作者要感谢克里斯托弗先生阮亚伦亨特和什里夫波特的Jumpstart，SMART和Biostart培训计划的援助以及生物物理，什里夫波特，LA的路易斯安那百年学院中文系。

1.直径为150mm剪切戒指建设（图1） 注:剪切环可以被构造通过改变内外培养皿大小，导致与不同的总表面积，细胞产量的设备和开发的剪切力范围以创建许多不同的尺寸。本报告描述了150毫米的菜具有内部100mm培养皿为98 平方厘米（ 图2）的总表面积相结合。 <img alt="图1" src="/files/ftp_upload/54632/54632fig1.jpg" /> 图1.剪切环组件。该图的顶部示出了部分组装的剪切环。通过3毫米的孔用移液管注入加入0.5ml二氯甲烷，如果一个紧密的密封还没有完全的内部和外部的菜肴之间形成。剪切环是通过将硅橡胶粘合剂的1mm厚的珠周围的内EDG密封关剪切环顶部电子。图的底部显示组装剪切环。蓝色代表镀内皮细胞的区域。外部和内部的红色箭头表示当置于轨道摇床剪切环内的剪切圈和媒体的轨道运动。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54632/54632fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54632/54632fig2.jpg" /> 图2.表面三围为150mm的剪切环（未按比例）。前面板显示对响应的轨道旋转外圈液离心式换档。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54632/54632fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>通过生成创建剪切环建筑模板在与放置在150 mm直径圈的中心100毫米的内直径的演示软件150毫米外缘轮廓。打印在A4白纸一张的模板。 </li><li>吸管的10ml二氯甲烷（二氯甲烷）至150毫米的玻璃培养皿。至关重要的是，该盘是玻璃，而不是聚苯乙烯（或其它的乙烯基塑料），二氯甲烷溶解大部分塑料并在这里用来加入塑料部件。 注意:使用手套所有建筑有足够的通风罩。二氯甲烷是接触刺激性和是肝毒性。 </li><li>对齐150毫米的塑料培养皿到外切环模板。 </li><li>使用旋转工具100毫米的菜的中心钻3毫米的孔。取下钻探产生的任何塑料刮屑。 </li><li>同时用戴手套的手保持，反转和浸来自前一步骤到二氯甲烷池100毫米的培养皿的顶部边缘约3秒。 </li><li>横贯FER的"湿"100mm皿边缘向下到150毫米盘的中央，仔细调整100毫米的菜到显着的模板。二氯甲烷将保险丝塑料，加入有100 mm和150 mm培养皿。轻轻顺时针旋转碟100mm的旋转和逆时针几次，以确保良好的附着力，以150毫米的菜。 注:应特别小心，以确保内部菜的外盘中央的正确对齐。偏心对准可能导致在在剪切环的不同位置的剪切应力的变化。小心不要让二氯甲烷到100mm皿的放置期间偶然滴到150毫米的培养皿的外轨道部分。这会熔化的底表面，其中的细胞将生长在塑料，产生不平坦的表面，可能会引起干扰。 </li><li>一旦二氯甲烷已经干涸，翻转新绑定的培养皿中，仔细检查的接触点，以确保紧密的密封已被两个培养皿中之间形成。 </li><li>如果一个密封还没有完全形成，通过3毫米的孔用移液管注入加入0.5ml二氯甲烷中。拿起菜并轻轻旋转，以允许二氯甲烷到达边缘。 注:如果旋转太快，二氯甲烷可以蔓延到150毫米的菜的外部分，表面细胞的地方将增长变形，破坏剪切环。泄漏的剪切戒指应该被丢弃。 </li><li>通过采用硅橡胶密封胶的3毫米珠封锁在100毫米的菜孔。 </li><li>使用配备有一台切割头旋转工具，切断一个15毫升的锥形聚苯乙烯组织培养试管前3 cm长的，造成至少1厘米以下帽管。抛光切割管的边缘，直到光滑和平坦使用切削头的平侧面。除去研磨产生的任何塑料刮屑。 </li><li>跟踪切断的15毫升锥形管到150毫米的菜用记号笔，从盘的边缘约为0.5厘米处的盖子。钻一个孔的描画圆内，留下一个余量大致从圆的边缘1-2毫米。 </li><li>将切断锥形管顶在钻孔。使用移液管，施加约250微升的二氯甲烷到锥形管的切断边缘与锥形管的150毫米盖结合。 </li><li>通过施加硅橡胶密封剂中的1毫米厚的珠周围的顶部培养皿的内边缘密封150毫米盖到150毫米的菜。 </li></ol> 2.剪切戒指的消毒<ol><li>吸取约10毫升磷酸盐通过15毫升锥形管口缓冲液进入新成立的剪环。漩涡周围去除剪切​​环内的任何杂物。用巴斯德吸管/真空抽吸器除去磷酸盐缓冲盐水。 </li><li>直到碎片被删除重复上述步骤。 </li><li>要STERI丽泽剪切环，使用用紫外线照射70％的乙醇漂洗的组合。 <ol><li>拧开盖子通风，吸管大约10mL，70％的乙醇通过接入端口，并重新拧紧瓶盖。旋转和剪切环多次翻转，确保剪切环的内部被充分洗涤。 </li><li>在通风橱，吸出多余的70％的乙醇。用喷雾瓶，彻底喷用70％乙醇在盖和进入端口。 </li><li>将下的组织培养罩内的紫外线辐射剪切环和帽直至完全干燥。 </li></ol></li><li>干燥后，拧在室温下回到了帽和消毒店剪戒指，直到在细胞培养电镀。 </li></ol> 3.剪应力研究<ol><li>板下通常使用1标准细胞培养协议消毒剪切环内皮细胞:转化的内皮细胞系4分流比。 注:大鼠视网膜microv在商业上获得ascular内皮细胞。人脑内皮细胞（hCMEC / D3）细胞提供从皮埃尔 - 奥利弗博士Couraud（INSERM，法国）一份厚礼，并在完全内皮基础培养基（EBM）进行培养。 </li><li>允许文化中流动的研究开始之前达到汇合。 USE 30 ml的组织培养基（10％胎牛血清，Dulbecco改良的Eagle与1％青霉素/链霉素/两性霉素培养基）。改变细胞培养基，每3天，并保持细胞在37℃用7.5％的CO 2和92.5％的室内空气。 </li><li>放置在孵化器的轨道摇床。 注:轨道摇床通常是重（&gt; 10公斤）。放置培养箱的底部货架上的轨道摇床，以尽量减少货架应力和振动。 </li><li>将实验剪切环对轨道振动筛。将静态对照组剪切环同样的培养箱内。 </li><li>在SH内估计最大剪应力耳环与方程<img alt="式（1）" src="/files/ftp_upload/54632/54632eq1.jpg" />哪里<img alt="公式（2）" src="/files/ftp_upload/54632/54632eq2.jpg" />是旋转的轨道摇床半径（厘米）， <img alt="公式3" src="/files/ftp_upload/54632/54632eq3.jpg" />为介质的粘度（泊）， <img alt="公式4" src="/files/ftp_upload/54632/54632eq4.jpg" />是介质的密度（以g / ml）和<img alt="公式5" src="/files/ftp_upload/54632/54632eq5.jpg" />是旋转频率（旋转/秒）24。 </li><li>发起于轨道摇床旋转设置到所需的转速（ 例如，90转），而使剪切环上的振动筛剪切应力应用程序（ 例如 ，72小时）的所需的持续时间。 注:轨道振动器能够产生的热量，所以培养箱温度最初应监测，以确保37℃保持整个实验的持续时间。另外，S听到环可转移到环境室（ 例如 ，模块化培养箱室），然后置于旋转培养箱内。 </li><li>后的细胞层被暴露于剪切为所需的时间长度，从孵化器中移除剪切环。拉断的剪切环盖和检索单元和/或媒体进行检查所需端点分析（ 例如，蛋白质印迹，荧光激活细胞分选等 ）25。 </li></ol>

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J.汶禹云已经从吕秀莲尼切洛基础研究基金。 J.亚历山大·史蒂芬拥有神经内科，LSUHSC-S系研究支持。

对暴露的内皮细胞剪切剪切环系统的建设是一个简单的方法来进行剪切应力的研究。然而，有一些是获得卓越的剪切戒指和更好的结果的关键几步之遥。一个完整的密封应在内和外环，以防止介质从泄漏可能创建样品之间不一致的剪切应力之间进行。如果完整的密封不进行，二氯甲烷最小量应通过在内圈的孔被加入到内和外碟用移液管之间的边缘。轻轻转动该环应该允许二氯甲烷以形成一个完整的密封。此外，塑料刮屑从切削无意中产生可以存在剪切环的轨道上，所以冲洗出来后施工剪切环应该删除任何碎片可能细胞生长产生不利的影响，并给出不一致的剪切应力的应用。 在S在文章中描述听到环体积相当庞大，导致了大批量每个样品的输出。然而，更小的版本，可以使用较小的培养皿来构造（ 如 100毫米的培养皿用60毫米插入）。构造多个较小剪切环可以允许较高的数字的研究复制与其他方法相比时，同时仍保持每个样品相对大的流体体积和表面积。 使用剪切环时的几个问题已悉。第一，一些轨道摇床产生过量的热，并且一些孵化失败来控制这个温度上升。这可以通过旋转器的适当的选择和使用非水夹套保温箱，其更容易地进行热交换的被避免。使用剪切戒指的时候，尤其是以下装配在露天环境中培养污染是另一个潜在的担忧。剪切戒指使用前必须彻底消毒。 <p类="jove_content"&gt;这里，我们已经描述了一种简单的方法，以暴露大量的内皮细胞的流体剪切应力。生产单向和周期性剪切的其它方法都可以，但也更昂贵的，复杂的，并考虑到表面区域和每个实验暴露的细胞的数目时费时。一种这样的方法涉及蠕动泵连接到平行板流动室，其可难以建立和相对昂贵的。而剪切环是简单构造和使用，剪切应力的应用是不太精确。轨道流量可产生复杂的流动的电流，其可以建模一些，但不是全部，在正常血管生理发现流的物理状态。内在不能精确地控制在剪切环流可以被看作是一个电流限制到模型中。此外，在上述协议中描述的数学公式可以被用作一种工具来估计在剪切环最大的流体剪切应力。然而，需要额外的和更复杂的实验和计算研究在剪切环明确表征流动模式。作为流动模式和流体剪切应力在剪切环更好的理解表征，应该能够更好地调整实验参数（剪切环的最佳尺寸，轨道摇床的转速，在剪切环的媒体音量等 ）以产生期望的周期性的，单向的剪切应力。

流体剪切应力已经显示出调节内皮基因方案1 -通过顺式调节元件6的活化，组蛋白乙酰转移酶活7和剪切应力响应元件（SSRE）8 5。通过调节组织因子9和组织型纤溶酶原激活剂（tPA）10表达的剪切应力的影响对凝血内皮的贡献。剪应力也影响通过调节PDGF-B的合成和反应8血管生成11和血管重塑的控制权。内皮衍生的血管活性介质肾上腺髓质素，内皮素-1，尾加压素II和松弛素也被剪切机12调节。内皮型一氧化氮合成酶的生产和一氧化氮产生的转录都是剪切依赖性10。剪切还控制内皮的ICAM-1的表达13。流动引起的剪切应力，因此可以powerfuLLY影响种类繁多的内皮细胞反应。重要的是，血管脉动现在也出现在血管性痴呆14的正常血管老化和形式的病理生理学中发挥重要作用，甚至可能导致其它神经变性疾病，如多发性硬化15。 静脉和动脉内皮细胞固有地暴露于不同的血流动力学流动模式在体内 ，许多不同的内皮细胞的表型可以显示出16。取决于幅度和流量的周期性，对内皮细胞的作用可以包括炎性细胞活化和细胞凋亡，其可反映在基因或蛋白质表达17,18变化。研究内皮细胞对剪切现象，因此仍然被困难复杂， 在能够充分产生这样的剪切模式体外模型制作。 许多不同的experimental协议已被开发应用的流体剪切应力内皮细胞单层。一个最常用的系统是平行板流动室，它创建室19内均匀的层流- 21。蠕动泵通常连接到创建的周期性流，这可以概括通常在体内 22的许多地点找到的流动特性。另一种常见的建立使用&#39;锥板"的模式，其中，流体剪切应力是由锥体23的旋转速度确定的。两个系统，和其他安排类似于它们，可能是很乏味的设置和需要，可以是相对昂贵并且不可访问的许多实验室的组件。 这些当前模型的另一个主要限制是，可以同时进行，每一个相对低的表面积复制研究的数量相对较少。这增加了时间和共<html>这种方法的mplexity。因此，诱导单向和周期性剪切的理想模型可能之一，可以很容易地设置研究重复的较高的数值，每个具有相对较大的表面积。此外，上述模式需要一个相当复杂的设置，其可以是成本高昂的许多用户。可使用的基本材料实验室产生的流体剪切扰动模型可能有几个优点。 施加单向的，周期性的剪切应力的一种简单和非常经济的方法涉及的圆形菜定轨振荡器上24的位置。这个协议是很简单的，并且可以按比例增加，以实现高的数字研究复制，每个具有相对较大的表面面积，根据需要。但是，设在盘的中心的细胞暴露于不同的流动模式比沿周细胞，产生在相同的培养皿混合蜂窝的表型应答。 _content"&gt;在当前的报告中，我们通过限制流量描述"剪切戒指&#39;，我们的模型创建单向和定期剪应力的建设和使用，设计的剪切环有效限制"混合"蜂窝剪切引起的表型通过内圈的位置的圆形培养皿至周边内途径。剪切环的结构和操作是简单和经济的，并且可以很容易地扩展使用广泛使用的组织培养供应以适应广泛范围的轨道摇床中。这模型可以在内皮细胞实验应用于生理和病理水平之内提供单向和周期性流动模式。

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">100 x 20 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430167</td>
			<td style="width:195px;">The dishes must be polystyrene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">150 x 25 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430599</td>
			<td>The dishes must be polystyrene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm glass petri dish&#160;</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">3160150BO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml polystyrene tissue culture plastic tubes</td>
			<td>Falcon</td>
			<td align="right">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Methylene chloride</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">D65100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">silicone rubber sealant</td>
			<td style="width:119px;">DAP</td>
			<td align="right" style="width:119px;">7079808641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ethanol</td>
			<td style="width:119px;">Decon</td>
			<td align="right" style="width:119px;">2701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">3 mL transfer pipette</td>
			<td style="width:119px;">Becton-Dickinson</td>
			<td align="right" style="width:119px;">357524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">printer paper</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">scissors</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">gloves</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool and set</td>
			<td style="width:119px;">Dremel</td>
			<td style="width:119px;">4000-6/50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool cutting head</td>
			<td>Dremel</td>
			<td>EZ476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool drill head</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">distilled water</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">orbital shaker</td>
			<td style="width:119px;">VWR</td>
			<td style="width:119px;">57018-754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">incubator</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat retinal microvascular endothelial cells</td>
			<td>Cell Biologics</td>
			<td>RA-6065</td>
			<td></td>
		</tr>
	</tbody>
</table>

the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress

通过诱导适应性以及在血管内皮表型和基因表达的病理变化，从正常水平，并在血管生理学和病理生理学的血管流体剪切中发挥重要作用的型态偏差。特别是，周期性的，单向流动引起的剪应力的适应不良效果可以触发各种几个血管细胞类型，尤其是血管内皮细胞的影响。而现在来自不同解剖起源的内皮细胞已经培养的，深入的其对流体剪切反应受到阻碍剪切模型的相对复杂性分析（ 例如，平行平板流动腔，锥板流速模型）。虽然这些都代表着优秀的方法，这种模式在技术上是复杂和缺点，包括相对漫长和复杂的设置时间，低表面积，泵和增压常常需要密封胶和密封垫要求受苦，创建对BOT的挑战^ h维护不育和无法运行多个实验。然而，如果流动和剪切的更高的吞吐量模型可用，对血管内皮的剪切响应更大的进步，在分子水平上尤其周期性剪切研究，可能会更迅速地先进。这里，我们描述了构造和使用剪切环:一种新颖的，简单到组装，和便宜的组织培养模型具有相对大的表面面积，可以轻松地允许在单向的，周期性的剪切应力在研究大量的实验重复的内皮细胞。

在这里，我们提出从两个hCMEC / D3脑内皮细胞和大鼠视网膜微血管内皮细胞单层，在剪切环培养代表性的结果。 允许hCMEC / D3脑内皮细胞单层生长在完整的EBM至汇合后，剪切环被放置在定轨摇床上72小时。从步骤3.5使用等式，计算出的最大剪应力<img alt="公式6" src="/files/ftp_upload/54632/54632eq6.jpg" />约为2.8达因/厘米2（与参数<img alt="公式（2）" src="/files/ftp_upload/54632/54632eq2.jpg" /> = 0.95厘米， <img alt="公式3" src="/files/ftp_upload/54632/54632eq3.jpg" /> = 0.0101风度， <img alt="公式4" src="/files/ftp_upload/54632/54632eq4.jpg" /> =0.9973克/ ml的24， <img alt="公式5" src="/files/ftp_upload/54632/54632eq5.jpg" /> = 1.5转/秒<html> ）。我们已发现，这些内皮细胞单层有时表现出对准平行于周期性流（ 图3）的方向上，虽然这不是均匀地观察到，因为电池层通常具有优良的粘合性剪切环表面在整个研究。 允许后大鼠视网膜微血管内皮细胞单层生长在完整大鼠内皮细胞培养基，包括EGF / VEGF的生长因子补充至汇合，剪切环被放置在定轨摇床上72小时。从步骤3.5使用等式，计算出的最大剪应力<img alt="公式6" src="/files/ftp_upload/54632/54632eq6.jpg" />为约12达因/厘米2（与参数<img alt="公式（2）" src="/files/ftp_upload/54632/54632eq2.jpg" /> = 0.95厘米， <img alt="公式3" src="/files/ftp_upload/54632/54632eq3.jpg" /> = 0.0101风度，尔斯/ ftp_upload / 54632 / 54632eq4.jpg"/&gt; =0.9973克/ ml的24， <img alt="公式5" src="/files/ftp_upload/54632/54632eq5.jpg" /> = 4转/秒）。 图4示出，相对于β肌动蛋白的上样对照，有从内皮细胞表面的血小板内皮细胞粘附分子（PECAM-1 / CD31）的大量显著损失。 PECAM-1是一种完整的膜蛋白，其是一个包含免疫受体酪氨酸依赖性抑制基序或&#39;ITIM&#39;26的免疫球蛋白（Ig）-superfamily的成员。 PECAM-1不仅表达在内皮细胞上，但也造血细胞上发现的。 PECAM-1起着内皮细胞 - 细胞粘附，白细胞交界轮回，细胞信号传导显著角色，和重要的是，剪切应力的机械性转导。 PECAM-1的在感应剪切应力作用是血管endotheli的功能临界<html>人的细胞和动态平衡17。当内皮单层暴露于剪切应力，PECAM-1直接响应通过改变其酪氨酸磷酸化在其上被施加的机械力，和ERK1 / 2的信号级联27的后续活化。此外，PECAM-1-eNOS的复杂的关联是通过干扰在剪切应力28中断。因此，PECAM-1使血管内皮细胞以感测在流体剪切应力的力的变化，可导致血管壁29的反应性扩张。 这些数据支持该模型显示出血管内皮细胞通过下调的重要交界和粘合剂的决定因素，其介导细胞间的接触，以及经血管细胞交换到曝光响应周期性的，单向的流体剪切力。 632fig3.jpg"/&gt; 在一个剪切环 图3. 脑内皮细胞形态。hCMEC / D3endothelial细胞单层的剪切环48h后定期流体剪切力或静态曝光的外观。文化的对齐并不总是遵守平行于流动方向（箭头所示）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54632/54632fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54632/54632fig4.jpg" /> 图4. 细胞对周期性剪切。大鼠视网膜微血管内皮细胞单层剪切戒指培养 显示出在/ CD31相对PECAM1减少对β-肌动蛋白（每个n = 3，学生非配对t检验，* -p &lt;0.05，误差条参考标准偏差），72小时的定期流体乳木果的R IN剪切环。

Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

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8.0K 次观看.  Louisiana State University Health Sciences Center in Shreveport. Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

视频: The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

科研

生物学

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

停流的动力学方法的应用研究的一种DNA修复蛋白的行动机制

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

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

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using the original fixed 45 &deg; or 90 &deg; cutting angle for testing shear force, a variable angle cutting box can be adjusted so the angles of the knives correspond to the fibre angle of each sample. Within 2 min after cooking to an internal temperature of 71 &deg;C on an open-hearth grill set at 210 &deg;C, a 1 cm by 5 cm core is cut from the steak, parallel to muscle fibre direction, using 2 knife blades set 1 cm apart. This warm core is then subjected to the Slice Shear Force protocol (SSF) to evaluate meat texture. The use of the variable angle cutting box and the SSF protocol provides an accurate representation of the maximal shear force, as the slice and muscle fibres are consistently parallel. Therefore, the variable angle cutting box, in conjunction with the SSF protocol, can be used as a high-throughput technique to accurately evaluate meat tenderness in different locations of the longissimus muscle and, potentially, in other muscles.

Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.

介绍一个角度可调切割盒切片肉的剪切力分析

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

新型杀虫化学对蚊子毒性测试规程

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...