期刊
METHODSX
卷 1, 期 -, 页码 239-243出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mex.2014.10.002
关键词
Spermatogenesis; Mouse; Testis; Flow cytometry; Vybrant DyeCycle Green
资金
- Agencia Nacional de Investigacion e Innovacion (ANII, Uruguay) [FCE-1-2011-16742]
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: center dot Shortening of the mechanical disaggregation time to optimize the integrity of the suspension. center dot Elimination of the 25 mm-filtration step to ensure the presence of large P cells. center dot 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 for the discrimination and purification of meiotic prophase I substages. (C) 2014 The Authors. Published by Elsevier B.V.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据