An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation

Detalhes bibliográficos
Autor(a) principal: Sana, Abel
Data de Publicação: 2023
Outros Autores: Rossi, Izadora Volpato, Sabatke, Bruna, Letícia Bassani, Bonato, Medeiros, Lia Carolina Soares, Ramirez, Marcel Ivan
Tipo de documento: Artigo
Idioma: por
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/61288
https://doi.org/10.3390/ life13091799
Resumo: Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.
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spelling Sana, AbelRossi, Izadora VolpatoSabatke, BrunaLetícia Bassani, BonatoMedeiros, Lia Carolina SoaresRamirez, Marcel Ivan2023-11-13T13:45:18Z2023-11-13T13:45:18Z2023SANA, Abel et al. An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation. Life. V.13, n. 1799, p. 1-10, 2023.2075-1729https://www.arca.fiocruz.br/handle/icict/61288https://doi.org/10.3390/ life13091799porMDPIGiardia lambliaExtracellular VesiclesCentrifugationHost-Parasite InteractionsVesículas ExtracelularesCentrifugaciónInteracciones Huésped-ParásitosVésicules extracellulairesInteractions hôte-parasiteGiardia intestinalisVesículas ExtracelularesCentrifugaçãoInterações Hospedeiro-ParasitaAn improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite’s interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles—derived from the plasma membrane) and small (exosomes—derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 ◦C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 ◦C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h.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/61288/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALlife-13-01799.pdflife-13-01799.pdfapplication/pdf1492718https://www.arca.fiocruz.br/bitstream/icict/61288/2/life-13-01799.pdf9deb65c684fafd40f40889de8d98b148MD52icict/612882023-11-13 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dc.title.en_US.fl_str_mv An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
title An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
spellingShingle An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
Sana, Abel
Giardia lamblia
Extracellular Vesicles
Centrifugation
Host-Parasite Interactions
Vesículas Extracelulares
Centrifugación
Interacciones Huésped-Parásitos
Vésicules extracellulaires
Interactions hôte-parasite
Giardia intestinalis
Vesículas Extracelulares
Centrifugação
Interações Hospedeiro-Parasita
title_short An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
title_full An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
title_fullStr An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
title_full_unstemmed An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
title_sort An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation
author Sana, Abel
author_facet Sana, Abel
Rossi, Izadora Volpato
Sabatke, Bruna
Letícia Bassani, Bonato
Medeiros, Lia Carolina Soares
Ramirez, Marcel Ivan
author_role author
author2 Rossi, Izadora Volpato
Sabatke, Bruna
Letícia Bassani, Bonato
Medeiros, Lia Carolina Soares
Ramirez, Marcel Ivan
author2_role author
author
author
author
author
dc.contributor.author.fl_str_mv Sana, Abel
Rossi, Izadora Volpato
Sabatke, Bruna
Letícia Bassani, Bonato
Medeiros, Lia Carolina Soares
Ramirez, Marcel Ivan
dc.subject.other.en_US.fl_str_mv Giardia lamblia
topic Giardia lamblia
Extracellular Vesicles
Centrifugation
Host-Parasite Interactions
Vesículas Extracelulares
Centrifugación
Interacciones Huésped-Parásitos
Vésicules extracellulaires
Interactions hôte-parasite
Giardia intestinalis
Vesículas Extracelulares
Centrifugação
Interações Hospedeiro-Parasita
dc.subject.en.en_US.fl_str_mv Extracellular Vesicles
Centrifugation
Host-Parasite Interactions
dc.subject.es.en_US.fl_str_mv Vesículas Extracelulares
Centrifugación
Interacciones Huésped-Parásitos
dc.subject.fr.en_US.fl_str_mv Vésicules extracellulaires
Interactions hôte-parasite
dc.subject.decs.en_US.fl_str_mv Giardia intestinalis
Vesículas Extracelulares
Centrifugação
Interações Hospedeiro-Parasita
description Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / EVAHPI—Extracellular Vesicles and Host–Parasite Interactions Research Group. Curitiba, PR, Brasil. / Universidade Federal do Paraná. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil.
publishDate 2023
dc.date.accessioned.fl_str_mv 2023-11-13T13:45:18Z
dc.date.available.fl_str_mv 2023-11-13T13:45:18Z
dc.date.issued.fl_str_mv 2023
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 SANA, Abel et al. An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation. Life. V.13, n. 1799, p. 1-10, 2023.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/61288
dc.identifier.issn.en_US.fl_str_mv 2075-1729
dc.identifier.doi.none.fl_str_mv https://doi.org/10.3390/ life13091799
identifier_str_mv SANA, Abel et al. An improved method to enrich large extracellular vesicles derived from Giardia intestinalis through differential centrifugation. Life. V.13, n. 1799, p. 1-10, 2023.
2075-1729
url https://www.arca.fiocruz.br/handle/icict/61288
https://doi.org/10.3390/ life13091799
dc.language.iso.fl_str_mv por
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dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
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/61288/1/license.txt
https://www.arca.fiocruz.br/bitstream/icict/61288/2/life-13-01799.pdf
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repository.name.fl_str_mv Repositório Institucional da FIOCRUZ (ARCA) - Fundação Oswaldo Cruz (FIOCRUZ)
repository.mail.fl_str_mv repositorio.arca@fiocruz.br
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