这项研究是由美国国立卫生研究院资助GM055360为SLB和U54HD068157到RGM支持。 JMB由T32遗传学培训格兰特在宾夕法尼亚大学（GM008216）的支持。

在STA-PUT协议包括三个阶段:1）设置仪器和试剂，2）准备从整个睾丸细胞悬液，以及3）小区负荷，沉淀和馏分收集。当一队的两名研究人员进行的，该协议需要八个小时的平均水平。 1。设立STA-PUT设备（图1） *** STA-PUT装置应放置在4℃的大冰箱或冷室，也可以容纳一个馏分收集器，如果是优选集合的该方法。 <ol><li>前一天晚上（或至少几个小时之前）你执行方法，清洗所有设备（特别是玻璃制品和管材）和消毒用70％乙醇。让干燥设备完全组装设备如图1之前。 </li><li>固定两个2升缸（ 图 1B和C）和细胞装载腔（ <str翁&gt;图1A）的顶级平台，并与两个小的管件与管夹连接所有。钳管子全部关闭。密封在最右边的2升缸嘴。 </li><li>放置在小区装载腔的小搅拌棒（ 图1A）和最左边的2升缸在一个较大的搅拌棒（ 图1B），其中将包含2％的BSA。 </li><li>将2L的沉降室的平台（ 图1D）上。将金属挡板（ 图1F）上直接开口在沉降室（ 图1D）的底部的上方。这一点至关重要，因为挡板防止馏分收集在细胞梯度的液体和破坏的涡旋。将盖子上的沉降室的顶部。 </li><li>施加非常少量的真空油脂的三路活塞（ 图1G）的磨口玻璃接头后，夹紧活塞向本身的底dimentation室，连接活塞和沉降室的磨口玻璃接头。 </li><li>小区装载腔（ 图1A）连接到与管子的活塞的右侧出口。关闭水龙头。 </li><li>装上电池分馏管连接到活塞的左侧出口。分馏管包括一个管件的用玻璃巴氏吸管连接到所述开口端。一块较小的孔管附着在玻璃移液管的窄端。窄移液器限制的细胞悬浮液中馏分收集的流动。钳这个小管在最底层。 </li><li>准备2升克雷布斯（1X）缓冲试验（ 表1）的日子。然后，准备550毫升2％BSA在1x克雷布斯，在1x克雷布斯550毫升4％BSA，并在1x克雷布斯50毫升0.5％的BSA。过滤并冷却这些溶液至4℃。 </li><li>倒入合适的2升缸（ 图1C）中的4％BSA溶液和2％BSA的解左2升缸（ 图1B）中。确保没有大的气泡在连接这些气瓶管路。 </li><li>倒Krebs缓冲液进入细胞装片室，并填写连接该室与沉降室的管道，确保没有大的气泡，因为这些可能会干扰梯度。挤压或轻弹管轻轻有助于消除气泡。确保所有的Krebs缓冲液的是在管内，而不是在细胞室中。 </li><li>允许少量的Krebs缓冲液流入沉降室，然后沥干几乎所有的缓冲区中，以便填充所述管，并移除任何较大的气泡的细胞分馏管。 </li><li>将馏分收集器下直接沉降室。确保所有的部分管到位，并用保鲜膜覆盖，以防止污染。 </li></ol> 2。从全隔离睾丸生精细胞<oL&gt; <li>从大约 12只成年小鼠（优选至少8周龄），并装在Krebs缓冲液中在室温下以洗掉任何污染材料解剖睾丸。这个协议被证实涉及使用实验室小鼠，并遵守宾夕法尼亚机构动物护理和使用委员会大学的指导方针被执行。 </li><li>添加45毫克胶原酶至50ml Krebs缓冲液的锥形管你打算添加的曲细精管的权利之前。让这个解决方案升温至33℃下几分钟。均匀地分割成两个50毫升锥形管。 </li><li>拆封睾丸中装有8毫升Krebs缓冲液中的单独板，弃去白膜和释放的曲细精管。白膜是薄膜包围的曲细精管。肾小管应容易从白膜脱落。要做到这一点，使白膜切口，持这种膜具有排Ř钳，并推动小管出来的，远离与另外一对钳子的膜。 </li><li>添加包含肾小管4毫升Krebs缓冲液到每个锥形管含有25ml的胶原酶溶液并孵育，振摇，在33℃10分钟。在结束时，小管应该有一个"意大利面条样"外观。 </li><li>允许细管沉降〜5分钟到管的底部。倾出上清液，并在25毫升的Krebs缓冲液（室温）洗涤2倍，使小管，以每次沉降到试管底部。留下约5ml Krebs缓冲液中的每个管中。 </li><li>边洗，加入30毫克胰蛋白酶至50ml Krebs缓冲液中的猎鹰管你打算添加的曲细精管的权利之前。让这个解决方案升温至33℃下几分钟。均匀地分割成两个50毫升锥形管。 </li><li>加25毫升胰蛋白酶溶液，以每片含细管两管。加3微克DNA酶（1μg/10ml）向每管，以防止细胞S来自结块。孵育，振摇，在33°C下10分钟。 </li><li>使用大口径吸管搅动含有小管的解决方案，吸取并排出约10倍。该解决方案应该开始看起来更像是一个单细胞悬液。 </li><li>孵育，振摇，在33℃下10分钟。使用大口径吸管驱散肾小管成单细胞悬液，吸取并排出约25倍。如果仍看到很多小管细胞团块，分散多用移液管和/或添加其他3微克DNA酶，以每管（双初始浓度）。 </li><li>通过100微米网孔的细胞过滤器（一个用于30毫升细胞悬浮液各管）筛选的单细胞悬浮液。结合的细胞悬浮液并计数细胞总数（应为7-8×10 8个细胞）。 ***不要装入超过800,000,000细胞上的梯度。 </li><li>基于在步骤2.10中得到的细胞计数，沉淀的appropr过滤后的细胞在450 RCF 5分钟的在Iate量和洗涤用30毫升Krebs缓冲液。重复。通常*** 22睾丸会产生超过800,000,000细胞，但不加载超过细胞的这个数字。 </li><li>仔细悬浮于25ml 0.5％BSA的DNA酶之间2.5和5微克含（取决于细胞聚集程度）不会产生泡沫细胞。确保细胞不聚集。通过100微米网孔的细胞过滤器过滤的单细胞悬浮液。细胞现在已经准备好装载。颠倒离心管数次在装货前拌匀。 </li></ol> 3。小区负载和沉积<ol><li>确保水龙头被关闭，但在位置，将允许液体从细胞室中（ 图1A）流入沉降室（ 图1D）。 </li><li>打开一个设置低（约70转），使搅拌棒不断移动两个搅拌棒板，但速度缓慢。 </li><li>倾细胞悬浮到细胞室中（ 图1A）和打开活塞，使细胞流慢慢进入沉降室，以10毫升/分钟（ 图1D）的速率。关闭水龙头。流速可以通过标记的沉降室体积的间隔来确定。 </li><li>倾将5ml 0.5％BSA的溶液进入细胞室和漏入沉降室，以10毫升/分钟的速率。关闭水龙头。这个步骤将洗涤细胞到细胞室。重复4倍。 </li><li>制备梯度:打开电池腔室（ 图1A）和两个2升缸（ 图1B和C）之间的夹具，并开始将液体排放到沉降室，在40毫升/分钟的速率（ 图1D）马上。调节流速，因此需要大约20-30分钟来加载BSA梯度进入沉降室。细胞趴在顶部薄，不受干扰层BSA梯度应该是可见的。一旦大多数BSA的加载，关闭活塞，并把它的位置，将允许液体从沉降室排入分馏管。 </li><li>关闭轰动板。 </li><li>使细胞沉淀于1小时45分钟。不打扰设备中，这时间内。任何机械搅拌会干扰梯度。 </li></ol> 4。馏分的分离与分析 ***当您执行下列步骤，要小心，不要打扰梯度。如果BSA梯度受到干扰，程序将无法正常工作！ <ol><li>一旦细胞沉淀，慢慢排水50毫升从沉降室到50ml锥形管中，备用。该馏分通常含有细胞和碎片的不必要的团块。 </li><li>附加组分管到馏分收集器。 ***另外，也可以通过手工收集的级分，如果一小部分科尔构造函数不可用。 </li><li>收集的级分:与上述活栓调节流速，使得10毫升（填充一个收集管中），收集每45秒。 </li><li>盖上馏分收集管和旋转5分钟，在450 RCF在4°C。倒掉上清轻轻地，重悬细胞，剩余的液体，并保持细胞在冰上。 </li><li>一旦所有的分数都收集，微观分析分数为不同的细胞群。不同类型的细胞可以根据细胞的大小和核形态8,15加以区别， 图2示出了"具有代表性的结果"为被富集（一）体细胞和减数分裂细胞和精原干细胞的3汇集级分，（B）圆精子细胞，并（三）延长和冷凝精子细胞。 <ul><li> A型精原细胞是12-14μm的CA。直径有圆形的细胞核是均匀的染色质组成。 </li><li> B型精原细胞是8-9μm的CA。直径并已ROUN这表明更多的异染色质致密沿核外周ð核。 </li><li>前细线期精母细胞为7.5-8.2μm的CA。直径，具有比B型精原细胞细胞质少，并且具有核看起来类似于在B型精原细胞。 </li><li>细线期初级精母细胞为8-10微米约。直径有细胞核，看起来类似于那些预细线期精母细胞，但似乎获得更密集的染色质在核膜。 </li><li>偶线期初级精母细胞为10-12微米约直径有细胞核，看起来类似于那些细线期初级精母细胞。 </li><li>粗线期精母细胞是从12-18微米长约任何地方。直径由一个薄的细胞质边缘周围的大核。更团块致密的染色质可以看出，在细胞核中。 </li><li>圆形精子细胞是10微米约。直径有一个圆形的细胞核，在中间一个密集的染色染色中心。 </li><li>残留身体是〜6.5微米，直径，圆形，并且没有细胞核。 </li><li>伸长和冷凝精子细胞的大小，以圆形精子细胞相似，但有一个独特的，镰刀形核。 </li></ul><ol><li>开始部分15和分析每五个分数高达85。通常情况下，15岁以下的馏分会过于混合块状，和分数高于85将包含几个到没有可用的细胞。 </li><li>为了分析的一小部分，取5ul的液体从馏分收集管（一旦细胞已离心后得到悬浮），并加入到在Krebs缓冲液8％的甲醛5UL。使固定的细胞在室温下放置五分钟。 </li><li>添加0.1％的Triton和DAPI（5μ/ ml的Krebs缓冲液）的5UL到固定细胞。使细胞在室温下放置5分钟。 </li><li>把10微升所得的溶液到滑动，盖上盖玻片，并用荧光显微镜进行分析，以确定各馏分的纯度。 </li><li>结合部分是在大小和核形态相似（参见"代表性成果"）来创建单元格的下列人群:减数分裂和体二倍体细胞，圆形精子细胞，和冷凝/长形精子细胞。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro,+short-term+culture+method+for+mammalian+haploid+round+spermatids+amenable+for+molecular+manipulation.">An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.</a> Mol. Reprod. Dev. 79, 19-30 (1002).</li></ol>

作者宣称，他们有没有竞争的财务权益。这个协议被证实涉及使用实验室小鼠，并符合所有相关的指南，法规和监管机构被处决。在这个协议中使用的动物宾夕法尼亚机构动物护理和使用委员会大学（IACUC协议＃804284）的指导和批准下维持。

那些谁研究精子依靠动物模型的生精细胞样本，作为一种可靠的细胞培养系统尚未产生所有的生精细胞类型3存在。虽然生精细胞是从整个睾丸容易收集，只有混居的结果。这就带来了一个问题，对于那些谁希望研究这些细胞的特异性亚型，如减数分裂的细胞，圆形精子细胞和精子细胞凝结。三种不同的方法，目前用于分离睾丸生精细胞的亚型:STA-PUT，流式细胞仪，和淘洗6-13。后两种方法需要使用昂贵件设备:细胞分选仪和一个淘析，分别。虽然FACS方法产生不同的精细胞类型的高纯度的人群中，该过程需要六个小时，并产生每个细胞类型每两到三个睾丸11只0.5-2.0×10 6个细胞。淘洗，像STA-PUT甲基OD，分离基于尺寸和密度的细胞，但是需要访问一个淘析，这是比在STA-PUT设备更加昂贵。 在STA-PUT过程使用一个简单的BSA梯度来分离不同大小的生精细胞具有相当高的收益率（〜10 8个/每群22〜睾丸）。相对于FACS方法，在STA-PUT过程产生更多的细胞/睾丸和花费少得多的分离细胞类型的时间。相较于FACS和淘洗，在STA-put过程是相对简单和便宜。在STA-PUT只需要在寒冷的房间或大冰箱和一组专门的玻璃器皿，使其成为实验室的理想方法无法获得细胞分选器或淘析。当正确执行，在STA-PUT可提供圆形精子细胞或精子细胞冷凝的约90％纯的人口。 在STA-PUT方法是非常有用的，但需要优化在几个不同的步骤，特别是Sedimentation。细胞类型的亚最佳分离可以通过几个不同的问题，最有关的BSA梯度而引起。要作出正确的梯度，打开在细胞装片室的搅拌棒和2升筒保持2％的BSA，而你正在创建的梯度。还清除气泡的所有管道和放置在挡板到位在沉降室装载细胞之前。降低了BSA的流速流入或流出的沉降室的可能有帮助。最重要的是，在STA-PUT设备不应该创建渐变，沉淀，或馏分收集的过程中受到干扰。 次优的细胞产量可睾丸的数量不足/大小，在胶原酶/胰蛋白酶处理不充分的细胞分离和细胞聚集的结果。如果一个人是无法获得的细胞为这个STA-PUT协议的适当数量由于使用新生小鼠或基因型会产生少量的生精CEL的LS，它可能有必要使用每个STA-PUT更多的动物或订购一个STA-PUT玻璃器皿试剂盒，较小的体积和细胞数量的优化（购自ProScience）18。为了获得细胞的最佳数目（700-800元），使用生育年龄的至少11只雄性小鼠，最好至少8-9周龄。然而，没有超过800万个单元格应该被装入一个STA表决。如果电池没有离解成胰蛋白酶处理后的单细胞悬浮液中，DNA酶的浓度可以增加高达1微克/ 5 ml溶液。 DNA酶是敏感的重复冷冻/解冻，并用新鲜的DNA酶，每次将导致小管的更有效的分散成单细胞悬浮液的循环。你还可以使用大口径吸管通过网状过滤之前，帮助打散细胞团块。 这将需要优化的STA-PUT方法的一个方面是时间的允许细胞沉淀通过BSA梯度的量。一小时45分钟，通常效果很好，但这次可能会有所不同从实验室到实验室。通常，细胞中的不同的层可以在视觉上整个BSA梯度看到。如果有几个细胞中收集到的第一个20级分和细胞组分80 +丰盈，沉降时间可能需要延长。如果在收集到的第一个20级分过多的细胞和有细胞类型的分离不充分时，沉降时间可能需要被降低。 与STA-put过程获得的细胞可用于许多不同类型的实验。在STA-PUT提供了充足的材料，免疫印迹分析，免疫荧光，和RNA分析，虽然大规模的生化实验，可能需要从几个不同的STA-PUT运行相结合的材料。当从其它细胞类型分开，单倍体精子细胞可以被培养，并进行体外分子操作长达三天，可以比更容易<em&gt;在体内治疗或创建一个基因敲除动物19。与所述STA-PUT协议获得的细胞已培养了一天没有污染的迹象明显，但如果细胞要用于更长的细胞培养实验中，设备应用乙醇灭菌，所有的解决方案应该被过滤灭菌，并且所有前小区负载和馏分收集之后的步骤应该在组织培养橱中进行。此外，应使用含有抗生素的培养基中。该STA-PUT方法不需要细胞固定的事实使得它适用于要求活细胞实验的理想方法。

哺乳动物的精子发生是发生在几个阶段的睾丸1的曲细精管一个复杂的分化过程。简单地说，干细胞样的精原细胞驻留附近的曲细精管分化的上皮细胞和分化为精母细胞，然后进行减数分裂。减数分裂完成后，所得到的单倍体细胞，或圆精子细胞，精子发生，分化过程，包括细胞质和细胞核的压实的脱落。精子逐渐发展鞭毛，并进行延伸细胞核和冷凝，产生拉长，然后冷凝精子细胞，分别。最终的产品是精子，它被释放到精曲小管，并最终成为他们进一步成熟附睾管腔。 因为精子的过程依赖于特殊的激素和分子条件睾丸，可靠的体外培养体系为精子的整个过程中尚未被开发2,3。培养的方法已经被开发用于从干细胞产生"原始生殖细胞样细胞"和单倍体，"圆精子细胞样细胞"，但这些方法中还没有能产生大量的这些细胞并不能产生更高精细胞类型4,5。幸运的是，精细胞类型中尺寸显著不同，它允许从整个睾丸与液体梯度进行分离而获得的单细胞悬浮液。在STA-PUT方法，在这里表现出来，使用线性渐变的BSA和简单的沉淀根据大小和质量6-9分离睾丸生精细胞。 在STA-PUT方法具有比其他两种最广泛使用的方法几个优点分离生精细胞类型:流式细胞仪和淘洗10-13。在STA-PUT设备需要ONLŸ几件玻璃器皿专业组装在一个寒冷的房间或大冰箱。因而，它比用细胞分选器或淘析器更便宜。在STA-PUT方法产生更高的金额每个细胞类型和睾丸比可以通过流式细胞仪在可比时间段进行排序的细胞，虽然每个细胞群的纯度不高那些与流式细胞仪11获得。细胞分选，利用磁珠（磁性激活细胞分选，MACS）最近已成功地用于从一混合睾丸细胞群体富集的精原细胞，但它是目前不适于分离精母细胞或精子细胞由于缺乏适当的表面的知识的标记物14。过FACS或MACS的STA-PUT方法的一个附加的优点是隔离适于后续培养物的活细胞，因为，相对于最FACS协议，它不要求任何DNA或其他类型的染色能力。对于需要升研究生精细胞类型的ARGE收益率〜90％的纯度，在STA-PUT是一种理想的方法。

<table><tbody><tr><td>BSA</td><td>Affymetrix/USB</td><td>10857 100MG</td><td>Alternate can be used.</td></tr><tr><td>0.5%, 2%, and 4% BSA solutions</td><td>Dissolve 0.25 g, 11 g, and 22 g (respectively) in total volumes of 50 ml, 550 ml, and 550 ml of 1x KREBS (respectively).</td><td>To be made day of the STA-PUT procedure. Filter sterilize.</td><td></td></tr><tr><td>KREBS (10x)</td><td>3.26 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; + 139.5 g NaCl +5.89 g MgSO&lt;sub&gt;4&lt;/sub&gt;/7H&lt;sub&gt;2&lt;/sub&gt;0 + 40 g Dextrose + 3.78 g CaCl&lt;sub&gt;2&lt;/sub&gt;/2H&lt;sub&gt;2&lt;/sub&gt;0 + 7.12 g KCl, bring to 2 L with ddH&lt;sub&gt;2&lt;/sub&gt;0</td><td>Autoclave/filter and store at 4 &amp;deg;C for several months.</td><td></td></tr><tr><td>KREBS (1x)</td><td>Dissolve 4.24 g NaHCO&lt;sub&gt;3 &lt;/sub&gt;in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;0. Add 200 ml 10x KREBS. Bring to 2 L with ddH&lt;sub&gt;2&lt;/sub&gt;0.</td><td>To be made day of the STA-PUT procedure. Filter sterilize.</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C9891-1G</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>DNAse</td><td>Sigma</td><td>DNEP-5MG</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T9201-1G</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Formaldehyde (~37%)</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td> </td><td></td><td></td><td></td></tr><tr><td>TABLE 2: EQUIPMENT</td><td></td><td></td><td></td></tr><tr><td> </td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td>Company</td><td>Catalog #</td><td>Comments</td></tr><tr><td>Complete STA-PUT apparatus</td><td>ProScience Glass Shop</td><td>STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td><td>Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td></tr><tr><td>10 &amp;mu;m mesh filter</td><td>Fisherbrand</td><td>22363549</td><td> </td></tr><tr><td>Mouse dissection tools</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>14 ml round bottom fraction collection tubes with caps</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Need approximately 100 tubes</td><td></td><td></td><td></td></tr><tr><td>Fraction collector</td><td>GE Healthcare Life Sciences</td><td>Model: Frac-920, Product code: 18-1177-40</td><td>Alternate can be used.</td></tr><tr><td>Microscope slides, cover glass</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Light/Fluorescent Microscope</td><td>Preference of researcher</td><td></td><td></td></tr></tbody></table>

separation of spermatogenic cell types using sta-put velocity sedimentation

哺乳动物的精子发生是发生在几个阶段的睾丸的曲细精管一个复杂的分化过程。目前，没有可靠的细胞培养系统，允许在体外分化精和精细胞的大多数生物学研究需要组织收获从象小鼠和大鼠的动物模型。因为睾丸含有大量的细胞类型 - 无论是无生精（睾丸间质，滋养，骨髓和上皮细胞）和生精（精原细胞，精母细胞，圆形精子细胞，精子细胞冷凝和精子） - 参与精子的生物学机制的研究需要隔离与这些不同类型的细胞的富集。在STA-PUT方法允许细胞的异质群体的分离 - 在这种情况下，从睾丸 - 通过一个线性BSA梯度。单个细胞类型具有不同的沉降速度根据CE滓LL大小，以及馏分富含不同类型的细胞可以被收集并在进一步的分析中使用。而STA-PUT方法不会导致细胞类型的高纯度的级分， 例如为可以与某些细胞分选方法获得，它不提供在每个级分高得多的产量总的细胞 （〜1×10 8个细胞/精细胞从7-8×10 8个细胞的起始种群类型）。这种高收益率的方法，只需要专门的玻璃器皿，可以在任何冷室或大型冷库中进行，使其成为具有有限的访问专门的设备，如荧光激活细胞分选（FACS）或淘析实验室的理想方法。

从STA-put过程的理想结果是细胞从睾丸根据细胞的大小和密度相当明显的分离。而细胞从睾丸中分离是通过BSA梯度沉淀，细胞的几个不同的频带可以观察到。细胞的任何团块往往会沉到梯度的底部，不会污染其他馏分。再往上升的梯度将是大体细胞和减数分裂的细胞。更远了梯度仍然会较小圆形精子细胞。在渐变的顶部将被浓缩精子细胞，精子和污染红细胞（这似乎是小圆细胞没有细胞核）。 馏分可迅速利用光及荧光显微镜的组合（图2）15进行分析。减数分裂，精原细胞和体细胞的二倍体细胞是在睾丸中发现的最大的细胞，将含有大量晶核该污点relatively均匀用DAPI。圆形精子细胞是小细胞，小圆形核，一般用明亮的染色染色中心。冷凝/长形精子细胞小细胞经常看长圆形，仿佛是一个小尾巴正在形成。这些细胞具有染色鲜艳用DAPI和形状像一个镰刀较小，紧凑的细胞核。一旦细胞级分合并，纯度可以进一步通过细胞裂解物的免疫印迹分析（图3）来确定。减数分裂细胞的共同标志物的联会复合体蛋白1 SCP1和SYCP2 16。冷凝精子细胞的共同标志物是过渡的蛋白质（ 例如 TP1）或鱼精蛋白17。 虽然每个STA-PUT来说可以是不同的，通常是减数分裂的细胞，精原细胞和体细胞二倍体细胞将零碎的CA被发现。25-40，圆形精子细胞中分数大约 55-65，和冷凝/零碎的CA长形精子细胞。65-75由于体/减数分裂细胞和圆形精子细胞之间的大小差异较小，分数约 45-50经常有体/减数分裂细胞和圆形精子细胞的偶百分比。用DAPI染色有助于区分细胞的这两个群体。还有就是圆形精子细胞较少重叠和冷凝/延长精子细胞组分，由于细胞的这两个种群之间的尺寸差别较大。通常情况下，15岁以下分数将包含上述85细胞和分数的许多大的团块将包含几个细胞和许多残余体，或者说是由精子细胞脱落的膜结合细胞浆。 通常，当从〜22的睾丸细胞分级分离与STA-put过程，它产生约 10 8个细胞/精细胞类型（减数分裂/体细胞的二倍体细胞，圆精子细胞，和冷凝/长形精子细胞）。馏分，它​​们被组合以建立细胞的最终群体应至少80％纯的细胞的有问题的类型。如果您没有看到这种程度的纯度或更高的，有可能是细胞分离或BSA梯度的问题。另外，如果有一些细胞在第20部分和细胞组分丰富80 +，或有细胞丰盈在第20部分和几乎没有任何零碎70 +，沉淀时间需要进一步优化。请参阅有关如何进行故障排除的讨论的建议。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50648/50648fig1.jpg" width="450px" /> 图1。设立STA-PUT设备:在STA-PUT装置的原理图和实际图像显示。所有玻璃器皿由塑料管，包括该装置连接到级分收集器的管相连。箭头表示夹具的位置。 A）在细胞装载室，包含了一个搅拌棒，B）2升气缸<html> 2％BSA，包含了搅拌棒，C），2升缸4％BSA，D）沉降室，E）搅拌板，F）隔板; G）旋塞阀<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50648/50648fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50648/50648fig2.jpg" width="450px" /> 图2。从STA把获得的细胞群 :流份合并为三个独立的细胞群体:减数分裂和体细胞，圆形精子细胞，而冷凝和长形精子细胞。每个人口用DAPI染色显示核的大小和形态的差异。相衬成像传达的细胞大小和形状差异。白色条代表10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50648/50648fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 _content"FO:保持together.within页="总是"&gt; <img alt="图3" fo:content-height="6in" src="/files/ftp_upload/50648/50648fig3.jpg" width="450px" /> 图3。从STA-PUT得到不同的细胞群的标志物:全细胞提取液，从图1中所示的每个细胞群体作出，并进行免疫印迹分析以显示每个人口蛋白表达的差异。联会复合体蛋白1（SCP 1）是一种蛋白在减数分裂过程中完全表达，并发现富集在减数分裂的分数，而精子冷凝馏分富含过渡蛋白1（TP1），表示在后期精子的蛋白质。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50648/50648fig3highres.jpg" target="_blank">点击这里查看大图像</a> 。

Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

在STA-PUT方法允许对生精细胞的不同人群根据大小和密度的分离。

生精细胞类型使用STA把速度沉降分离

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

separation-spermatogenic-cell-types-using-sta-put-velocity

Nanyang Technological University

Research

JoVE Journal

Biology

25.7K 次观看.  University of Pennsylvania. 在STA-PUT方法允许对生精细胞的不同人群根据大小和密度的分离。 

视频: Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

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 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.

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. ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

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.

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

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

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.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

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.

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 ...

生物学

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射程序研究胚胎发育

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

小鼠圆形精子细胞注射

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

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 ...

生精细胞类型使用STA把速度沉降分离

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此视频中的章节

步特定老鼠的精子细胞分选流式细胞仪

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

用小鼠模型对精子发生细胞学分析的精管壁球技术

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

流动小鼠减数分裂细胞的流式细胞仪净化

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

简单和高效的睾丸细胞悬液的制备技术