这项工作的支持，R01-HD058629 IEZ

1。制备ECM包被的培养皿或盖玻片<ol><li>准备ECM原液中溶解：ECM，衬底在PBS（Dulbecco氏磷酸盐缓冲盐水，＃14040-133，Invitrogen公司）。使用为0.2mm的注射器过滤器（VWR，＃28145-495）和冻结以等分试样在-80℃下进行筛选</li><li> ECM原液稀释在PBS中，以所需的浓度。对于MaxGel，DMEM（Dulbecco改良的Eagle培养基，Invitrogen公司，＃11995-065）中稀释。 </li><li>如果将进行免疫荧光染色，灭菌通过浸渍在乙醇中的12毫米的圆形玻璃盖玻片（VWR，＃89015-724）和在空气中晾干。这项工作应在无菌层流罩。如果免疫荧光法，将不会执行跳到步骤1.5。 </li><li>地点灭菌盖玻片在24孔板的各孔中（Corning公司，＃3526）。 </li><li> 0.5毫升稀释的底物溶液加入到24孔板，在室温下孵育3小时温度的各孔中。 </li><li>吸出的ECM解决方案，并用THRE在PBS的e时代。 </li><li>吸干PBS及立即使用或包裹在封口膜，并储存于4℃下进行长达2个星期。 </li></ol> 2。准备解剖工具 Sylgard的，添加木炭准备每板制造商的协议。这些板的耐药黑色背景提供支持牵制胚胎和颜色提供良好的对比度，便于可视化的透光性的胚胎。 <ol><li>为了准备板，权衡直接进入三倒入烧杯中的平衡150 G部分的液体15克B部分木炭粉和1 g（世界精密仪器，＃SYLG184）。混合在一起，用一个木制的冰棒棍或压舌板。倒入玻璃或塑料100 cm培养皿。用一个小丁烷火炬，举行了约10至12英寸以上的菜烧的气泡。将平板上的盖子，让坐在至少24小时来设置。 </li><li>准备清扫工具插入一个Minutien针（0.1毫米diameteR）进入的销保持器（精细科学工具，＃26002-10和＃26018-17），分别交替26表针（BD，＃309597），与借助钳子弯曲成直角，可以用作解剖仪器。锋利的学生杜蒙＃5镊子（精细的科学工具，＃91150-20）用磨刀石（精细的科学工具，＃29008-22）。 </li><li>所有清扫工具，在使用前用70％乙醇消毒。 </li></ol> 3。收集胚胎<ol><li>根据使用动物委员会条例安乐死的时间为8.5天coitum孕鼠和取出子宫。 </li><li>将子宫在塑料培养皿中，用无菌PBS。 </li><li>用无菌PBS洗净子宫一次。 </li><li>在解剖显微镜下小心地取出胚胎蜕膜组织，用一个钳子。 </li><li>删除胚外膜和基因分型，如果需要保存。 </li><li>计数体节的胚胎阶段。理想的阶段，使外植体，分析细胞BEHavior在神经褶海拔之间的6和10体节阶段时，神经褶才刚刚开始提升的颅间充质细胞的主要形态发生之前。 </li><li>胚胎在PBS中洗涤。在一个黑色的SYLGARD板用DMEM包含没有酚红（Invitrogen公司，＃21063-029）的地点。删除的胚外膜和基因分型，如果需要保存。 </li></ol> 4。外植体的制备<ol><li>再聚焦在头部区域的胚胎在最高可能的放大倍率的显微镜。 </li><li>用解剖针在每手削减。使用一针保持在地方和其他组织作出削减。 </li><li>剪去头的菱前的胚胎。 </li><li>切沿中线平分头，分成两等分。 </li><li>切的头部和中线周围的外边缘。这些削减将删除的premigratory的的波峰和外边框和表面外胚层的在中线prechordal板。其余的生活将是外植体，将包含迁移颅神经嵴旁轴中胚层和表面外胚层。 </li><li>当组织被切断，除去解剖的碎片从培养皿用无菌巴斯德吸管。这可以防止混淆了外植体解剖的碎片。 </li><li>轻轻冲洗外植体，由pipeting向上和向下20微升移液器吸头和PIPETMAN。这将删除松散贴壁细胞和防止外植体边缘呈锯齿状。 </li><li>小心地拿起每个外植体，在约10微升的液体，使用移液管尖20微升和PIPETMAN和地点的中心处的包被的培养以及。 </li><li>中加入20μl含15％FBS补充的培养基中，以覆盖外植体。 </li><li>地点板在湿润的组织培养箱（5％CO 2，37℃）1-2小时，以允许附加到外植体的培养皿的底部。定期查看，以确保外植体不干燥了。 </li><li>外植体已经附着时，小心地加至每个孔中加入250μl含10％FBS DMEM培养液已被温热至37℃的水浴中。一旦外植体连接时不会移动板在显微镜下轻轻摇动。 </li><li>返回菜组织培养的孵化器。 </li><li> 24小时后，将外植体牢固地附着和细胞就会开始移民。此时药理剂可添加到介质中。 </li><li> 48小时后，电镀，已经从外植体细胞。可以可视化和成像的距离迁移。我们通常会停止在这一点上，因为文化外植体开始出现较长的孵育的生存能力下降。如吖啶橙或台盼蓝甲活力染色也可用于判断是否将外植体的可行性。 </li><li>在这一点上，可以获取图像文件从外植体的细胞迁移的距离和数量。另外，如果外植体涂布于盖玻片上，它们可以是固定的，并执行的可视化感兴趣的蛋白质的免疫组织化学分析。 </li></ol>在协议中的关键步骤<ol><li>把外植体的大致相同的大小。 </li><li>清除所有清扫污物解剖板解剖，以避免混淆所需的移植组织碎片。 </li><li>当电镀外植体井或盖玻片上是一定要放在液滴的媒体中心以及与外植体。 </li><li>允许植足够的时间来连接洪水以及与媒体之前，。 </li><li>每一个测试基板和药理活性剂处理条件应该是标准化的。 </li><li>两个每个胚胎外植体可以由（一个从左边和从右侧的神经管中的一个）。 </li><li>当测试迁移的细胞突变小鼠品系不同实验条件下，把一个突变的外植体在对照组和治疗组。 </li></ol>故障排除<ol><li>如果外植体无法连接，增加附件规定的时间。根据我们的经验，约一半的外植体不附加和较大的外植体更有效地附着。 </li><li>如果外植体附加到井的周围，它们将难以图像。请务必将外植体中心的良好。 </li><li>理想的情况下，外植体是大致相同的尺寸。然而，外植体大小的差异可以被校正为在quanitating从外植体的细胞的迁移时，使用的外植体的直径作为校正因子。 </li><li>如果发生污染确保消毒工作条件正在使用。所有的工具和工作表面应用70％乙醇消毒。移液管和培养皿应该是新的和不育。 </li><li>如果外植体迷路的解剖过程中，要定期清除杂物，以防止丢失的外植体。 </li><li>掌握解剖非常耗时，而且需要练习。优化正常的放大倍率，锐性剥离的工具和针帮助。 </li></ol>

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没有利益冲突的声明。

采用的方法提供了一个功能强大的检测，检查颅骨间充质细胞的行为。在除了这里介绍的静态分析，在明亮的字段或组合住成像实验与GFP-标记的蛋白质的表达，可以采用实时检查细胞的行为，因为它们从外植体中迁移。对于实时成像实验中，外植体可以用DiI标记或分化的神经嵴细胞的迁移，从近轴中胚层，ROSA-YFP Mesp1创建或ROSA-YFP; WNT1中铁快运或其他转基因株系可以使用。颅间充质细胞-ECM相互作用也可以利用此外植体的分析研究。在这里，我们，本的颅间充质中表明，细胞对这些迁移到不同的程度，ECM的影响的细胞的形态的不同的ECM基板的板外植体对。此外，外植体可以被嵌入在一个三维矩阵存取的行为大公LS在这种情况下。此外植体检测是可以改变的遗传学和药理学的操作的颅间充质行为来分析内在的和外在的线索，可以调节和修改迁移的影响分析。例如，我们在我们的研究中利用该测定辨别Hectd1突变颅间充质15中的异常细胞的行为的过量的热休克蛋白90（Hsp90）分泌的作用。在这些实验中，我们处理的外植体与抗-Hsp90的抗体，格尔德霉素和DMA（二甲基amelioride），热休克蛋白90（Hsp90）蛋白阻止分泌的热休克蛋白90（Hsp90），表明异常行为的Hectd1突变的细胞是由于过量的细胞外热休克蛋白90（Hsp90）15。类似的方法可用于药理学解剖颅间充质细胞的正常或异常的形态发生的基本的附加通路。测定完成后，外植体和迁移的细胞可以分析由一个主机的方法。例如，免疫组化分析C可检查本地化的关键细胞运动的蛋白质。转录组分析也可以治疗，以确定如何影响基因的表达。 这种技术的一个显着的限制是，它不会在三维模型的行为，因为它们将在体内发生。的协议，其中外植体被嵌入在一个三维的矩阵（ 例如，基质胶）以及表达荧光蛋白标记的细胞区室的变形例，可以采用必要的，以解决这个问题。即使进行这些修改，这与在体内的体外测定中看到的行为相关联的进一步的实验来将是必要的。其他限制包括大量的胚胎需要产生足够数量的来生成静态显著数据。最重要的是，夹层是相对简单的，它需要练习才能熟练掌握。

在胚胎8.5天（E8.5）和9.5在小鼠胚胎神经管闭合发生在颅地区。未能正确关闭的神经管头无脑儿，一个共同的结构在人类出生缺陷和不符合生活。驱动头的神经管闭合的力量，会产生神经组织本身和周围的表皮细胞和间充质3。特别是扩大的颅间充质被认为是必不可少的海拔的颅神经褶1,2。的颅间充质是含有丰富的细胞外基质蛋白，特别是糖基化蛋白，硫酸肝素糖蛋白，硫酸软骨素，透明质酸4-8。 不像在鸡胚胎神经嵴细胞的移民从背神经管，神经管闭合后，在小鼠胚胎神经嵴迁移的神经褶的同时，开始为rISE（后5体节期）。因此，在小鼠胚胎神经胚形成过程中，脑神经细胞来源于神经嵴和近轴中胚层间充质细胞组成的。诱导神经嵴旁轴中胚层的人口在不同时期的发展定位在不同的位置在胚胎发育成不同的结构9,10。近轴中胚层起源于原始的条纹，并迁移到前牙区的胚胎的的推定神经板的基础。交界处的神经板和表皮外胚层的神经嵴细胞是诱导，经历了一个上皮细胞向间质细胞转变，脱层只是之前的啮齿类动物胚胎的神经褶海拔。神经嵴细胞迁移以及刻板的路径中的subectodermal的旁轴中胚层的鳃弓，额鼻和眼周的间充质。近轴中胚层，将有助于一些的头骨穹窿的骨骼和面部肌肉;而日E神经嵴将有助于其他的头骨和面部骨骼，除了颅神经9-11。差异的近轴中胚层和神经嵴细胞谱系标记的Mesp1创建和WNT1的表达Cre重组酶转基因小鼠的线，分别为9。 治疗的ECM干扰剂，如透明质酸，硫酸软骨素酶ABC，肝素酶或重氮-氧代-亮氨酸（DON）在神经胚形成减值的神经管闭合7啮齿动物胚胎神经管闭合的颅间充质细胞中的重要作用已推断实验， 12-14。在这些实验中，后的静态部分神经胚形成的组织学分析显示相关颅间充质7,12-14 dysmorphogeneis。然而，由于致畸剂获得多个组织，但它仍然被确定，如果颅间充质是真正的靶组织。在支持的结论，该组织是必不可少的神经胚形成，的颅间充质出现异常后，一些的小鼠突变与exencephaly 15日至17日组织学分析。尽管如此，在大多数情况下，突变的颅间充质细胞的细胞行为的效果没有得到解决。 我们已经制定了一个体外植试验，直接研究基因突变的结果或药物处理的行为的的颅间充质细胞15。此法的基础，是类似访问的颅间充质细胞的分化潜能公布的2003年Tzahor 等的菱18，除非我们修改了外植体解剖学习的迁移性能更前的人群的颅骨间充质前神经网络的基础板。我们的方法是在鸡胚进行修改外植体实验分析的神经嵴细胞的迁移行为与科Ÿ差异。前的准备工作外植神经嵴或更后的近轴中胚层19,20。此外，在海拔在鸡胚神经褶，神经嵴尚未移居海外，从背神经管和，从而植前的近轴中胚层将不包含神经嵴细胞。在我们的试验中，颅间质组成的近轴的中胚层，神经嵴和表面外胚层的外植体被制备和镀在基板上。可以进行实验操作，包括隔离的外植体，基因突变，电镀外植体，在不同的ECM或药物治疗。与细胞迁移的外植体和的距离，数量和行为可以分析和治疗组之间的比较。此外，该制剂是可以改变的，以通过实时成像技术中的细胞迁移分析。迁移实验后，外植体可以被固定，并进行免疫组化分析，裘THER阐明的治疗效果。从总体上看，这里介绍的协议是一个简单的体外实验调查的颅间充质细胞的行为。作为一个代表性的实验中，我们利用这个试验，以检查不同的细胞外基质的的颅间充质迁移。

an explant assay for assessing cellular behavior of the cranial mesenchyme

是来自中枢神经系统的神经板，经过一系列复杂的形态发生变动导致的神经管形成一个被称为神经胚形成过程中。在神经胚形成过程中，被认为是推动神经褶海拔形态的间充质细胞，神经板的基础。颅间充质是由近轴中胚层和神经嵴细胞。细胞的颅间充质形式组成星状状细胞和细胞外基质（ECM）股神经褶支持混杂的一个pourous小梁。在神经胚形成过程中，的颅间充质进行定型重排，从而导致其扩张和神经褶海拔，这些动作被认为是提供动力。然而，途径和细胞的行为，带动颅间充质形态仍然很差研究。 ECM的的颅间充质underly THES的细胞之间的相互作用的E细胞的行为。在这里，我们描述了一个简单的体外植试验设计的特点，这些细胞的行为。此法是可以改变的药理操作解剖的信号通路参与和实时成像分析，以进一步鉴定这些细胞的行为。我们提出了一个代表性的实验表明本测定中的实用程序的特征的颅间充质细胞的迁移性能上的各种ECM组分。

从颅间充质细胞的外植体的细胞迁移的外观如图2所示。我们测试了在不同的的ECM基板，是在颅间充质细胞在神经胚形成包括纤维连接蛋白，层粘连蛋白（100毫克/毫升;西格玛L2020），（100毫克/毫升;西格玛F1141）MaxGel ECM（1:500稀释的DMEM迁移;西格玛＃E0282）和透明质酸（1毫克/毫升，Sigma公司，＃53747）。细胞不能迁移从外植体时，没有ECM存在（数据未示出）和测试的所有基板支持的迁移的细胞。的形状，以及从外植体的细胞迁移的大小取决于基板。这是预期的，因为以前的研究表明，细胞与不同的ECM的相互作用所产生的机械力的影响的形状和迁徙性的细胞21。 <img alt="图1" src="/files/ftp_upload/4245/4245fig1.jpg" fo:contenT-宽度=“5.5英寸”的FO：src =“/ files/ftp_upload/4245/4245fig1highres.jpg”/&gt; 图1。外植体的制备。（A）编制外植体，E8.5胚胎蜕膜组织和胚外组织的解剖。 （B）胚胎头从身体左前菱解剖。外植体的制备是通过从中线和头部的外边缘的移除prechordal的板分别在中线和premigratory的神经嵴和在边界表面外胚层，除去组织。 （C）的外植体的制备是通过切去组织从中线边缘和外边缘的头部移除prechordal板中线和premigratory的神经嵴和在边界表面外胚层。 （D）的外植体放置在一个小体积培养基在ECM涂覆的菜/盖玻片和允许连接之前，除了更多的介质。 <img alt="图2" src="/files/ftp_upload/4245/4245fig2.jpg" /> 图2。从不同衬底上镀的外植体的颅间充质细胞的迁移。外植体接种于纤维连接蛋白，层粘连蛋白，MaxGel ECM或透明质酸和文化拍下后48小时。大小酒吧= 200毫米。

Watch this Scientific Journal Video about An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme at JoVE.com

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

颅间充质经历了戏剧性的形态发生变动，可能提供的驱动力升高的神经褶<sup1,2</sup&gt;。在这里，我们描述一个简单的<em&gt;体外</em&gt;外植体试验，在神经胚形成的颅间充质细胞的行为特征。此法有许多应用，包括适合药物处理和实时成像分析。

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

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JoVE Journal

Biology

9.7K 次观看.  Children's National Medical Center. 颅间充质经历了戏剧性的形态发生变动，可能提供的驱动力升高的神经褶

视频: An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Nanogenomedicine在MCF - 7细胞的细胞毒性：MTT法

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

用于评估细胞运动和相关信号事件的机械控制的单细胞Durotaxis测定

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...