A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green

Prieto Mena, Daniel - Aparicio Díaz, Hector Gonzalo - Morande, Pablo E. - Zolessi, Flavio R.

Resumen:

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.


Detalles Bibliográficos
2014
Methyl green
DNA
Flow cytometry
Confocal microscopy
Electrophoresis
Fluorescence
Inglés
Universidad de la República
COLIBRI
https://hdl.handle.net/20.500.12008/22055
Acceso abierto
Licencia Creative Commons Atribución – No Comercial – Sin Derivadas (CC –BY-NC-ND 4.0)
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author Prieto Mena, Daniel
author2 Aparicio Díaz, Hector Gonzalo
Morande, Pablo E.
Zolessi, Flavio R.
author2_role author
author
author
author_facet Prieto Mena, Daniel
Aparicio Díaz, Hector Gonzalo
Morande, Pablo E.
Zolessi, Flavio R.
author_role author
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dc.contributor.filiacion.es.fl_str_mv Prieto Mena, Daniel. IIBCE
Aparicio Díaz, Hector Gonzalo. Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología
Morande, Pablo E. Instituto Pasteur (Montevideo)
Zolessi, Flavio R. Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología
dc.creator.none.fl_str_mv Prieto Mena, Daniel
Aparicio Díaz, Hector Gonzalo
Morande, Pablo E.
Zolessi, Flavio R.
dc.date.accessioned.none.fl_str_mv 2019-10-02T22:12:05Z
dc.date.available.none.fl_str_mv 2019-10-02T22:12:05Z
dc.date.issued.es.fl_str_mv 2014
dc.date.submitted.es.fl_str_mv 20190930
dc.description.abstract.none.fl_txt_mv The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.
dc.description.es.fl_txt_mv Preprint presentado a Springer
dc.format.mimetype.es.fl_str_mv application/pdf
dc.identifier.citation.es.fl_str_mv Prieto, D., y otros. "A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green" [Preprint]. Publicado en: Histochemistry and Cell Biology, 2014, 142 (3): 335-345. doi: 10.1007/s00418-014-1215-0
dc.identifier.uri.none.fl_str_mv https://hdl.handle.net/20.500.12008/22055
dc.language.iso.none.fl_str_mv en
eng
dc.rights.license.none.fl_str_mv Licencia Creative Commons Atribución – No Comercial – Sin Derivadas (CC –BY-NC-ND 4.0)
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
dc.source.none.fl_str_mv reponame:COLIBRI
instname:Universidad de la República
instacron:Universidad de la República
dc.subject.es.fl_str_mv Methyl green
DNA
Flow cytometry
Confocal microscopy
Electrophoresis
Fluorescence
dc.title.none.fl_str_mv A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
dc.type.en.fl_str_mv Preprint
dc.type.none.fl_str_mv info:eu-repo/semantics/preprint
dc.type.version.none.fl_str_mv info:eu-repo/semantics/submittedVersion
description Preprint presentado a Springer
eu_rights_str_mv openAccess
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identifier_str_mv Prieto, D., y otros. "A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green" [Preprint]. Publicado en: Histochemistry and Cell Biology, 2014, 142 (3): 335-345. doi: 10.1007/s00418-014-1215-0
instacron_str Universidad de la República
institution Universidad de la República
instname_str Universidad de la República
language eng
language_invalid_str_mv en
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publishDate 2014
reponame_str COLIBRI
repository.mail.fl_str_mv mabel.seroubian@seciu.edu.uy
repository.name.fl_str_mv COLIBRI - Universidad de la República
repository_id_str 4771
rights_invalid_str_mv Licencia Creative Commons Atribución – No Comercial – Sin Derivadas (CC –BY-NC-ND 4.0)
spelling Prieto Mena, Daniel. IIBCEAparicio Díaz, Hector Gonzalo. Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de BiologíaMorande, Pablo E. Instituto Pasteur (Montevideo)Zolessi, Flavio R. Universidad de la República (Uruguay). Facultad de Ciencias. Instituto de Biología2019-10-02T22:12:05Z2019-10-02T22:12:05Z201420190930Prieto, D., y otros. "A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green" [Preprint]. Publicado en: Histochemistry and Cell Biology, 2014, 142 (3): 335-345. doi: 10.1007/s00418-014-1215-0https://hdl.handle.net/20.500.12008/22055Preprint presentado a SpringerThe increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.Made available in DSpace on 2019-10-02T22:12:05Z (GMT). No. of bitstreams: 5 101007s0041801412150.pdf: 3672120 bytes, checksum: 6b58577ddb18cbbba34972c6cd796920 (MD5) license_text: 38300 bytes, checksum: 098d76773c7b7afafb04cabc04ea8a56 (MD5) license_url: 47 bytes, checksum: 966d4a1cc97b2c4389b5142dd97d3c7f (MD5) license_rdf: 9754 bytes, checksum: ffcba5f515f45166c8d3bb6aa02e3123 (MD5) license.txt: 4194 bytes, checksum: 7f2e2c17ef6585de66da58d1bfa8b5e1 (MD5) Previous issue date: 2014application/pdfenengLas obras depositadas en el Repositorio se rigen por la Ordenanza de los Derechos de la Propiedad Intelectual de la Universidad De La República. (Res. Nº 91 de C.D.C. de 8/III/1994 – D.O. 7/IV/1994) y por la Ordenanza del Repositorio Abierto de la Universidad de la República (Res. 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- Universidad de la Repúblicafalse
spellingShingle A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
Prieto Mena, Daniel
Methyl green
DNA
Flow cytometry
Confocal microscopy
Electrophoresis
Fluorescence
status_str submittedVersion
title A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
title_full A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
title_fullStr A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
title_full_unstemmed A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
title_short A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
title_sort A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green
topic Methyl green
DNA
Flow cytometry
Confocal microscopy
Electrophoresis
Fluorescence
url https://hdl.handle.net/20.500.12008/22055