Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye
Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accompl...
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| その他の著者: | , , , , |
| フォーマット: | article |
| 言語: | 英語 |
| 出版事項: |
2014
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| 主題: | |
| オンライン･アクセス: | http://hdl.handle.net/20.500.12381/130 http://dx.doi.org/10.1016/j.mex.2014.10.002 |
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| _version_ | 1868890170784219136 |
|---|---|
| author | Rodríguez-Casuriaga, Rosana |
| author2 | Santiñaque, Federico F. Folle, Gustavo A. Souza, Elisa López-Carro, Beatriz Geisinger, Adriana |
| author2_role | author author author author author |
| author_browse | Folle, Gustavo A. Geisinger, Adriana López-Carro, Beatriz Rodríguez-Casuriaga, Rosana Santiñaque, Federico F. Souza, Elisa |
| author_facet | Rodríguez-Casuriaga, Rosana Santiñaque, Federico F. Folle, Gustavo A. Souza, Elisa López-Carro, Beatriz Geisinger, Adriana |
| author_role | author |
| collection | IIBCE en REDI |
| dc.creator.none.fl_str_mv | Rodríguez-Casuriaga, Rosana Santiñaque, Federico F. Folle, Gustavo A. Souza, Elisa López-Carro, Beatriz Geisinger, Adriana |
| dc.date.none.fl_str_mv | 2014 2019-10-16T15:04:31Z 2019-10-16T15:04:31Z |
| dc.format.none.fl_str_mv | 5 p. |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/20.500.12381/130 FCE_1_2011_1_6742 http://dx.doi.org/10.1016/j.mex.2014.10.002 |
| dc.language.none.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | Elsevier |
| dc.relation.none.fl_str_mv | MethodsX 1:239-243. doi: 10.1016/j.mex.2014.10.002 |
| dc.rights.none.fl_str_mv | Acceso abierto info:eu-repo/semantics/openAccess Reconocimiento 4.0 Internacional. (CC BY) |
| dc.source.none.fl_str_mv | MethodsX. 2014; 1: 239-243 reponame:IIBCE en REDI instname:Instituto de Investigaciones Biológicas Clemente Estable instacron:Instituto de Investigaciones Biológicas Clemente Estable |
| dc.subject.none.fl_str_mv | Citometría de flujo Espermatogénesis Meiosis Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| dc.title.none.fl_str_mv | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| dc.type.none.fl_str_mv | Artículo info:eu-repo/semantics/article Publicado info:eu-repo/semantics/publishedVersion |
| description | Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting. Using that method we could distinguish and purify early meiocytes (leptotene/zygotene stages, L/Z) from more advanced ones (pachytene, P) in guinea pig, which presents an unusually high content of early stages. Here we present an upgrade of that method with improvements that have enabled the obtainment of high-purity meiotic substages also from mouse testis, namely: • Shortening of the mechanical disaggregation time and elimination of a 25µm-filtration step, to optimize the integrity of the suspension and ensure the presence of large P cells. • Inclusion of a non-cytotoxic, DNA-specific, 488_nm-excitable vital fluorochrome (Vybrant-DyeCycle-Green [VDG], Invitrogen) instead of Hoechst 33342 (requires UV laser, which can damage nucleic acids) or propidium iodide (usually related to dead/damaged cells). As far as we know, this is the first report on the use of this fluorochrome on testicular cells. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | anni_b35e6d37d8108aebb99e2f5b71d94ac0 |
| identifier_str_mv | FCE_1_2011_1_6742 |
| instacron_str | Instituto de Investigaciones Biológicas Clemente Estable |
| institution | Instituto de Investigaciones Biológicas Clemente Estable |
| instname_str | Instituto de Investigaciones Biológicas Clemente Estable |
| language | eng |
| network_acronym_str | anni |
| network_name_str | oai-lr-anni |
| oai_identifier_str | oai:redi.anii.org.uy:20.500.12381/130 |
| publishDate | 2014 |
| publishDateSort | 2014 |
| publisher.none.fl_str_mv | Elsevier |
| reponame_str | IIBCE en REDI |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | Acceso abierto Reconocimiento 4.0 Internacional. (CC BY) |
| spelling | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dyeRodríguez-Casuriaga, RosanaSantiñaque, Federico F.Folle, Gustavo A.Souza, ElisaLópez-Carro, BeatrizGeisinger, AdrianaCitometría de flujoEspermatogénesisMeiosisCiencias Naturales y ExactasCiencias BiológicasBioquímica y Biología MolecularAvailability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting. Using that method we could distinguish and purify early meiocytes (leptotene/zygotene stages, L/Z) from more advanced ones (pachytene, P) in guinea pig, which presents an unusually high content of early stages. Here we present an upgrade of that method with improvements that have enabled the obtainment of high-purity meiotic substages also from mouse testis, namely: • Shortening of the mechanical disaggregation time and elimination of a 25µm-filtration step, to optimize the integrity of the suspension and ensure the presence of large P cells. • Inclusion of a non-cytotoxic, DNA-specific, 488_nm-excitable vital fluorochrome (Vybrant-DyeCycle-Green [VDG], Invitrogen) instead of Hoechst 33342 (requires UV laser, which can damage nucleic acids) or propidium iodide (usually related to dead/damaged cells). As far as we know, this is the first report on the use of this fluorochrome on testicular cells.Agencia Nacional de Investigación e InnovaciónElsevier2019-10-16T15:04:31Z2019-10-16T15:04:31Z2014Artículoinfo:eu-repo/semantics/articlePublicadoinfo:eu-repo/semantics/publishedVersion5 p.http://hdl.handle.net/20.500.12381/130FCE_1_2011_1_6742http://dx.doi.org/10.1016/j.mex.2014.10.002MethodsX. 2014; 1: 239-243reponame:IIBCE en REDIinstname:Instituto de Investigaciones Biológicas Clemente Estableinstacron:Instituto de Investigaciones Biológicas Clemente EstableengMethodsX 1:239-243. doi: 10.1016/j.mex.2014.10.002Acceso abiertoinfo:eu-repo/semantics/openAccessReconocimiento 4.0 Internacional. (CC BY)oai:redi.anii.org.uy:20.500.12381/1302026-06-16T05:25:44Z |
| spellingShingle | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye Rodríguez-Casuriaga, Rosana Citometría de flujo Espermatogénesis Meiosis Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| status_str | publishedVersion |
| title | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| title_full | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| title_fullStr | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| title_full_unstemmed | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| title_short | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| title_sort | Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye |
| topic | Citometría de flujo Espermatogénesis Meiosis Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| url | http://hdl.handle.net/20.500.12381/130 http://dx.doi.org/10.1016/j.mex.2014.10.002 |