Quantitation of yeast cell-cell fusion using multicolor flow cytometry

Autores
Salzman, Valentina; Porro, Valentina; Bollati Fogolín, Mariela; Aguilar, Pablo Sebastián
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.
Fil: Salzman, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; Uruguay
Fil: Porro, Valentina. Instituto Pasteur de Montevideo; Uruguay
Fil: Bollati Fogolín, Mariela. Instituto Pasteur de Montevideo; Uruguay
Fil: Aguilar, Pablo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; Uruguay
Materia
Bi-Molecular Complementation
Fertilization
Flow Cytometry
Mating
Microscopy
Saccharomyces Cerevisiae
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/49348
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/49348
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Quantitation of yeast cell-cell fusion using multicolor flow cytometrySalzman, ValentinaPorro, ValentinaBollati Fogolín, MarielaAguilar, Pablo SebastiánBi-Molecular ComplementationFertilizationFlow CytometryMatingMicroscopySaccharomyces Cerevisiaehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.Fil: Salzman, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; UruguayFil: Porro, Valentina. Instituto Pasteur de Montevideo; UruguayFil: Bollati Fogolín, Mariela. Instituto Pasteur de Montevideo; UruguayFil: Aguilar, Pablo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; UruguayWiley-liss, Div John Wiley & Sons Inc2015-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/49348Salzman, Valentina; Porro, Valentina; Bollati Fogolín, Mariela; Aguilar, Pablo Sebastián; Quantitation of yeast cell-cell fusion using multicolor flow cytometry; Wiley-liss, Div John Wiley & Sons Inc; Cytometry Part A; 87; 9; 9-2015; 843-8541552-4922CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22701/abstractinfo:eu-repo/semantics/altIdentifier/doi/10.1002/cyto.a.22701info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:49:18Zoai:ri.conicet.gov.ar:11336/49348instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:49:19.144CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Quantitation of yeast cell-cell fusion using multicolor flow cytometry
title Quantitation of yeast cell-cell fusion using multicolor flow cytometry
spellingShingle Quantitation of yeast cell-cell fusion using multicolor flow cytometry
Salzman, Valentina
Bi-Molecular Complementation
Fertilization
Flow Cytometry
Mating
Microscopy
Saccharomyces Cerevisiae
title_short Quantitation of yeast cell-cell fusion using multicolor flow cytometry
title_full Quantitation of yeast cell-cell fusion using multicolor flow cytometry
title_fullStr Quantitation of yeast cell-cell fusion using multicolor flow cytometry
title_full_unstemmed Quantitation of yeast cell-cell fusion using multicolor flow cytometry
title_sort Quantitation of yeast cell-cell fusion using multicolor flow cytometry
dc.creator.none.fl_str_mv Salzman, Valentina
Porro, Valentina
Bollati Fogolín, Mariela
Aguilar, Pablo Sebastián
author Salzman, Valentina
author_facet Salzman, Valentina
Porro, Valentina
Bollati Fogolín, Mariela
Aguilar, Pablo Sebastián
author_role author
author2 Porro, Valentina
Bollati Fogolín, Mariela
Aguilar, Pablo Sebastián
author2_role author
author
author
dc.subject.none.fl_str_mv Bi-Molecular Complementation
Fertilization
Flow Cytometry
Mating
Microscopy
Saccharomyces Cerevisiae
topic Bi-Molecular Complementation
Fertilization
Flow Cytometry
Mating
Microscopy
Saccharomyces Cerevisiae
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.
Fil: Salzman, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; Uruguay
Fil: Porro, Valentina. Instituto Pasteur de Montevideo; Uruguay
Fil: Bollati Fogolín, Mariela. Instituto Pasteur de Montevideo; Uruguay
Fil: Aguilar, Pablo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Instituto Pasteur de Montevideo; Uruguay
description Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.
publishDate 2015
dc.date.none.fl_str_mv 2015-09
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/49348
Salzman, Valentina; Porro, Valentina; Bollati Fogolín, Mariela; Aguilar, Pablo Sebastián; Quantitation of yeast cell-cell fusion using multicolor flow cytometry; Wiley-liss, Div John Wiley & Sons Inc; Cytometry Part A; 87; 9; 9-2015; 843-854
1552-4922
CONICET Digital
CONICET
url http://hdl.handle.net/11336/49348
identifier_str_mv Salzman, Valentina; Porro, Valentina; Bollati Fogolín, Mariela; Aguilar, Pablo Sebastián; Quantitation of yeast cell-cell fusion using multicolor flow cytometry; Wiley-liss, Div John Wiley & Sons Inc; Cytometry Part A; 87; 9; 9-2015; 843-854
1552-4922
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22701/abstract
info:eu-repo/semantics/altIdentifier/doi/10.1002/cyto.a.22701
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Wiley-liss, Div John Wiley & Sons Inc
publisher.none.fl_str_mv Wiley-liss, Div John Wiley & Sons Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.070432

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