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

这部分工作是由中国船舶重工集团公司（I + D项目C022）和PEDECI​​BA支持。作者要感谢玛丽拉博拉蒂和瓦伦蒂娜普罗图1从细胞生物学部（巴斯德研究所蒙得维的亚）慷慨协作有关MoFlo的细胞分选，轻轻提供在这个项目中使用的所有豚鼠标本梅里亚蒙得维的亚。转载BioMed Central的许可; 图2和图3，表1与许可S.卡格尔AG，巴塞尔和图4和图5分别与约翰·威利父子公司（版权拥有者许可，复制或改编威利-布莱克韦尔，2011）。

1。细胞悬液的制备<ol><li> IACUC或同等专业委员会（乌拉圭全国委员会动物实验的建议使用牺牲标本[CNEA）。在我们的情况下，过量的巴比妥给药。 </li><li>解剖睾丸标准批准程序后，将它们放置在一个96毫米的玻璃培养皿上冰，冰冷的DMEM含10毫升辅以10％胎牛血清。 </li><li>删除白膜和削减解封的睾丸，每侧的2 - 3毫米见方的小块。 </li><li>然后，这些碎片被处理在Medimachine，自动机械粉碎机组织里?...

<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>

这里描述的优化方法使细胞悬液的制备从啮齿动物睾丸组织在一个非常快速和可重复的方式，避免了酶和洗涤剂处理和维护良好的细胞的完整性和类型比例。的简洁的程序（跨度15分钟包括睾丸切除，组织切割和处理），涉及最少的处理，酶处理的情况下的一些主要优点。所有这些将占短暂的生命大分子保存好，这是关键的时候，具有代表性的化合物存在于原始细胞人口需要。 <p class="jove_conten...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Medimachine system</td>
 <td>BD</td>
 <td>340587</td>
 <td></td>
 </tr>
 <tr>
 <td>Medicon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340591</td>
 <td></td>
 </tr>
 <tr>
 <td>Filcon unit (50 &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>Hoechst 33342</td>
 <td>Sigma-Aldrich</td>
 <td>14533</td>
 <td>Stock solution 5 mg/ml; used at a final concentration of 5 &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 &mu;g/ml</td>
 </tr>
 </tbody>
</table>

simple and efficient technique for the preparation of testicular cell suspensions

哺乳动物睾丸是非常复杂的器官，包含超过30种不同类型的细胞，包括体睾丸细胞和生殖细胞的不同阶段。这种异质性是一个重要的缺点，关于哺乳动物精子发生的研究的基础，作为纯的或富集的细胞群，在精子发育的某些阶段需要1分子分析。 各种策略，如Staput离心淘洗1，2,3，和流式细胞仪（FC）4,5，为了使基因差异表达的研究已获得丰富或纯化睾丸细胞群。 它是必需的细胞是在悬浮液中大多数浓缩/净化方法。理想的情况下，将细胞悬浮液，将代表原始组织，具有高比例的活细胞和少数多核 - 这往往会形成，因为合胞性质的生精上皮6,7 -和缺乏细胞团块。以前的报告已经证明，由一个专门的机械方法编制的睾丸细胞悬液成群更容易比胰酶消化1。另一方面，酶的RNA酶处理的和/或分解的酶，如胰蛋白酶和胶原酶导致特定的大分子的降解，这是不希望的目标的下游应用。理想的过程中应尽可能短，涉及最少的操作，从而实现了良好的保存大分子的利益，如基因。目前的协议，从固体组织细胞悬液的制备通常费时，高度取决于运营商，并且可以选择性地破坏某些类型的细胞1,8。 这里介绍的协议结合了高度重复性和极其短暂的机械分解的优势酶处理bsence，导致以良好的品质，可以用于流式细胞仪分析和分选4，不可告人的基因表达研究9的细胞悬浮液。

以及分列的细胞悬浮液从大鼠睾丸制备与这里描述的协议的一个例子是图1中所示。 在酶处理6,8和前面描述的机械分解方法2相比，这里介绍的是速度更快，处理涉及较少，很容易重现（取决于运营商），并呈现稀缺的细胞碎片（特别是相对于其它机械方法）和非常少的多核（已经描述了形成作为广泛的组织操纵1,2的结果）。此外，由于相?...

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

睾丸细胞悬浮液从啮齿动物材料，避免酶和洗涤剂，机械制备一种新的协议进行说明。方法很简单，速度快，重复性好，并呈现良好的品质，这是适合流式分选和RNA提取的细胞悬液。

简单和高效的睾丸细胞悬液的制备技术

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

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