Name: a standardized approach for multispecies purification of mammalian male germ cells by mechanical tissue dissociation and flow cytometry
Uploaded: 2017-07-12T14:00:00
Description: Watch this Scientific Journal Video about A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry at JoVE.com

作者感谢山坡动物医院（圣路易市，MO）进行狗睾丸; Jason Arand和Ted Cicero博士在圣路易斯华盛顿大学（WashU）的实验室提供大鼠睾丸和Brianne Tabers来协助收集;洗手间的Jared Hartsock和Salt实验室为豚鼠睾丸;和迈克尔·塔尔科特博士在WashU的比较医学科为微型猪睾丸。作者还承认华盛顿大学医学院的Alvin J. Siteman癌症中心和密苏里州圣路易斯的Barnes-Jewish医院，使用Siteman流式细胞术核心，该核心提供了人员操作的细胞分选服务。 Siteman癌症中心由NCI癌症中心支持拨款＃P30 CA91842部分支持。 该研究由FCT博士研究生资助[SFRH / BD / 51695/2011至ACL]，美国国家卫生研究院的赠款[R01HD078641和R01MH101810至DFC]和FCT研究合同[IF / 01262/2014 to AML]。

以下所述的所有程序符合圣路易斯华盛顿大学动物研究委员会的规定。 机械解离协议的准备工作<ol><li> 预湿50μm一次性组织解聚筒，用于解离整个鼠睾丸。 注意：这种一次性组织解聚筒包含设计用于切割组织的微刀片和具有大约100个六角形孔的不动钢网。用于组织解聚筒的不同尺寸的网可用于基于物种和期望的细胞类型来适应该方案。本文中提到的所有哺?...

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考虑到在哺乳动物精子发生过程中高度保守的染色质动力学，这项工作的目的是制定一个方案来分离不同哺乳动物睾丸组织分化阶段的雄性生殖细胞（ 图1 ）。将单一工作流程应用于不同动物模型的主要障碍之一是需要物种特异性调整，特别是在组织解离方案方面。目前的方法主要依赖于酶组织消化，甚至在物种12内也有协议。为了克服这个问题，本文描?...

鉴于缺乏代表精子发生进展的体外系统，以及睾丸中存在巨大的细胞异质性，对雄性生殖细胞生物学的研究需要强大的技术来分离特定细胞类型的富集群体。荧光激活细胞分选（FACS）已被广泛地用于此目的1，2，3，4，5，因为它提供了高产率和纯度，并超过在生殖细胞类型，它可以识别并的数量的其他分离方法选择6个 ，7个 ，8。流式细胞仪分析的原理是基于单细胞激光束激发后差分光图案的检测。当细胞通过激光时，反射/散射光与细胞大小（前向散射; FSC）和细胞内复杂性（侧向散射; SSC）成比例。有关流式细胞术的详细信息，请参阅Ormerod 9 。 雄性生殖细胞在精子发生的不同阶段经历DNA含量，染色质结构，大小和形状的特异性修饰。因此，不同的细胞群可以被识别并且通过光散射和DNA染色组合用荧光染料10，11分离。几种染料可以用于此目的，如Hoechst的-33342（Hoechst公司）（以格伊辛格和罗德里格斯Casuriaga 3中综述），其在流动方面被频繁使用的流式细胞仪的睾丸细胞的分析在过去的十年1，2，4，10 ， 12 。牛浦Ñ激励用UV光，Hoechst的发射蓝色荧光正比于细胞DNA含量，而远红荧光反映在染色质结构和压实1，13，14可变性。其结果是，在不同分化阶段的雄性生殖细胞的Hoechst染色的单细胞悬浮液的FACS期间表现出特定模式（HO-FACS; 1，12）。有趣的是，由于染色体排出的机制仅在精原细胞阶段才有活性，Hoechst蓝色荧光的强度与这些细胞中的染色质含量成正比，并且在Ho-FACS 15期间它们作为侧群聚簇。另外，Hoechst染色与非渗透性染料碘化丙啶（PI）组合使用户可以在FACS 1期间区分活体（PI阴性）与死亡（PI阳性）细胞SUP&gt;，2，10，12。睾丸生殖细胞已经在流式细胞以前使用这种策略分析和在小鼠广泛优化以区分多达9种生殖细胞类型，包括在减数分裂I 1，2，4，16的4个不同阶段的细胞。为了这项工作的目的，Hoechst染色有三个主要优点。首先，何-FACS已成功地应用于雄性生殖细胞在小鼠模型1，2，12，和其它啮齿动物，例如大鼠和豚鼠17，18，19的分离。第二，Hoechst是一种细胞渗透性染料，不需要膜透化，因此保留ves细胞完整性。最后，由于Hoechst的优先结合的聚（D [AT]）的DNA序列1，20，这意味着，RNA被保留并且，除了DNA和蛋白质不需要RNA酶处理，可用于细菌的进一步的下游分子研究细胞分化。 尽管在哺乳动物物种的流式细胞术分析中观察到的DNA倍体和/或可染色性有相似性（在Geisinger和Rodriguez-Casuriaga 3中综述），但通过流式细胞术对雄性生殖细胞分离描述的方案存在很大的变异性。不同的研究已经使用组织解离的具体方案，并且在不同的模型生物体，主要是小鼠，大鼠和豚鼠中使用不同的DNA结合染料（单独或组合）和FACS门控策略。因此，对不同物种收集的数据的直接比较可能会受到不确定性的影响由方法之间的差异导致的未技术工件。重要的是，整个哺乳动物精子发生（2N-4N-2N-1N）中染色质动力学的显着保护表明，标准化方案可以横向应用于各种哺乳动物物种。 这项研究的目的是建立一个单一的工作流程，适用于不同的哺乳动物物种，通过组合和调整此前公布的技术2,20。通过进行机械解离以克服酶消化所需的物种特异性调节的需要来实现组织处理方法的标准化5 。值得注意的是，啮齿动物睾丸组织的机械解离已被证明比两种方法产生的单细胞悬浮液的酶组织消化20和Ho-FACS表现更好可比的结果5 。作为原理证明，该协议描述了用于隔离多达5个生殖细胞群体的设置：精原细胞（SPG）;原发性（SPC I）和次级精母细胞（SPC II）和精子细胞（SPD） - 圆形（rSPD）和伸长（eSPD）。重要的是，它在实验室中易于实施，主要要求是用于组织解离和进入配备有UV激光的细胞分选器的系统。该工作流程（ 图1 ）是快速和直接的，允许在不到2小时内从新鲜睾丸组织中同时分离4个生殖细胞群体。缩短的处理时间对于维持进一步下游程序的细胞完整性至关重要。此外，其在5种不同物种中的成功表现可以广泛地应用于哺乳动物进化枝内，使其成为分离生殖细胞的理想方法，用于哺乳动物雄性生殖生物的比较研究奥日。 该方案由三个主要部分组成，除了准备步骤外：（1）睾丸组织的机械解离和（2）用Hoechst和PI染色睾丸细胞，然后（3）对相关的精原细胞进行FACS分选。一旦收集，这些丰富的不同哺乳动物睾丸生殖细胞的群体可以用于广泛的应用。该协议描述了一种“一刀切”的解离方法，以从许多不同的哺乳动物物种中纯化雄性生殖细胞。根据用户希望用分离的生殖细胞进行的研究类型，可以使用其他介质或缓冲液。以下方案步骤用于从一个完整的鼠睾丸产生单细胞悬浮液。

<table border="0" cellpadding="0" cellspacing="0" width="731">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">4% paraformaldehyde</td>
			<td style="width:115px;">VWR</td>
			<td style="width:101px;">#15710</td>
			<td style="width:279px;">4% PFA, For fixing sorted cells&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medimachine</td>
			<td>BD Biosciences</td>
			<td>#340588;</td>
			<td>System for testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1X Dulbecco modified Eagle medium</td>
			<td>Life Technologies</td>
			<td>#31053</td>
			<td>DMEM, for testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medicon</td>
			<td>BD Biosciences</td>
			<td>#340591</td>
			<td>Disaggregation cartridge for testis dissociation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>#10082139</td>
			<td>FBS, for FACS ceollction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>#H3570</td>
			<td>Hoecsht, For FACS staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40&#181;m cell strainer</td>
			<td>GREINER BIO-ONE</td>
			<td>#89508-342</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100x 15mm petri dish</td>
			<td>Falcon</td>
			<td>#351029</td>
			<td>For testis dissection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL polypropylene round-bottom tubes</td>
			<td>Falcon</td>
			<td>#352063</td>
			<td>For FACS collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>#20170-650</td>
			<td>For FACS collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3mL needless syringe</td>
			<td>BD Biosciences</td>
			<td>#309657</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50mL conical tube</td>
			<td>MIDSCI</td>
			<td>#C50R</td>
			<td>For testis dissociation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>#L7011</td>
			<td>PI, For FACS staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MoFlo&#160;Legacy cell sorter</td>
			<td>Beckman Coulter&#160;</td>
			<td>#ML99030</td>
			<td>For FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Summit Cell Sorting software</td>
			<td>Beckman Coulter&#160;</td>
			<td>#ML99030</td>
			<td>For FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>#AM9625</td>
			<td>PBS, For microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscopic glass slide</td>
			<td>Fisher Scientific</td>
			<td>#12-544-4</td>
			<td>For microscopy&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific&#160;</td>
			<td>#12-548-87</td>
			<td>For microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable transfer pipette (3mL)&#160;</td>
			<td>Samco Scientific</td>
			<td>#225</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I&#160;</td>
			<td>Roche</td>
			<td>#10104159001</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbon steel surgical blade</td>
			<td>Miltex</td>
			<td>#4-111</td>
			<td>For testis dissection&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Countess automated cell counter&#160;</td>
			<td>Invitrogen</td>
			<td>#C10227&#160;</td>
			<td>For automatic cell counting&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>#T8154</td>
			<td>For viability staining&#160;</td>
		</tr>
	</tbody>
</table>

a standardized approach for multispecies purification of mammalian male germ cells by mechanical tissue dissociation and flow cytometry

荧光激活细胞分选（FACS）已经成为分离哺乳动物睾丸生殖细胞富集群体的首选方法之一。目前，它允许以高产率和纯度区分多达9种鼠生殖细胞群体。鉴别和纯化中的这种高分辨率是可能的，这是由于精子发生过程中染色质结构和数量的独特变化。这些模式可以通过用荧光DNA结合染料如Hoechst-33342（Hoechst）染色的雄性生殖细胞的流式细胞术来捕获。本文是最近开发的用于分离哺乳动物睾丸生殖细胞的方案的详细描述。简单地说，通过机械解离从睾丸组织产生单细胞悬浮液，用Hoechst和碘化丙啶（PI）双重染色，并通过流式细胞术进行处理。连续门控策略，包括选择不同DNA含量的活细胞（PI阴性）（Hoechst强度），i在FACS分选期间使用，以区分多达5种生殖细胞类型。这些包括相应的平均纯度（通过显微镜评估确定）：精原细胞（66％），原发性（71％）和次级（85％）精母细胞和精子细胞（90％），进一步分为圆形（93％）和延长（87％）亚群。整个工作流的执行是直接的，允许与适当的FACS机器同时隔离4个单元格类型，并且可以在不到2小时内执行。由于减少的处理时间对于保留离体细胞的生理学是至关重要的，所以该方法对于雄性生殖细胞生物学的下游高通量研究是理想的。此外，用于哺乳动物生殖细胞的多特异性纯化的标准化方案消除了变量的方法学来源，并且允许将单一试剂用于不同的动物模型。

单细胞悬液从睾丸组织的机械解离 图2比较了在不同条件下通过小鼠睾丸组织的机械解离获得的单细胞悬浮液。通过加工新鲜组织，未染色（ 图2A ）或用Hoechst染色（ 图2B ）获得的样品显示在不同分化阶段存在单细胞，重要的是，细胞结构似乎被保留，包括精子?...

Watch this Scientific Journal Video about A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry at JoVE.com

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

这项工作描述了从不同哺乳动物物种的睾丸组织获得纯化的生殖细胞群体的方法的标准化。结合机械睾丸解离，染色与Hoechst-33342和碘化丙啶和FACS分选是一个简单的方案，广泛应用于男性生殖生物学的比较研究。

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

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

Research

JoVE Journal

Developmental Biology

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

科研

发育生物学

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing

组织清除后使用3D重建方法检测和定量小鼠门牙中的标记保留细胞

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the ...