Name: flow cytometry purification of mouse meiotic cells
Uploaded: 2011-04-15T11:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

这个项目是支持的，部分款项从佛罗里达州斯克里普斯和奖项数目从国家普通医学科学研究所和国家儿童健康和人类发展研究所，分别R01GM085079和R21HD061304。这是手稿斯克里普斯研究所20917。

此协议是可以分开在两个主要步骤：（1）小鼠睾丸细胞的分解和Hoechst 33342染色其次，如果有必要，（2）排序有关减数分裂的分数，包括减数分裂的所有阶段，从生殖细胞的流式细胞仪圆形精子细胞。一旦收集，这些高浓缩铀的减数分裂的人群，可用于广泛的分析。本协议描述了一个成人睾丸的解离;卷可以据此改编为未成年人或额外的睾丸。 1。睾丸离解<ol><li>放置在一个1...

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	<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>Grabske, R. J., Lake, S., Gledhill, B. L., 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=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell. Physiol. 86, 177-189 (1975).</li><li>Purification, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=culture,+and+fractionation+of+spermatogenic+cells.">culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, 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.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Meistrich, M. L., Trostle, P. K. <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+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr. Biol. 16, 660-667 (2006).</li><li>Lassalle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+Population'+cells+in+adult+mouse+testis+express+Bcrp1+gene+and+are+enriched+in+spermatogonia+and+germinal+stem+cells.">Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.</a> Development. 131, 479-487 (2004).</li><li>Bastos, H. <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+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Chowdhury, R., Bois, P. R., Feingold, E., Sherman, S. L., Cheung, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+variation+in+human+meiotic+recombination.">Genetic analysis of variation in human meiotic recombination.</a> PLoS Genet. 5, e1000648-e1000648 (2009).</li><li>Roig, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+TRIP13/PCH2+is+required+for+recombination+and+normal+higher-order+chromosome+structure+during+meiosis.">Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis.</a> PLoS Genet. 6, (2010).</li><li>Getun, I. V., Wu, Z. K., Khalil, A. M., 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=Nucleosome+occupancy+landscape+and+dynamics+at+mouse+recombination+hotspots.">Nucleosome occupancy landscape and dynamics at mouse recombination hotspots.</a> EMBO Rep. 11, 555-560 (2010).</li><li>Romanienko, P. J., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Spo11+gene+is+required+for+meiotic+chromosome+synapsis.">The mouse Spo11 gene is required for meiotic chromosome synapsis.</a> Mol. Cell. 6, 975-987 (2000).</li></ol>

本协议允许同时净化成年雄性小鼠的整个范围内具有非常高的纯度的减数分裂期细胞，使调查研究这一基本过程的动态。纯净细胞，可用于众多应用，核小体映射 9,10 RNA 提取11日 ，重组分子探测，蛋白质分析和多不等。然而，检测方法要适应纯化减数分裂细胞的数量。此外，这种方法的高重复性，它是可能的，池中的多个独立的各种表现在不同的日子里增加一个特定的实验所需?...

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<td>Collagenase Type-1</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>CLSS1</td>
<td>Dissolve to 12,000U/ml in GBSS, store at 4&deg;C</td>
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<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>TRL3</td>
<td>Dissolve in 1mM HCl to 50mg/ml, store at 4&deg;C</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Arcos</td>
<td>230001000</td>
<td>Saturated solution (10mg/ml in DMSO, store at 4&deg;C</td>
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<tr>
<td>DNAse I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>DNEP</td>
<td>Dissolve in water/50% glycerol to 1mg/ml, store at -20&deg;C</td>
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<td>Gey&#x2019;s Balance Salt Solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G9779</td>
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flow cytometry purification of mouse meiotic cells

在睾丸细胞类型的异质性和减数分裂的细胞培养模型的情况下已表现减数分裂过程中的独特差异化的方案，研究的重大障碍。已经开发了两种主要方法净化，在不同程度上，各种成人和未成熟动物的减数分裂分数：淘洗或Staput（沉淀）BSA和/或percoll梯度。这些方法都依赖于细胞的大小和密度分离减数分裂细胞 1-5 。总体而言，除少数细胞群6，这些协议未能产生足够的纯度许多减数分裂的细胞群，都需要详细的分子生物学分析。此外，这种方法通常减数分裂细胞类型可以在特定的时间，这将增加额外的复杂性水平的重复性和均匀性进行比较时，减数分裂的细胞样本进行纯化。 在这里，我们描述了一个精致的方法，使一个轻松地可视化，识别，并净化减数分裂细胞，生殖细胞，圆形精子细胞，使用流式细胞仪结合用Hoechst 33342染色7,8。这种方法，提供了一个在整个减数分裂过程中的整体快照，并允许一个高度净化可行的细胞从最减数分裂期。然后可以将这些纯化的细胞分子的变化，伴随通过减数分裂的进展，例如在基因表达9,10和核小体占用的动态变化，在减数分裂重组11热点详细分析。

Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

Flow Cytometry Purification of Mouse Meiotic Cells

一种有效的方法，从小鼠睾丸中获得高度纯化的可行减数分裂分数描述，一个精细胞分解协议相结合的荧光激活细胞分选（FACS）。这种方法需要利用DNA含量和核密度的离散减数分裂分数的差异。

流动小鼠减数分裂细胞的流式细胞仪净化

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 ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Meiosis

SCIENTISTS IN ACTION

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

LED Thermo Flow ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Combining Optogenetics with Flow Cytometry

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

小鼠精减数分裂染色体的制备

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...