Name: simple and efficient technique for the preparation of testicular cell suspensions
Uploaded: 2013-08-04T14:00:00.000+00:00
Duration: 15 min 33 s
Description: Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

本研究の一部はCSIC（I + DプロジェクトC022）とPEDECI​​BAによってサポートされていました。著者は優しく、このプロジェクトで使用されているすべてのモルモット標本を提供するために細胞生物学MoFloのセルソーターに関する彼らの寛大なコラボレーションのためのユニット（パスツール研究所デモンテビデオ）、およびメリアル·モンテビデオからマリエラBollatiとヴァレンティナポロに感謝したいと思います。 図1図2と図3、およびS. KargerのAG、バーゼルの許可を得て、表1;; BioMedの中央の許可を得て再現しましたし、 図4と図5は、ジョン·ワイリー＆サンズ株式会社（が所有する著作権の許可を得て複製したり、適応したワイリーブラックウェル、2011）。

1。細胞懸濁液の調製<ol><li>このような動物実験委員会または同等の専門委員会の勧告後に使用する試験片を生け贄に捧げる（ウルグアイ、国家委員会における動物実験のための[CNEA]）。我々のケースでは、ペントバルビタールの過剰摂取は、投与した。 </li><li>標準的な承認さ​​れた手順に従って、精巣を解剖し、氷冷DMEM、10 mlを入れ、氷の上に96ミリメートルのガラスペトリ皿に配置し、10％ウシ胎児血清を添加した。 </li><li>白膜を取り外し、2の平方根の部分にカプセル開放精巣をカット - それぞれの側に3ミリメートル。 </li><li>それらの作品は、その後Medimachine、組織があきステンレススクリーン、金属ローターを含む使い捨てユニットの内側に細分化され自動化された機械的な粉砕機で処理されます。 50μmの使い捨てユニット、disaggregatorのスイッチ、その過程で、これらの作品の5 - そのためには、風邪の代わりに1ミリリットルはDMEMと4を補足<html>メーカーからの簡単な指示に従う50秒のために。 </li><li>針なしの5ミリリットルの注射器 - 3を使用して分解ユニットから得られた細胞懸濁液を回復。 </li><li>以前にDME​​Mを補足0.5mlの浸し、50μmのナイロンメッシュを通して濾過する。 </li><li>氷の上で浸した25μmのナイロンメッシュ、と場所を使用して再度サスペンションをフィルタリング。 </li><li>ノイバウアー室にカウントし、1に対する細胞濃度を調整- 2×10 7細胞/ ml添加したDMEMで。精巣材料の少なくとも4×10 7細胞/グラムは、通常得られる。 </li><li>最後に、細胞凝集を防止するために0.2％の最終濃度にNDA（2 - ナフトール-6,8 - ジスルホン酸、カリウム塩）を追加する。 </li><li>オプション：製造者の指示に従って、動物細胞のための市販のキットに生存精巣細胞懸濁液の細胞生存率を確認する。 </li></ol> 2。フローサイトメトリー分析ve_content &quot;&gt;我々は、ヨウ化プロピジウムの高濃度（PI）により染色した細胞の分析のために488nmで発光するように調整されたコヒーレントアルゴンイオンレーザーを備えたベクトン·ディッキンソンFACSVantageフローサイトメーターを使用している。（未定着にPI入り口の問題ストレス下の細胞は、他の場所で9,10）対処されている。 <ol><li> PI染色のため、細胞懸濁液に50μgの/ mlの最終濃度で蛍光色素追加し、0℃で10分間インキュベート℃の暗所でC。 </li><li>レーザパワーは100mWに設定され、26分の575バンドパスフィルタFL2にPI-発せられる蛍光を収集するために使用される。 </li><li>私たちは、70μmのノズルをFC測定を行う。精母細胞集団を選別するため、エンベロープとして3並べ滴を使用して、通常の-Rやノーマル-Cモードでソート設定。 3でのサンプルとコレクションチューブ保つ - 4°冷却ユニットを使用してC。毎秒500〜1,500の割合で細胞を分析するサンプルの差を調整します。 </li><li>分析するセルクエストソフトウェア（BD）を使用次のパラメーター：前方散乱（FSC-H）、側方散乱（SSC-H）、総放出される蛍光またはパルス面積（FL2-）、および蛍光発光またはパルス幅（FL2-W）が持続。 </li></ol>代わりに、我々は5μgの/ mlの最終濃度に生体染色色素ヘキスト33342を採用し、37時10分°C、暗所でインキュベートした。細胞分析はMoFloサイトメーター（DakoCytomation）を用いて25mWでのUV励起波長のレーザを動作させて搭載し、70μmのノズルを用いて行った。データ操作はサミットv4.3のソフトウェア（DakoCytomation）を用いて行った。

<ol>
	<li>Meistrich, M. L., Prescott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+and+nuclei+from+rodent+testes.">Separation of spermatogenic cells and nuclei from rodent testes.</a> Methods of Cell Biology. 15, 15-54 (1977).</li><li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation,+physical+characterization,+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse.">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li><li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li><li>Geisinger, A., Rodr&#237;guez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis.">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+velocity+sedimentation.">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry.">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., Santi&#241;aque, F., L&#243;pez, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells.">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li><li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+but+not+dead?+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide.">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., L&#243;pez-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis.">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li><li>Span&#242;, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+for+reproductive+biology.">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li></ol>

ここで説明する最適化された方法は、酵素と界面活性剤処理を回避し、良好な細胞の完全性と型の比率を維持し、非常に高速かつ再現可能な方法で、げっ歯類の精巣組織からの細胞懸濁液の調製を可能にする。手順（15分スパンが精巣解剖、組織の切断、および処理が含まれます）、関係する最小限の処理、酵素処理の不在の簡潔さは、主な利点のいくつかである。これらはすべて、元の細?...

<table><tbody><tr><td>Medimachine system</td><td>BD</td><td>340587</td><td></td></tr><tr><td>Medicon unit (50 &amp;mu;m)</td><td>BD</td><td>340591</td><td></td></tr><tr><td>Filcon unit (50 &amp;mu;m)</td><td>BD</td><td>340603</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>430-2100</td><td></td></tr><tr><td>Fetal calf serum</td><td>PAA</td><td>A11-151</td><td></td></tr><tr><td>NDA</td><td>Chemos BmbH</td><td>277081</td><td></td></tr><tr><td>H&amp;#8211;chst 33342</td><td>Sigma-Aldrich</td><td>14533</td><td>Stock solution 5 mg/ml; used at a final concentration of 5 &amp;mu;g/ml</td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>287075</td><td>Stock solution 1 mg/ml; used at a final concentration of 50 &amp;mu;g/ml</td></tr></tbody></table>

simple and efficient technique for the preparation of testicular cell suspensions

哺乳動物の精巣は、体精巣細胞と生殖細胞のさまざまな段階を含む、30以上の異なる種類の細胞が含まれている非常に複雑な器官である。精子開発の特定の段階で純粋なまたは濃縮された細胞集団は最も分子解析1のために必要とされるので、この不均一性は、哺乳類の精子形成の拠点の研究に関する重要な欠点である。 例えばStaput 2,3、遠心水簸1、およびフローサイトメトリー（FC）4,5等の各種戦略が異なる遺伝子発現研究を可能にするために、富化または精製された精巣細胞集団を得るために用いられてきた。 これは、細胞が最も濃縮/精製アプローチが懸濁液であることが必要である。理想的には、細胞懸濁液は、元の組織の代表となり、生細胞と少数multinucleatesの高い割合を持っている - ために形成する傾向があると不足細胞塊1 -精上皮6,7の合胞体性質。以前の報告では、もっぱら機械的な方法によって調製精巣細胞懸濁液は、トリプシン処理したもの1よりも容易に凝集していることも明らかでした。 RNアーゼおよび/または特定のダウンストリームアプリケーションのために望ましくない特定の高分子の劣化へのトリプシン及びコラゲナーゼ鉛のように分解する酵素で、一方、酵素処理。理想的なプロセスは、mRNAのようなその他の巨大分子の良好な保存を達成するために、可能な限り短くし、最小限の操作を含むべきである。固形組織からの細胞懸濁液の調製のための現在のプロトコルは、通常、時間がかかり、非常にオペレータに依存しており、選択的に特定の細胞型1,8に損傷を与えることができる。 ここで紹介するプロトコルは、と再現性の高い、非常に簡単な機械的な分解の利点を兼ね備えていますフローサイトメトリー分析および選別4、及び不純な遺伝子発現研究9に用いることができる良質の細胞懸濁液につながる酵素処理のbsence。

ここで説明されたプロトコルを用いて調製したラット精巣からよく分解された細胞懸濁液の例は、図1に示されている。 酵素処理6,8へと前述した機械的な分解方法2と比較すると、ここで紹介する1（特に他の機械に比べて、はるかに高速です少ないハンドリングを伴う、（not演算子に依存）を簡単に再現可能であり、希少細胞の破片をレンダ?...

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

酵素および洗剤を回避するげっ歯類の材料から精巣細胞懸濁液を機械的に調製するための新規なプロトコルは、記載されている。この方法は非常に簡単、迅速、再現性があり、フローソーティングおよびRNA抽出のために適している良質の細胞懸濁液を、レンダリングする。

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

simple-efficient-technique-for-preparation-testicular-cell

Research

JoVE Journal

Biology

18.7K ビュー.  Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). 酵素および洗剤を回避するげっ歯類の材料から精巣細胞懸濁液を機械的に調製するための新規なプロトコルは、記載されている。この方法は非常に簡単、迅速、再現性があり、フローソーティングおよびRNA抽出のために適している良質の細胞懸濁液を、レンダリングする。

ビデオ: Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

機械的組織解離およびフローサイトメトリーによる哺乳動物雄胚細胞のマルチシード精製のための標準的アプローチ

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

発生生物学

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

標準的な実験装置を用いてマウス精巣からパチテン精子細胞と精子を濃縮

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

ラット精巣からのセルトリ細胞と尿細管周囲細胞の単離

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

免疫・感染学

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

生きた無傷組織における細胞分裂解析のためのショウジョウバエ・ラーバルとプパル精巣の調製

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

細胞外マトリックスベースおよび細胞外マトリックスフリーのマウス精巣オルガノイドの生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

バイオレット励起細胞透過性DNA結合色素を用いたマウス精子細胞の分離

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

マウス減数分裂細胞のフローサイトメトリーの精製

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

生物学

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

長期成体マウスのSSCラインの導出のために文化の精原幹細胞や前駆細胞の濃縮シリアル（SSCS）

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUTの速度沈降を使用して造精細胞型の分離

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

精巣生殖細胞の三次元クロマチンアーキテクチャを保存するためのスライド調製方法

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...