Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

Autores
Stockert, Juan C.; Carou, María Clara; Casas, Adriana Gabriela; Garcia Vior, María Cecilia; Ezquerra Riega, Sergio Dario; Blanco, María M.; Espada, Jesús; Blázquez Castro, Alfonso; Horobin, Richard W.; Lombardo, Daniel Marcelo
Año de publicación
2020
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.
Fil: Stockert, Juan C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina
Fil: Carou, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina
Fil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina
Fil: Garcia Vior, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina
Fil: Ezquerra Riega, Sergio Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Blanco, María M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina
Fil: Espada, Jesús. Universidad Bernardo O'Higgins; Chile
Fil: Blázquez Castro, Alfonso. Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología; España
Fil: Horobin, Richard W.. University of Glasgow; Reino Unido
Fil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Materia
BIOCHEMISTRY
BIOLOGICAL SCIENCES
BIOMOLECULES
CELL BIOLOGY
CELL CULTURE
FLUORESCENT LABELING
LIPID DROPLETS
PMS
QSAR
REDOX REAGENTS
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:11336/142223
Acceder
id CONICETDig_72f06a3a7ae18b0fb12f33653972ca08
oai_identifier_str oai:ri.conicet.gov.ar:11336/142223
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfateStockert, Juan C.Carou, María ClaraCasas, Adriana GabrielaGarcia Vior, María CeciliaEzquerra Riega, Sergio DarioBlanco, María M.Espada, JesúsBlázquez Castro, AlfonsoHorobin, Richard W.Lombardo, Daniel MarceloBIOCHEMISTRYBIOLOGICAL SCIENCESBIOMOLECULESCELL BIOLOGYCELL CULTUREFLUORESCENT LABELINGLIPID DROPLETSPMSQSARREDOX REAGENTShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.Fil: Stockert, Juan C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Carou, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; ArgentinaFil: Garcia Vior, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Ezquerra Riega, Sergio Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Blanco, María M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; ArgentinaFil: Espada, Jesús. Universidad Bernardo O'Higgins; ChileFil: Blázquez Castro, Alfonso. Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología; EspañaFil: Horobin, Richard W.. University of Glasgow; Reino UnidoFil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaElsevier2020-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/142223Stockert, Juan C.; Carou, María Clara; Casas, Adriana Gabriela; Garcia Vior, María Cecilia; Ezquerra Riega, Sergio Dario; et al.; Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate; Elsevier; Heliyon; 6; 6; 6-2020; 1-62405-8440CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S2405844020310264info:eu-repo/semantics/altIdentifier/doi/10.1016/j.heliyon.2020.e04182info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-12-23T13:16:48Zoai:ri.conicet.gov.ar:11336/142223instacron: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:34982025-12-23 13:16:49.169CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
title Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
spellingShingle Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
Stockert, Juan C.
BIOCHEMISTRY
BIOLOGICAL SCIENCES
BIOMOLECULES
CELL BIOLOGY
CELL CULTURE
FLUORESCENT LABELING
LIPID DROPLETS
PMS
QSAR
REDOX REAGENTS
title_short Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
title_full Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
title_fullStr Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
title_full_unstemmed Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
title_sort Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate
dc.creator.none.fl_str_mv Stockert, Juan C.
Carou, María Clara
Casas, Adriana Gabriela
Garcia Vior, María Cecilia
Ezquerra Riega, Sergio Dario
Blanco, María M.
Espada, Jesús
Blázquez Castro, Alfonso
Horobin, Richard W.
Lombardo, Daniel Marcelo
author Stockert, Juan C.
author_facet Stockert, Juan C.
Carou, María Clara
Casas, Adriana Gabriela
Garcia Vior, María Cecilia
Ezquerra Riega, Sergio Dario
Blanco, María M.
Espada, Jesús
Blázquez Castro, Alfonso
Horobin, Richard W.
Lombardo, Daniel Marcelo
author_role author
author2 Carou, María Clara
Casas, Adriana Gabriela
Garcia Vior, María Cecilia
Ezquerra Riega, Sergio Dario
Blanco, María M.
Espada, Jesús
Blázquez Castro, Alfonso
Horobin, Richard W.
Lombardo, Daniel Marcelo
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv BIOCHEMISTRY
BIOLOGICAL SCIENCES
BIOMOLECULES
CELL BIOLOGY
CELL CULTURE
FLUORESCENT LABELING
LIPID DROPLETS
PMS
QSAR
REDOX REAGENTS
topic BIOCHEMISTRY
BIOLOGICAL SCIENCES
BIOMOLECULES
CELL BIOLOGY
CELL CULTURE
FLUORESCENT LABELING
LIPID DROPLETS
PMS
QSAR
REDOX REAGENTS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.
Fil: Stockert, Juan C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina
Fil: Carou, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina
Fil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina
Fil: Garcia Vior, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina
Fil: Ezquerra Riega, Sergio Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
Fil: Blanco, María M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica; Argentina
Fil: Espada, Jesús. Universidad Bernardo O'Higgins; Chile
Fil: Blázquez Castro, Alfonso. Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología; España
Fil: Horobin, Richard W.. University of Glasgow; Reino Unido
Fil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina
description Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 μg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.
publishDate 2020
dc.date.none.fl_str_mv 2020-06
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/11336/142223
Stockert, Juan C.; Carou, María Clara; Casas, Adriana Gabriela; Garcia Vior, María Cecilia; Ezquerra Riega, Sergio Dario; et al.; Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate; Elsevier; Heliyon; 6; 6; 6-2020; 1-6
2405-8440
CONICET Digital
CONICET
url http://hdl.handle.net/11336/142223
identifier_str_mv Stockert, Juan C.; Carou, María Clara; Casas, Adriana Gabriela; Garcia Vior, María Cecilia; Ezquerra Riega, Sergio Dario; et al.; Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate; Elsevier; Heliyon; 6; 6; 6-2020; 1-6
2405-8440
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S2405844020310264
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.heliyon.2020.e04182
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
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
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 12.952241

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