程实验室的电子显微镜的工作部分是支持由NIH（1R01GM082893，1R01GM098672，1S10RR026814 - 01和P50GM082250（A.弗兰克尔）），和来自加州大学旧金山分校的生物医学研究的突破，机会奖和新技术奖项目给予资助。

1。电磁电网碳涂层的制备<ol><li>填充用蒸馏水的大烧杯。 </li><li>使用玻璃吸管砸在水面单一的火棉胶（〜20μL，2％戊酯，电子显微镜学，PA，美国）下降。火棉胶将蔓延在水/空气表面，形成一个薄薄的塑料层;注意：可用于任何尘埃颗粒清除水面的火棉胶的首次下降。从第一个下拉膜中取出后，水面上的灰尘清晰。 </li><li>广场的EM电网，400或200目吉尔德铜电网购特德佩拉公司（美国），一个个浮于水面上的塑料层，我们通常的地方​​50〜100电网在一个紧密的包装图案，与光泽方电网面临的塑料层（图1A）。注：400目网格常规负染的EM电网;而200目网格收集倾斜对图像。 </li><li>剪下一个馅饼CE纸张尺寸比电网的塑料层上放置的面积略大一些。仔细将纸张顶部的网格（图1B），等到纸张变得完全湿（图1C）。注：不同类型的纸张有不同的水的吸收率。重要的是，纸张不吸收水分太快。例如，定期的滤纸是不是为此目的，因为它吸收水分过快使得很多皱纹。碳薄膜皱纸不平整，从而不能收集随机的锥形倾斜三维重建倾斜对图像良好。来测试不同的文件，以找到一个合适的的。在我们的实验室，我们一直使用从当地的杂货店买的记事本文件。有时报纸的纸张也发现是合适的。另一种选择是使用相对厚的宣纸用于中国/日本书法。 </li><li>迅速作出周围切口上的塑料薄膜和接近浸泡过的纸。紧接着，使用一对镊子，拿起纸与塑料薄膜和纸张之间夹着网格。放置在培养皿纸与电网所面临的空气干燥（图1D）;注意：重要的是让纸张风干。干燥过快产生许多皱纹的文件。 </li></ol> 2。碳涂层网格<ol><li>在我们的实验室中使用的碳涂布机是一个Cressington 208C高真空涡轮增压碳镀膜机（泰德佩拉）。我们的系统配备一个Cressington薄膜厚度的显示器，该措施的原则的基础上涂碳薄膜的厚度，石英晶体的振荡频率是在其表面沉积薄膜的质量改变。然而，协议在这里不需要厚度的显示器，因为它往往不是在每一个实验室。 </li><li>锐化使用卷笔刀（泰德佩拉）和负载炭棒中碳涂层米碳棒achine（图2A）。两棒，用锋利的尖端和其他与平坦的放置互相反对。 </li><li>应用真空润滑脂磨砂载玻片面积的一半，并把它旁边电网（图2B）。只有一半的载玻片磨砂面积应包括真空润滑脂。注：真空润滑脂面积不会改变碳涂层后的颜色。真空润滑脂与邻近地区之间的对比提供了一种涂碳的厚度估计。 </li><li>广场与舞台上的碳涂布机电网的纸张。将载玻片电网（图2C）。注：你也可以用塑料薄膜载玻片涂电网首次转让涂装前，很多情况下电网下降从纸张。 </li><li>泵的真空室，等到真空达到10-5托的水平。注：低真空镀膜往往会产生许多火花，导致许多大型的碳粒子网格。 </li><li>增加当前非常缓慢。碳棒的锐利尖端会先变成红色，然后作为当前增加的白色。不要用肉眼直接观看高亮度提示。停止增加涂层启动时的电流。载玻片上会改变颜色从白色，浅灰色和深灰色。黑暗幻灯片的关联被涂碳薄膜的厚度。关闭当前的关闭停止涂层时所需的厚度已经达到（图2D）。注：1）经过反复的碳涂层工艺，玻璃钟罩内涂碳，其颜色变暗。这使得涂碳膜厚度大于它实际上是。 2）一个削尖的碳棒可用于几个涂层。每次涂后逐渐减少提示的清晰度。最大电流是影响碳棒的清晰度。一个直径较小的新鲜削尖杆需要一个相对较低的当前蒸发碳。后来涂层可能需要略高的电流。 </li><li>放空真空室，取出后用涂电网。 注1：控制碳厚度更精确的方法是使用薄膜的厚度监视器。或者可以定性校准对精确的厚度测量显示器的颜色。这种技术的一个有用的碳厚度为3至7纳米。 </li></ol>这是一个简单的方法，准备大量的碳包覆电网，通常〜100％准备网格。网格包含一个薄薄的塑料层，它通常不会影响负染图像的质量。然而，在此方法制备的网格是不适合用于cryoEM。这层塑料可以去掉浸泡了几个小时到氯仿电网。 3。准备铀甲酸溶液（0.75％）为负染色的EM。详细的协议此前5介绍<ol><li>到小烧杯称取37.5mg铀酸盐; </li><li>添加5毫升的开水，搅拌5分钟在黑暗中; </li><li>加入10μL5M NaOH溶液，继续搅拌5分钟; </li><li>过滤器的解决方案，使用注射器过滤器（Fisherbrand，13毫米注射器过滤器，0.2μm的尼龙非无菌，目录编号09-720-5），并存储在一个覆盖着铝箔的8毫升猎鹰管过滤的解决方案; 注意。铀酸盐粉末，就可以买到从电子显微镜科学（哈特菲尔德，PA），目录号22451 。铀酸盐的解决方案是光敏感，应尽量远离直射光线，例如，通过覆盖猎鹰管铝箔。新鲜配制的解决方案有一个浅黄色（图3A）。染色解决方案，可以保持一到两周之前开始沉淀在板凳上。铀酸盐是酸性的pH〜4。加入NaOH的pH值增加，但增加速度太快或过量可导致色斑沉淀。除了产生对比，铀酸盐也修复的蛋白质样品。由于固定利率是如此之快，低pH值通常不影响整体的蛋白质构象或蛋白质复合物的形成 7 。 </li></ol> 4。准备负染色EM网格的议定书“。该协议是此前5详细描述<ol><li>辉光放电的碳镀膜网格，使用Pelco easiGlow GlowDischarge系统（特德佩拉公司，美国）。当前使用的是15 mA和网格为30秒。 注意出院焕发。辉光出院电网，辉光放电后不久，应使用。我们经常使用的辉光放电后在不到2个小时的网格。 </li><li>将两个20μL滴蒸馏水和两个铀酸盐溶液25μl滴在一块的封口膜（图3B）。 </li><li>保持辉光放电的网格，与汽车使用反向力抗毛细管钳碳涂层的一面朝上。注：这是有必要使用抗毛细管钳。否则，液体样品将被吸走从电网由毛细管力之间的镊子。 </li><li>应用2μL蛋白质样品辉光放电的电网，并等待30秒。注：等待时间和辉光放电的长度影响吸附在网格蛋白的浓度。非常稀释的样本，需要更长的吸附倍。然而，长时间吸附，可能会改变，因为水蒸汽的缓冲液浓度。为了达到适当的粒度分布在电网需要有些试验和错误做法。 </li><li>用滤纸吸干样本。 </li><li>触摸网格表面的水滴，然后水用滤纸吸干。重复与水的第二次下降。 </li><li>触摸网格表面的污渍简要的首次下降。用滤纸吸干污渍的解决方案。 </li><li>触摸网格表面的色斑20秒第二个下拉。印迹ØFF染色溶液用滤纸。 </li><li>使用真空干燥电网。注：也可以留在板凳上，或在空气干燥的化学物质罩网格。真空干燥的好处是可以立即观察在EM电网。快干产生可能的神器是不均匀的色斑。 </li></ol> 5。透射电子显微镜<ol><li>在这个演示中使用的显微镜​​（FEI公司）与一个加坦的4K x 4K CCD相机配备一个Tecnai的T12。 </li><li>简单插入样品前检查显微镜对齐;注意：这是只需要每一次的EM会议。在一个模型显微镜中，近乎完美的对齐可以保存。载入这样的一个对齐文件应恢复显微镜对准了近乎完美的条件。 </li><li>装入单倾斜的标准试样座EM网格。 </li><li>插入CompuStage的T12的持有人。抽水周期，等待完成。 </li><li>插入列持有人。&lt;/ LI&gt; <li>焦点标本，并设置所需的散焦，通常1.5um。注：基础，可以实现手动对焦使用最低对比的方法，或使用CCD相机，捕捉到的实时图像和计算生活傅立叶功率谱。 </li><li>使用CCD相机记录图像; </li><li>在图像的负面染色的蛋白质样品，蛋白质往往被与白的对比，即对一个比较暗的背景色的白色密度。请注意，而不同地区的同一电网中的蛋白质含量通常都颇为相似，不观察不同领域不同层次的染色在同一电网。有时地区可能有不均匀染色或染色阳性（蛋白质出现白底黑字），而其他一些地区有完善的污渍。它往往是必要的网格搜索找到一个良好的成像面积的。 </li></ol> 6。代表性的成果典型的良好形象产生负面STA的例子独立非执行董事的样本，马脾铁蛋白（图4A），厂（图4B），古20S蛋白酶体（图4c）和核小体（图4D），显示。 <img alt="图1" src="/files/ftp_upload/3227/3227fig1.jpg" /> 图1。制备碳包覆电网为负染色的EM。）配售EM电网在火棉胶薄膜表面上的封闭包装图案由一。 b）将一块略高于网格面积较大的纸张。三）等待，直到纸张得到完全湿。四）拿起纸连同空气干燥的电网。 <img alt="图2" src="/files/ftp_upload/3227/3227fig2.jpg" /> 图2。 。涂层的EM电网与碳一）设置的碳蒸发器：在涂料仪器的两个碳杆，以一杆削尖点提示（左）。其他杆（右）在平坦的表面，并在接触与在对面s的一个点提示IDE。 b）放置在一个培育的载玻片的一侧少量的真空油脂。黑框内的面积真空润滑脂。 c）设置为涂层的电网。玻片用于保存的文件位置。箭头指向真空润滑脂的区域。 d）在涂层，覆盖与真空油脂的幻灯片的颜色不改变颜色。对比表明涂碳的厚度。 <img alt="图3" src="/files/ftp_upload/3227/3227fig3.jpg" /> 图3。说明负染色过程A）刚准备（左）铀酸盐的解决方案是透明无沉淀的迹象。 1〜2周后，沉淀在试管底部的底部开始积累。 b）将蒸馏水和铀在一块的封口膜甲酸溶液滴两滴（约25μl）。使用一对反毛细管钳与碳端辉光排出的碳包覆EM网格起来。下降的样品（〜2μL）是放置在网格中。 c）在等待30秒，印迹滤纸的解决方案。四）翻转网格网格的样品侧接触水的首次下降。重复步骤C和D来完成染色过程。 <img alt="图4" src="/files/ftp_upload/3227/3227fig4.jpg" /> 图4。负染蛋白质样品EM图像的例子。）马脾铁蛋白中的B）片段的抗原结合（FAB），C）古细菌20S蛋白酶体和D）核小体。所有图像记录具有相同的放大倍率。比例尺是20nm的。

<ol>
	<li>Cheng, Y., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advent+of+near-atomic+resolution+in+single-particle+electron+microscopy.">The advent of near-atomic resolution in single-particle electron microscopy.</a> Annu. Rev. Biochem. 78, 723-742 (2009).</li><li>Grigorieff, N., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-atomic+resolution+reconstructions+of+icosahedral+viruses+from+electron+cryo-microscopy.">Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy.</a> Curr. Opin. Struct. Biol. 21 (2), 265-273 (2011).</li><li>Zhou, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+resolution+cryo+electron+microscopy+of+macromolecular+complexes.">Atomic resolution cryo electron microscopy of macromolecular complexes.</a> Adv. Protein Chem. Struct. Biol. 82, 1-35 (2011).</li><li>Adrian, M., Dubochet, J., Lepault, J., McDowall, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+of+viruses.">Cryo-electron microscopy of viruses.</a> Nature. 308 (5954), 32-36 (1984).</li><li>Ohi, M., Li, Y., Cheng, Y., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Staining+and+Image+Classification+-+Powerful+Tools+in+Modern+Electron+Microscopy.">Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.</a> Biol. Proced. Online. 6, 23-34 (2004).</li><li>Rabl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+gate+opening+in+the+20S+proteasome+by+the+proteasomal+ATPases.">Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases.</a> Mol. Cell. 30 (3), 360-368 (2008).</li><li>Zhao, F. Q., Craig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+time-resolved+changes+in+molecular+structure+by+negative+staining.">Capturing time-resolved changes in molecular structure by negative staining.</a> J. Struct. Biol. 141 (1), 43-52 (2003).</li><li>Radermacher, M., Wagenknecht, T., Verschoor, A., Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+from+a+single-exposure,+random+conical+tilt+series+applied+to+the+50S+ribosomal+subunit+of+Escherichia+coli.">Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli.</a> J. Microsc. 146 (Pt. 2), 113-136 (1987).</li><li>Zhao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+conditions+of+human+leukotriene+C4+synthase+requiring+adjustment+of+a+particularly+large+combination+of+specific+parameters.">Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring adjustment of a particularly large combination of specific parameters.</a> J. Struct. Biol. 169 (3), 450-454 (2010).</li><li>Nishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+leukocyte+beta2+integrins+by+conversion+from+bent+to+extended+conformations.">Activation of leukocyte beta2 integrins by conversion from bent to extended conformations.</a> Immunity. 25 (4), 583-594 (2006).</li></ol>

没有利益冲突的声明。

负染色EM是一个强大的工具，可以用来研究纯化的蛋白质样品的三维结构，如使用随机的锥形倾斜8方法，它需要从试样倾斜至60 °，并收集来自同一地区的一对图像，但untilted，计算大分子复合体的三维重建。此外，负染EM可以有许多其他应用，如二维（2D）的膜蛋白9晶体筛选，观察在不同的构象10，或者干脆进行可视化从而纯化的蛋白质样品的均匀性和/或异质性的蛋白质复合体提供反馈，以提高蛋白质纯化程序，等它可以是一个强大的生物化学检测。在这篇文章中，我们提出的协议，从准备收集在电子显微镜负染的蛋白质样品图像的碳包覆电网。如下图所示，程序比较简单，易于遵循。如果试图计算由随机的锥形倾斜方法观察到的样品的三维重建，这是必要的收购倾斜对图像。但是，如果进行单粒子分析的目的是，多个untilted图像就足够了。在这两种情况下，进一步的图像处理是必要的，这超出了这个视频文章的范围。 任何方法，有变化的协议。铀酸盐作品非常好，在大多数情况下为负染色试剂。醋酸铀是另一种常用的负染试剂。它的工作原理非常相似铀酸盐，但更大的晶粒尺寸，并在许多情况下，低于铀酸盐的对比。使用醋酸铀的好处是，解决方案的持续时间长于铀酸盐。一个并不需要每隔一至两个星期准备一个新的解决方案。两个铀甲酸和醋酸的酸性。一个明显的关注的是，低pH值可诱导在被研究的蛋白质样品的变化。两种染色的解决方案也可作为蛋白固定剂。早先的研究已经表明，这两个修复蛋白样品迅速 7 。这种快速的固定低pH值不太重要。在染色过程中，成为一个蛋白质样品固定前低的pH值可诱导任何更改。然而，其他如钼酸铵和磷钨酸负染试剂，可能会产生更好的效果染色样品时，低pH值是关注 7 。钼酸铵和磷钨酸约中性pH值，但这些污渍产生的蛋白质样品的对比度通常比铀酸盐。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 产品编号 </td><td> 评论 </td></tr><tr><td>火棉胶</td><td>电子显微镜科学，PA，美国</td><td> 12620-30 </td><td> 30ML，2％戊酯</td></tr><tr><td>吉尔德铜电网</td><td>泰德佩拉公司，美国</td><td> G400/G200 </td><td>二百分之四百网格</td></tr><tr><td> Cressington 208C高真空涡轮碳涂布机</td><td>泰德佩拉公司，美国</td><td> 9260 </td><td></td></tr><tr><td>双指出碳棒</td><td>泰德佩拉公司，美国</td><td> 58 </td><td></td></tr><tr><td>铀酸盐</td><td>电子显微镜科学，PA，美国</td><td> 16984-59-1 </td><td>纯度为99.9％ </td></tr><tr><td> PELCO easiGlow GlowDischarge系统</td><td> T美国版佩拉公司， </td><td> 91000 </td><td>在当地的药店购买</td></tr><tr><td>用Whatman＃1滤纸</td><td> Fisher Scientific则</td><td> 09 - 805E </td><td></td></tr></tbody></table>

visualizing proteins and macromolecular complexes by negative stain em: from grid preparation to image acquisition

负染色或冷冻水合生物样品的单粒子电子显微镜（EM），已成为一个多功能的工具，在结构生物学 1 。近年来，这种方法已取得了巨大的成功，研究蛋白质和大分子复合物2，3结构。电子低温显微镜（cryoEM），其中冷冻水合蛋白样品是在一层薄薄的玻璃体冰4的嵌入式相比，负染色是一个简单的样品制备方法，其中蛋白质样品嵌入一层薄薄的干重金属盐，以增加标本的对比5。负染色增强对比EM允许相对较小的生物样品的检查。此外，以确定三维（3D）纯化蛋白质或蛋白质复合物6的结构，这种方法可以用于更广泛的用途。例如，负染色的EM可以很容易地使用可视化纯化蛋白质样品，获得信息，如同质/异质性的样品，蛋白质复合物或大型集会的形成，或简单地评价一种蛋白质制剂的质量。 在这个视频文章中，我们提出一个完整的协议使用的EM观察负染蛋白质样品，准备为负碳包覆电网收购在加速电压在120KV经营电子显微镜负染样品图像污渍的EM。这些协议已在我们的实验室常规新手用户可以很容易地遵循。

Watch this Scientific Journal Video about Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition at JoVE.com

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

负染色电子显微镜（EM）的可视化蛋白质样品已成为一种流行的结构分析方法。它是有用的定量结构分析，如计算一个正在研究的分子的三维重建，也为蛋白质制剂质量的定性检查。在这篇文章中，我们目前准备的EM电网，染色样品，并在电子显微镜下的可视化样品的详细协议。新手用户可以轻松地遵循这些协议，并利用负染作为常规检测EM，除了其他生化分析评估其蛋白质样品，。

负染色的EM可视化蛋白质和大分子复合物：从电网的制备图像采集

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

visualizing-proteins-macromolecular-complexes-negative-stain-em-from

Research

JoVE Journal

Bioengineering

58.3K 次观看.  University of California San Francisco. 负染色电子显微镜（EM）的可视化蛋白质样品已成为一种流行的结构分析方法。它是有用的定量结构分析，如计算一个正在研究的分子的三维重建，也为蛋白质制剂质量的定性检查。在这篇文章中，我们目前准备的EM电网，染色样品，并在电子显微镜下的可视化样品的详细协议。新手用户可以轻松地遵循这些协议，并利用负染作为常规检测EM，除了其他生化分?...

视频: Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

科研

生物工程

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

去耦力学传递线索的影响制备羟基聚丙烯酰胺水凝胶的

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

使用磁力学监测化学合成氧化铁纳米粒子的细胞合并和后续生物降解

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...