Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining
Autor(a) principal: | |
---|---|
Data de Publicação: | 2021 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFRGS |
Texto Completo: | http://hdl.handle.net/10183/233833 |
Resumo: | Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. |
id |
UFRGS-2_59732dc2391de8f9736bcac8dea606b2 |
---|---|
oai_identifier_str |
oai:www.lume.ufrgs.br:10183/233833 |
network_acronym_str |
UFRGS-2 |
network_name_str |
Repositório Institucional da UFRGS |
repository_id_str |
|
spelling |
Lemos, Natália EmerimDieter, CristineFarias, Mariela GraneroRheinheimer, JakelineSouza, Bianca Marmontel deBauer, Andrea CarlaCrispim, Daisy2022-01-07T04:27:00Z20212357-9730http://hdl.handle.net/10183/233833001135178Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability.application/pdfengClinical and biomedical research. Porto Alegre. Vol. 41, no. 4 (2021), p. 325-331Transplante das ilhotas pancreáticasIlhotas pancreáticasSeparação celularSobrevivência celularCitometria de fluxoIslet transplantationIslet isolationViabilityFlow cytometryComparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staininginfo:eu-repo/semantics/articleinfo:eu-repo/semantics/otherinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFRGSinstname:Universidade Federal do Rio Grande do Sul (UFRGS)instacron:UFRGSTEXT001135178.pdf.txt001135178.pdf.txtExtracted Texttext/plain28157http://www.lume.ufrgs.br/bitstream/10183/233833/2/001135178.pdf.txta27f15b2878f27ad8571a1df3ad15a8bMD52ORIGINAL001135178.pdfTexto completo (inglês)application/pdf624505http://www.lume.ufrgs.br/bitstream/10183/233833/1/001135178.pdf1a018be809d01807072d449da9b1269eMD5110183/2338332022-02-22 04:47:16.940172oai:www.lume.ufrgs.br:10183/233833Repositório de PublicaçõesPUBhttps://lume.ufrgs.br/oai/requestopendoar:2022-02-22T07:47:16Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS)false |
dc.title.pt_BR.fl_str_mv |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
title |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
spellingShingle |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining Lemos, Natália Emerim Transplante das ilhotas pancreáticas Ilhotas pancreáticas Separação celular Sobrevivência celular Citometria de fluxo Islet transplantation Islet isolation Viability Flow cytometry |
title_short |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
title_full |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
title_fullStr |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
title_full_unstemmed |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
title_sort |
Comparison of two techniques for assessing pancreatic islet viability : flow cytometry and fluorescein diacetate/propidium iodide staining |
author |
Lemos, Natália Emerim |
author_facet |
Lemos, Natália Emerim Dieter, Cristine Farias, Mariela Granero Rheinheimer, Jakeline Souza, Bianca Marmontel de Bauer, Andrea Carla Crispim, Daisy |
author_role |
author |
author2 |
Dieter, Cristine Farias, Mariela Granero Rheinheimer, Jakeline Souza, Bianca Marmontel de Bauer, Andrea Carla Crispim, Daisy |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Lemos, Natália Emerim Dieter, Cristine Farias, Mariela Granero Rheinheimer, Jakeline Souza, Bianca Marmontel de Bauer, Andrea Carla Crispim, Daisy |
dc.subject.por.fl_str_mv |
Transplante das ilhotas pancreáticas Ilhotas pancreáticas Separação celular Sobrevivência celular Citometria de fluxo |
topic |
Transplante das ilhotas pancreáticas Ilhotas pancreáticas Separação celular Sobrevivência celular Citometria de fluxo Islet transplantation Islet isolation Viability Flow cytometry |
dc.subject.eng.fl_str_mv |
Islet transplantation Islet isolation Viability Flow cytometry |
description |
Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. |
publishDate |
2021 |
dc.date.issued.fl_str_mv |
2021 |
dc.date.accessioned.fl_str_mv |
2022-01-07T04:27:00Z |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/other |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10183/233833 |
dc.identifier.issn.pt_BR.fl_str_mv |
2357-9730 |
dc.identifier.nrb.pt_BR.fl_str_mv |
001135178 |
identifier_str_mv |
2357-9730 001135178 |
url |
http://hdl.handle.net/10183/233833 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.pt_BR.fl_str_mv |
Clinical and biomedical research. Porto Alegre. Vol. 41, no. 4 (2021), p. 325-331 |
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.source.none.fl_str_mv |
reponame:Repositório Institucional da UFRGS instname:Universidade Federal do Rio Grande do Sul (UFRGS) instacron:UFRGS |
instname_str |
Universidade Federal do Rio Grande do Sul (UFRGS) |
instacron_str |
UFRGS |
institution |
UFRGS |
reponame_str |
Repositório Institucional da UFRGS |
collection |
Repositório Institucional da UFRGS |
bitstream.url.fl_str_mv |
http://www.lume.ufrgs.br/bitstream/10183/233833/2/001135178.pdf.txt http://www.lume.ufrgs.br/bitstream/10183/233833/1/001135178.pdf |
bitstream.checksum.fl_str_mv |
a27f15b2878f27ad8571a1df3ad15a8b 1a018be809d01807072d449da9b1269e |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS) |
repository.mail.fl_str_mv |
|
_version_ |
1801225048080515072 |