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乳动物物种都使用了50微米的药筒。 <ol><li>将1mL冰冷酚醛游离1x Dulbecco&#39;s Modified Eagle Medium（DMEM）加到50μm的一次性组织解聚筒上。 注意:苯酚红可能会干扰红色荧光的检测在FACS期间。 </li><li>从组织解聚筒吸取1x DMEM，使用一次性3毫升无针注射器从注射器端口。 </li></ol></li><li>按"ON"按钮打开组织分解系统。该系统不需要"预热"时间。 </li><li>预湿三个40μm一次性过滤器，冰冷1×DMEM。将每个40μm一次性过滤器放在50mL锥形管上。移取1 mL 1x DMEM，并允许液体通过。 </li><li>准备一个100毫米x 15毫米培养皿收集睾丸组织。移取500μL的1x DMEM到培养皿上。 </li></ol> 2.用于机械解离方案的睾丸组织的制备<ol><li> 解剖雄性小鼠，用手术剪刀和镊子小心地清除新鲜的整个睾丸或睾丸碎片（如果与其他哺乳动物物种处理）。 注意:确保组织没有脂肪和坏死。神父<html> esh睾丸组织比冷冻组织产生优异的单细胞悬浮质量。 <ol><li>将收集的睾丸/片段转移到制备的含有500μL1x DMEM的100mm×15mm培养皿中。 </li><li>轻轻冲洗1x DMEM中的组织以除去红细胞（如果存在）。 </li></ol></li><li> 仔细清除白喉白蛉的厚度在不同的物种中会有差异。 <ol><li>用镊子抓住睾丸的一端。 </li><li>用手术刀刺穿睾丸的另一端，同时用上一步骤的镊子夹住睾丸的一端。 </li><li>使用手术刀擦拭睾丸小管，同时用镊子将睾丸的一端固定在步骤2.2.1中。 </li></ol></li><li>使用手术刀将睾丸小管切割成〜2-3 mm 3 。 </li></ol> 3.通过睾丸组织的机械解离获得单细胞悬液注意:下面描述的解离步骤是针对一只成年小鼠睾丸，来自幼体小鼠或非鼠类的睾丸组织的体积应相应调整，少年动物可能不包含生殖细胞的所有阶段，一些哺乳动物物种与非交配季节相比，繁殖季节睾丸组织组成发生变化。 <ol><li>使用镊子将小睾丸小管块转移到预先润湿的50μm的组织解聚筒，并加入1mL的1mM DMEM。 </li><li>将组织解聚筒装载到组织解聚系统，并通过将旋钮从"待机"转到"运行"模式，处理5分钟。 </li><li> 从组织解聚系统中取出组织解聚筒，并用注射器端口的一次性3毫升无针注射器吸出细胞悬液。 <ol><li>吸出数次以从组织解聚筒中除去所有液体。不所有的1mL可以从组织解聚筒回收。 </li></ol></li><li> 将抽吸的细胞悬浮液通过两个一次性预先润湿的40μm过滤器。过滤两次以除去任何细胞聚集体。 <ol><li>将一个预先润湿的40μm过滤器放在50mL锥形管中。将注射器中的吸入细胞直接添加到40μm过滤器上。用一次性移液管吸取直通单细胞悬液。 </li><li>取出使用的预先润湿的40μm过滤器，并将另一个预先润湿的40μm过滤器放入50mL锥形管中。将收集的单细胞悬液移至干净的40μm过滤器上。 </li><li>用干净的一次性移液管收集过滤的单细胞悬液。 </li></ol></li><li>将经过滤的细胞悬浮液移回到50μm的组织解聚筒中，并在组织解聚系统中再处理5分钟。 </li><li>从组织分解中恢复所有液体在墨盒上。 </li></ol> 4.用Hoechst和碘化丙啶染色（PI） <ol><li>将回收的细胞悬浮液从50μm的组织解聚筒转移到干净的1.5mL管中。将回收大约1-1.5mL的单细胞悬浮液。 </li><li> 将回收的单细胞悬浮液分成四个1.5 mL或5 mL管。 <ol><li>对于前三个管，将150μL单细胞悬浮液和移液管的单细胞悬浮液移至最后四个管中（约550μL的单细胞悬液）。 注意:最后一个管中单细胞悬浮液的量可能会根据步骤3.6中细胞悬浮液恢复的效率而变化。 </li></ol></li><li>将四管的第一管作为FACS会话的未染色控制。 </li><li>准备单染染色（PI或Hoechst）细胞悬浮液作为对照。向每个管中加入2.5μLHoechst或1μLPI（第二次和第RD）。 </li><li>准备一个双重染色的管。这将用于收集生殖细胞亚群。向最后一个管中加入2.5μLHoechst和1μLPI。 注意:Hoechst的保质期为2-3个月。使用较老的染料会在FACS会议期间引起重大改变。 </li><li>在黑暗中室温孵育30分钟。将样品置于管旋转器中，或每5-10分钟倒置1.5 mL管。 </li><li>用预先润湿的40微米过滤器过滤细胞悬浮液，并将过滤的溶液保持在冰上并在黑暗中直到FACS会话。 注意:过滤步骤对于防止FACS的细胞团是必要的。另外，可以使用DNase（ 见图1 ）或2-萘酚-6,8-二磺酸二钾盐（NDA 20 ）进行处理以限制细胞团聚。延长的等待期将影响在FACS期间检测到的荧光信号，并可能导致细胞死亡。 </li&gt; </ol> 5.荧光活化细胞分选（FACS）设置和纯化睾丸细胞<ol><li> 准备FACS收集管。 <ol><li>涂5毫升聚丙烯圆底管或1.5毫升管，400μLFBS用于细胞收集。涂层后含量超过FBS。 </li><li>将FACS收集培养基（1×DMEM + 10％胎牛血清，FBS）加入管中:1mL用于5mL试管或100μL用于1.5mL试管。 </li></ol></li><li> 在细胞分选器和软件中设置适当的排序条件。 注意:其他细胞分选机和分析软件可以与下面描述的类似设置和门控策略一起使用。这里描述的条件改编自Gaysinskaya和Bortvin 2 ，Geisinger和Rodriguez-Casuriaga 3以及Getun 等人。 4 <ol><li>加载463/2的紫外线激光5 nm带通滤波器检测Hoechst蓝色和680 nm LP带通滤波器，以检测Hoechst红色并检测PI。 </li><li>使用555DLP分色镜将蓝色与红色荧光区分开来。 </li><li>使用70微米的喷嘴，并以1000-2000个/秒的速度对细胞进行分类。 注意:排序效率直接受流量的影响。高流量（&gt; 3500事件/ s）会提高排序速度，但会导致人口污染。有关详细信息，请参阅参考12 。 </li></ol></li><li> 使用控制样品设置门。 <ol><li>加载并运行未染色样品。 </li><li>排除基于FSC与SSC图谱的细胞碎片。细胞碎片将弹出阴谋的左下象限。任意设定用户或细胞分选技术员对细胞碎片的阈值。 </li><li>通过调整FSC和脉冲宽度的阈值对单个单元进行门控。不同的细胞分选机有不同的区分方式贵族单身随着脉冲宽度反映细胞穿过激光的时间，当与多细胞聚集体相比时，单细胞具有较低的值。可以使用FSC与脉冲宽度的曲线图上的调整阈值来选择单体。 </li><li>设置最佳的光电倍增管（PMT）电压。 </li><li>使用未染色和单染染色细胞来建立PI或Hoechst荧光信号的阈值。 注意:为了确定每种染料的适当荧光范围，优化感兴趣的细胞的基线PTM电压是很重要的。这对于最佳信号检测和灵敏度至关重要。未染色和单染色的对照细胞用于利用PI和/或Hoechst染料建立一系列负性和阳性染色的细胞，并使信号中的噪声最小化。有关使用控制电池优化PMT电压的进一步说明，请参阅Gaysinskaya和Bortvin 2 </li><li>活细胞门基于PI染色（PI阴性）和FSC图。死细胞对于PI荧光将是阳性的。 </li><li>通过绘制基于Hoechst蓝色荧光的细胞计数直方图来设置DNA含量门。应出现Hoechst蓝色荧光浓度增加的3个峰，表示单倍体（1C），二倍体（2C）和四倍体（4C）细胞。设置3个门，每个峰值一个。 </li><li>在进行门控生殖细胞群体之前，先观察FSC至SSC图上至少50万次事件。 </li></ol></li><li> 门不同的生殖细胞群体。 <ol><li>负载Hoechst / PI染色样品。 </li><li>设置所有种群共同的前3个父门。 <ol><li>根据FSC vs SSC图来排除细胞碎片。根据FSC和脉冲宽度图选择单片。通过选择PI阴性细胞选择活细胞。 </li></ol></li><li>定义精原细胞门。 </li><li>绘制基于Hoechst蓝色和红色荧光的PI阴性细胞强度。精原细胞看起来像侧面群体（ 见图1 ）。 </li><li>为剩下的生殖细胞群定义门。注意:有关可视化细节，请参见图1和补充图2。 </li><li>在DNA含量门中绘制PI阴性细胞。 <ol><li>对于精子细胞，以Hoechst最低荧光（1C）为最高峰。 </li><li>对于精母细胞II，使用中间Hoechst荧光（2C）门极化。 </li><li>对于精母细胞I，以最高Hoechst荧光（4C）门极化。 </li></ol></li><li>绘制Hoechst蓝色和红色荧光强度，以从DNA含量门精细胞精子细胞和精子细胞群体。 </li></ol></li><li>将选定的亚群门收集到预先准备的收集管中。每个睾丸平均需要45分钟至1.5小时才能收集每个亚群约0.5-6.0×10 6个细胞。 注意:长时间的细胞暴露于Hoechst ma随着时间的推移，人口的位置略微移动。在FACS 20-30分钟后刷新设置。每个亚群的最大产量将取决于解离步骤的效率。 </li></ol>纯化细胞的显微镜评估<ol><li>在4℃下以500-600xg旋转收集细胞10分钟。 </li><li>用1mL冰冷的1x磷酸盐缓冲溶液（PBS）或1×DMEM再悬浮细胞沉淀。 注意:可以根据分选的细胞数量和所需的最终浓度调整再悬浮体积。 </li><li>将40μL经洗涤的细胞移至干净的玻璃载玻片上并放置盖玻片。 </li><li> 用4％多聚甲醛（PFA）固定剩余的细胞，并将固定的细胞在4℃下保存在黑暗中，以备将来参考。 <ol><li>在收集管中直接吸取100μL4％PFA。 </li><li>简单地涡旋管或移液管几次以确保细胞悬液的充分混合。 </li></ol></li><li>在装有紫外灯的显微镜的显微镜下可视化制备的载玻片，以在63X物镜下检测Hoechst荧光。参考结果部分 - 分类生殖细胞群体的形态学评估 - 有关如何鉴定特定生殖细胞类型的更多细节。 </li></ol>

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A. <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 (8), 625-634 (2011).</li><li>Rodriguez-Casuriaga, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+preparation+of+rodent+testicular+cell+suspensions+and+spermatogenic+stages+purification+by+flow+cytometry+using+a+novel+blue-laser-excitable+vital+dye.">Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye.</a> MethodsX. 1, 239-243 (2014).</li><li>Rodriguez-Casuriaga, R., et al. <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>Hess, R. A., Renato de Franca, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+and+cycle+of+the+seminiferous+epithelium.">Spermatogenesis and cycle of the seminiferous epithelium.</a> Adv Exp Med Biol. 636, 1-15 (2008).</li><li>Zhao, H., Traganos, F., Dobrucki, J., Wlodkowic, D., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+DNA+damage+response+by+the+supravital+probes+of+nucleic+acids.">Induction of DNA damage response by the supravital probes of nucleic acids.</a> Cytometry A. 75 (6), 510-519 (2009).</li><li>Gassei, K., Ehmcke, J., Dhir, R., Schlatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+activated+cell+sorting+allows+isolation+of+spermatogonia+from+adult+primate+testes+and+reveals+distinct+GFRa1-positive+subpopulations+in+men.">Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1-positive subpopulations in men.</a> J Med Primatol. 39 (2), 83-91 (2010).</li><li>Hayama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+selection+methods+for+rat+and+mouse+round+spermatids+without+DNA+staining+by+flow+cytometric+cell+sorting.">Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.</a> Mol Reprod Dev. 83 (6), 488-496 (2016).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+selection+of+valuable+mutant+mouse+round+spermatids+and+strain+rescue+via+round+spermatid+injection.">FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection.</a> Zygote. 23 (3), 336-341 (2015).</li></ol>

所有作者声明没有竞争的兴趣。

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. ...

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Developmental Biology

11.7K 次观看.  Washington University School of Medicine. 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。 

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

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

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 the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

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

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

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

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

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

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

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

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

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

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

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

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

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

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

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

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.