作者想感谢博士。里克瑞安Umesh制作帕特尔，杰夫蒂尔，马修许，汉吉尔卡Mistry在发展这些项目和协议的支持和Michele Hatler。

细胞制备： <ol><li>直到〜70％至80％融合（细胞检测，文化生长介质中的星形胶质细胞神经​​元/播种前，除非细胞从解冻或隔离直接播种）。 </li><li>通过方法适合于细胞类型的兴趣分离细胞培养瓶/板。如果有必要，大衣检测井盘与D -聚赖氨酸或细胞外基质蛋白，增强细胞的粘附。联合培养的星形胶质细胞和神经元可能同时或顺序号种子。对于连续的种子队，电镀的第一层作为“基础”的星形胶质细胞是建议，由神经元的电镀和分化，如有必要。细胞类型，随后的培养时间，感兴趣的参数进行适当调整细胞密度等，根据文化，年龄，细胞来源和播种密度，小学文化的差异很大，GFAP表达或突起生长增殖率 - 是很重要的表征和优化您的电池系统，提供为您的实验模型中大多数生物的相关性，以及提供有效的成像，分割和使用的HCS（见图1）分析。加入细胞板（种子细胞可能在90μL的媒体，以促进毒素治疗，在下面的步骤3中所述）后，使板在室温下在水平表面上坐15-30分钟，使即使是细胞分布。在此期间，孵育细胞生长介质中至少24小时（37 ° C / 5％的CO 2），然后切换到分化培养条件下（例如，低血清/ NGF对PC12细胞），适当的。继续文化，直到细胞达到所需的合流或分化水平，改变在适当的时间间隔为细胞类型的媒体。 </li><li>细胞治疗（控制化合物，测试化合物等）可在任何时候，这种文化期间，利息的处理时间，当然适当。丙烯酰胺，双氧水和K - 252A是作为神经毒性控制化合物。提供了充足的试剂是重复的12点的剂量反应曲线（包括一个 生署2 O或DMSO控制范围内设定的剂量反应）所有五个96孔板。该化合物是提供250X的浓度（K - 252A，假设为1μm的最大处理）或10X（丙烯酰胺和过氧化氢，假设分别为100毫米和10毫米，最大的治疗）。推荐的治疗准备工作涉及半的日志（1：√10）连续稀释250X化合物在DMSO，复方稀释液稀释10X（10X控制卫生署2的化合物可能会连续稀释后，直接在无菌的dh 2 O或复方稀释液）。每次治疗10μL，然后可能被加入到90μL，每口井已经存在的文化传媒，作最后的1X浓度（0.4％DMSO或10％DH 2 O ）。样本数据是在37 ° C之前，以固定为24或96小时复方治疗。 </li></ol>细胞固定和免疫荧光染色： 注：染色时间约2.5小时后固定。不要让井干染色步骤之间。试剂的愿望和豁免应进行低流速，以减少由于流体剪切任何细胞的损失 。 所有建议“是指每以及”卷单井的96孔微孔板。所有“建议每96孔板”卷包括液体处理量损失超过25％。所有的染色步骤是在室温（RT） 。所有缓冲区和抗体的解决方案是稳定的，在室温下至少24小时。 <ol start="4"><li>在文化时期的结束，预温HCS固定的解决方案（2X）室温（RT）或37 ° C，如果需要的话（12 mL/96-well板）。在化学通风柜，加入100μL/孔直接向文化传媒，并允许在室温下30分钟的修复。删除固定液/毒素包含媒体，并在符合危险废物的规定处置（见MSDS）。如果立即着手染色，冲洗两次，每孔200μL的HCS免疫缓冲区。另外，如果板沾在稍后的时间，冲洗两次与200μL洗涤缓冲液，然后离开井和存储板，紧紧密封在4 º C，直到染色第二次冲洗量。 </li><li>如果固定样本已储存在4℃染色前，200μL的HCS免疫缓冲区冲洗两次，然后进行染色协议。 </li><li>准备工作βIII兔抗微管蛋白/鼠标抗GFAP HCS（6 mL/96-well板）主要抗体的解决方案如下：每个解冻的抗体加入60微升至5.88毫升的HCS免疫缓冲区。拌匀。删除以前的免疫缓冲液冲洗。加入50μL小学抗体溶液到每口井和1小时在室温下孵育。 </li><li>删除第一抗体解决方案。用200μLHCS免疫缓冲区冲洗三次。 </li><li>准备工作的HCS中抗体/赫斯特的HCS核染色（6 mL/96-well板）的解决方案如下：每个解冻二级抗体加入30μL和30μL解冻赫斯特的HCS核染色5.91毫升的HCS免疫缓冲区。拌匀，由轻保护的解决方案。删除以前的HCS免疫缓冲区冲洗。加入50μL的HCS中抗体/赫斯特的HCS核染色溶液，孵育1小时，在避光RT。 </li><li>删除的HCS中学抗体/赫斯特HCS核染色解决方案。用200μLHCS免疫缓冲液冲洗两次。 </li><li>删除以前的HCS免疫缓冲区冲洗。用200μL的HCS洗涤缓冲液冲洗两次，留下第二次冲洗量在井。 </li><li>立即密封板和形象，或存放在4℃板避光，直到成像准备。 </li></ol>图像采集和分析： <ol start="12"><li>染色板的成像和分析，可能是各种可用的HCS平台，包括细胞分析仪（通用电气医疗集团），ArrayScan（ThermoFisher科学）或歌剧（Perkin Elmer公司）后执行。在表1提供的成像和分析的一些指导方针： </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222图像采集准则 </td></tr><tr><td> 检测试剂 </td><td> 物镜 </td><td> 激发滤光片范围[峰/带宽（NM）] </td><td> 排放过滤范围[峰/带宽（nm）] </td></tr><tr><td>赫斯特的HCS核染色</td><td> 20X </td><td>四十○分之三百六十○ </td><td>四十零分之四百六十○（或五十分之五百三十五如有必要） </td></tr><tr><td> HCS二抗，FITC标记的驴抗兔IgG </td><td> 20X </td><td>四十零分之四百八</td><td>五十零分之五百三十五</td></tr><tr><td> HCS二抗，Cy3标记的驴抗小鼠IgG </td><td> 20X </td><td>五十零分之五百三十五</td><td>五十○分之六百</td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222图像分析准则 </td></tr><tr><td> 晶胞参数 </td><td> 检测 </td><td> 分割/测量 </td><td> 理由 </td></tr><tr><td>细胞数，核特性</td><td>赫斯特的HCS核染色</td><td>核区（460 nm发射通道）。计算原子核的数目。 DNA含量（核武器强度）或核领域的分析，也有可能。 </td><td>使用细胞数，核特性，以确定细胞的损失，毒性表型等，可以“过滤器”βIII-微管蛋白或GFAP表达获得单独的神经元和星形胶质细胞计数/刻画（强烈推荐）的原子核。 </td></tr><tr><td> βIII-微管蛋白的表达，突起生长</td><td> HCS二抗，FITC标记的</td><td>胞内区（535 nm发射通道）。 FITC标记的信号可能是用来区分突起的神经元细胞体（例如，通过最小/平均细胞的身体部位，最小/最大突起的长度和宽度）。确定参数，如总长度突起，突起计数/细胞等</td><td>突起生长的测量可在神经细胞分化过程中或化学性损伤，疾病状态等调制可以“过滤”βIII-微管蛋白的表达，获得不同的神经元的特性（强烈推荐）的胞体。 </td></tr><tr><td> GFAP的表达，星形胶质细胞区 </td><td> HCS二抗，Cy3标记的共轭 </td><td>胞内区（600 nm发射通道）。 Cy3标记的信号可以用来定义星形胶质细胞的细胞质分割。确定参数，如平均细胞质的信号强度，细胞面积，等</td><td> GFAP的表达和星形胶质细胞面积的星形胶质细胞的发展，或作为一种化学性损伤，疾病状态的结果可能会在调制，等“过滤器”与GFAP的表达，获得独特的星形胶质细胞的特性（强烈推荐）的细胞体。 </td></tr></tbody></table> 表1图像采集和分析指南- HCS222βIII-Tubulin/GFAP（联合培养）含量代表性的成果： <img src="/files/ftp_upload/1173/figure31173.png" alt="图3" /> 图1。 βIII-微管蛋白和GFAP immunofluorescence的混合大鼠神经细胞培养。 镀前几天神经播种在文化，星形胶质细胞所产生的原发性大鼠海马神经元或大鼠PC12细胞的原代大鼠海马星形胶质细胞共培养。主神经元培养一个额外的11天，而PC12s培养分化条件下（低血清/ NGF）增加2天。根据HCS222检测协议进行处理，固定和免疫细胞。细胞在细胞分析仪1000（3.3），20X（主神经元）或10X（PC12）物镜成像上的GE和分析细胞分析仪1000工作站（3.4）轴突生长和多目标分析算法中使用了GE顶板。赫斯特的HCS核染色（蓝色），βIII-微管蛋白（绿色）和GFAP（红色）荧光的融合图像。单独分析βIII-微管蛋白的荧光通道，允许突起生长分割（中间面板：概述蓝（初级神经元的胞 ​​体）或黄色（PC12），绿色（主神经元）或淡蓝色（PC12 ）的突起）。 GFAP的渠道分析允许星形胶质细胞的分割（ 底部面板：细胞核，细胞质蓝色概述绿色）。 GFAP的原代大鼠海马神经元/星形胶质细胞的共同文化的分割演示的能力，区分核/胞体，GFAP（+），（绿色概括）和GFAP（ - ）（红色），使不同的细胞计数神经元和星形胶质细胞混合培养设置。 <img src="/files/ftp_upload/1173/figure41173.png" alt="图4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="图4第2部分" /> 图2。海马神经元/星形胶质细胞共同培养有毒强调的原代大鼠的剂量反应。 原代大鼠海马星形胶质细胞接种于96孔板（六）在生长介质和培养6天。原代大鼠海马神经元（直接从解冻），然后上方的镀前的星形胶质细胞，接种于培养基中培养11天。细胞经连续稀释的丙烯酰胺或过氧化氢的文化在过去24小时（最大浓度= 100mm和10mm的分别）。根据HCS222检测协议进行处理，固定和免疫细胞。细胞成像上的GE细胞分析仪1000（3.3），20X（10场/孔）和分析（核/胞浆/突起分割）使用GE的细胞分析仪1000工作站（3.4）轴突生长和多目标分析算法。所列数据是平均± SEM，N = 3。多个参数（突起生长，GFAP的强度，星形胶质细胞面积和神经元或星形胶质细胞计数）为剂量依赖性的发展趋势进行了调查。

<ol>
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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

作者Millipore公司的所有员工。

迄今为止， 在体外实验旨在研究使用混合培养的神经元和星形胶质细胞神经毒性风险评估中已有限。这是广为接受的神经胶质细胞中不可分割的神经元提供了空间和代谢支持，并在调节神经功能，包括神经细胞迁移，分化和突触活动的发挥至关重要的作用。胶质细胞的星形胶质细胞也释放细胞因子和其他可溶性因子，这是许多毒素的神经诱导周围神经组织的不良反应，以及促进双方的宽容能力。展示的神经胶质细胞相互作用的深入，合作文化的研究，星形胶质细胞已证明，以保护神经元免受氧化应激的毒性，而星形胶质细胞的功能，如抗氧化防御和细胞能量水平的维护，减值已被证明看房影响神经元的存活。最近的研究表明，星形胶质细胞在体外培养的神经毒性测试系统的使用可能被证明更切合人体中枢神经系统的结构和功能的神经细胞，单独。尽管越来越多的神经元，星形胶质细胞相互作用的生理意义的认识，以前一直难以执行这些相互作​​用的定量分析，在完整的细胞。高通量筛选的到来，使发展的强大的新的分析方法来解决这个问题。 在这项研究中，我们已经表明，HCA的可能是由多个神经元和星形胶质细胞在一个单一的检测表型的定量分析。在初级神经元，我们已经完成了的神经元的数量，神经轴突计数和突起的长度，如神经毒性标志的定量测量。在星形胶质细胞，反应性胶质化是一个已知的神经毒性的生物标志物的特点，GFAP的表达，星形胶质细胞的数量和星形胶质细胞形态的改变。我们已经证明，这些端点的每个人都可以很容易地通过HCA的评估。这些研究支持神经特异性的生物标志物HCA，可在体外试验中使用的电池的一部分，迅速 ​​屏幕为神经中毒的端点化学品的大量的概念。 Millipore公司的HCS222为神经元和星形胶质细胞共培养高含量分析试剂盒是由高品质的检测试剂和协议，同时在多种神经毒性的细胞模型的分析βIII-微管蛋白和胶质纤维酸性蛋白（GFAP）。高度验证，该试剂盒提供的试剂，允许用户规范检测，减少检测，检测变异，再现产生一个高信号背景比的图像。

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

高含量的分析（HCA）的分析相结合细胞自动成像和强大的图像分析算法和检测试剂，允许在一个单一的检测多个细胞表型的测量。在这项研究中，我们利用HCA的发展为神经毒性的新的分析。神经毒性评估的药物安全性评价的重要组成部分，以及作为一个环保工作的重要重点。此外，神经也是一个广为接受的的<em&gt;在体外</em&gt;标记的神经退行性疾病如阿尔茨海默氏症和帕金森氏病的发展。最近据报道，HCA的神经元的筛查中的应用。 HCA的检测标签βIII-微管蛋白的神经细胞，可以提供高通量，非主观的神经元的数量，神经轴突计数和突起的长度，所有这些都表明神经毒性作用，如参数的定量测量。然而，星形胶质细胞的作用仍然未开发的，在这些车型。星形胶质细胞具有不可或缺的作用，在维护中枢神经系统（CNS）的动态平衡，当他们在应对有毒物质或疾病状态激活的神经保护作用和neurodegradation关联。 GFAP的是一种中间丝蛋白，主要表达在中枢神经系统的星形胶质细胞。星形胶质细胞的活化（胶质细胞增生），GFAP的表达上调，通常伴有星形胶质细胞增生和肥大。这种反应性胶质化的过程中已经提出的神经系统造成损害的早期标志。 GFAP的定量分析的传统方法是通过免疫分析。这种方法无法提供信息，对细胞定位，形态和细胞数量是有限的。我们确定，HCA可以用来克服这些限制，并同时测量多个与胶质细胞增生相关的功能 - GFAP表达的变化，星形胶质细胞肥大，和星形胶质细胞的增殖 - 在一个单一的检测。在共同的文化研究，星形胶质细胞已被证明，以防止有毒侮辱的几种类型的神经元和批判性的影响神经元的存活。最近的研究表明，在使用的星形胶质细胞<em&gt;在体外</em&gt;神经毒性测试系统可能证明比单独的神经细胞对人体的中枢神经系统的结构和功能。因此，我们已经开发出神经元和星形胶质细胞共培养HCA的检测，包括协议和验证，目标具体分析βIII-微管蛋白和胶质纤维酸性蛋白（GFAP）检测试剂。此实验可同时分析神经轴突生长，神经胶质增生，神经元和星形胶质细胞的形态和在多种细胞模型的神经元和星形胶质细胞的发展，一种新型的，非主观的，高通量检测神经毒性评估。该试剂盒拥有增强检测神经毒性和神经科学的研究和药物发现提高生产力的巨大潜力。

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

本文介绍了一种新的协议和试剂，为敏感的神经毒性作用的化合物和治疗联合培养的神经元和星形胶质细胞使用高含量的分析测量设计。结果表明，高含量的分析，代表着一个令人兴奋的新技术，神经毒性评估。

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

25.0K 次观看.  Millipore Inc.  日本語 

视频: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

科研

生物学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...