Abstract
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 enabled the obtainment of high-purity meiotic substages also from mouse testis, namely:•Shortening of the mechanical disaggregation time to optimize the integrity of the suspension.•Elimination of the 25μm-filtration step to ensure the presence of large P cells.•Inclusion of a non-cytotoxic, DNA-specific, 488nm-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 for the discrimination and purification of meiotic prophase I substages.
Highlights
Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level
Spermatogenic stages purification by FACS using a blue-laser-excitable vital dye Heterogeneity of mammalian testis is a major difficulty for the understanding of spermatogenesis bases, since pure or enriched cell populations representing the different stages of sperm development are required for most molecular analyses [1]
The cell suspension should represent as much as possible the original tissue, lack cell clumps [1], and have a high proportion of viable cells as well as few multinucleates, which tend to form as a consequence of the syncytial nature of the seminiferous epithelium [7,8]
Summary
Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye Rosana Rodrıguez-Casuriaga a,*, Federico F. Elisa Souza a, Beatriz Lopez-Carro b, Adriana Geisinger a,c a Departamento de Biologıa Molecular, Instituto de Investigaciones Biologicas Clemente Estable (IIBCE), Montevideo, Uruguay b Servicio de Citometrıa de Flujo y Clasificacion Celular (SECIF), IIBCE, Montevideo, Uruguay c Seccion Bioquımica, Facultad de Ciencias, Universidad de la Republica, Uruguay
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