Singular human monocyte
Protocol
Isolation
Dendritic cells
description Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Clínica. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Programa de Pós-Graduação em Vigilância Sanitária. Rio de Janeiro, RJ, Brasil.
publishDate 2020
dc.date.accessioned.fl_str_mv 2020-04-23T13:28:54Z
dc.date.available.fl_str_mv 2020-04-23T13:28:54Z
dc.date.issued.fl_str_mv 2020
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.citation.fl_str_mv CHOMETON, Thaize Quiroga et al. A protocol for rapid monocyte isolation and generation of singular human monocytederived dendritic cells. PLoS ONE, v. 15, n. 4, p. 1-16, 9 Apr. 2020.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/40931
dc.identifier.issn.pt_BR.fl_str_mv 1932-6203
dc.identifier.doi.pt_BR.fl_str_mv 10.1371/journal.pone.0231132
identifier_str_mv CHOMETON, Thaize Quiroga et al. A protocol for rapid monocyte isolation and generation of singular human monocytederived dendritic cells. PLoS ONE, v. 15, n. 4, p. 1-16, 9 Apr. 2020.
1932-6203
10.1371/journal.pone.0231132
url https://www.arca.fiocruz.br/handle/icict/40931
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
instname:Fundação Oswaldo Cruz (FIOCRUZ)
instacron:FIOCRUZ
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
instacron_str FIOCRUZ
institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
bitstream.url.fl_str_mv https://www.arca.fiocruz.br/bitstream/icict/40931/1/license.txt
https://www.arca.fiocruz.br/bitstream/icict/40931/2/PauloRZuquim_Antas_etal_IOC_2020.pdf
https://www.arca.fiocruz.br/bitstream/icict/40931/3/PauloRZuquim_Antas_etal_IOC_2020.pdf.txt
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