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...

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 border="0" cellpadding="0" cellspacing="0" width="856">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:109px;">Affymetrix/USB</td>
			<td style="width:255px;">10857 100MG</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">0.5%, 2%, and 4% BSA solutions</td>
			<td colspan="2" style="width:364px;">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 style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="91">
			<td height="91" style="height:91px;width:311px;">KREBS (10x)</td>
			<td colspan="2" style="width:364px;">3.26 g KH2PO4 + 139.5 g NaCl +5.89 g MgSO4/7H20 + 40 g Dextrose + 3.78 g CaCl2/2H20 + 7.12 g KCl, bring to 2 L with ddH20</td>
			<td style="width:183px;">Autoclave/filter and store at 4 &#176;C for several months.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">KREBS (1x)</td>
			<td colspan="2" style="width:364px;">Dissolve 4.24 g NaHCO3 in 100 ml ddH20. Add 200 ml 10x KREBS. Bring to 2 L with ddH20.</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Collagenase</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">C9891-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">DNAse</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">DNEP-5MG</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Trypsin</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">T9201-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Triton-X100</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Formaldehyde (~37%)</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">TABLE 2: EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:255px;">Catalog #</td>
			<td style="width:183px;">Comments</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:311px;">Complete STA-PUT apparatus</td>
			<td style="width:109px;">ProScience Glass Shop</td>
			<td style="width:255px;">STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td>
			<td style="width:183px;">Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10 &#956;m mesh filter</td>
			<td style="width:109px;">Fisherbrand</td>
			<td style="width:255px;">22363549</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mouse dissection tools</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:311px;">14 ml round bottom fraction collection tubes with caps</td>
			<td colspan="3" style="width:547px;">Preference of researcher </td>
		</tr>
		<tr height="22">
			<td colspan="3" height="22" style="height:22px;width:547px;">Need approximately 100 tubes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:311px;">Fraction collector</td>
			<td style="width:109px;">GE Healthcare Life Sciences</td>
			<td style="width:255px;">Model: Frac-920, Product code: 18-1177-40</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Microscope slides, cover glass</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Light/Fluorescent Microscope</td>
			<td colspan="3" style="width:547px;">Preference of researcher</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）。 <...

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

Research

JoVE Journal

Biology

25.5K 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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

两种类型的检测，检测青蛙精子Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

一种新型的全基因组分析的贝叶斯变点算法的多元ChIPseq数据类型

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

分离的小鼠胚胎面外胚层和间充质

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...