硅主模具制造 光刻技术 材料： <ul><li> 75毫米的硅晶片</li><li> SU - 8 2负光阻（Microchem，波士顿，MA） </li><li>氮气</li></ul>方法： <ol><li> 2个小时，脱水在120 ° C的硅晶片。 </li><li> 7分钟的紫外线臭氧治疗表面，以除去有机物。 </li><li>氮气吹表面，以去除微粒。 </li><li>应用SU - 8 2，覆盖面70％（约10毫升）75毫米晶圆。 </li><li>在2000年的20秒的转速旋转300转/ S加速。 </li><li>晶圆放在一个烤盘和坡道从室温至95 ° C。 </li><li> Softbake光致抗蚀剂为15分钟，在95 ° C。 </li><li>洪水暴露晶圆为20 - 365纳米（我行）25 mJ/cm2剂量形式的底层。 </li><li>应用SU - 8 2第二光阻层。 </li><li> 550 20秒的转速旋转，300转/秒，加速约11微米厚的薄膜。 </li><li>晶圆放在一个烤盘和坡道从室温至65 ° C。 </li><li> Softbake晶圆在65℃5分钟。 </li><li>斜坡的电磁炉，从65 ° C至95 ° C。 </li><li> Softbake晶圆55分钟，在95 ° C。 </li><li>酷晶圆室温20分钟。 </li><li>对准晶圆mPAD面具和揭露为95 ​​- 100 mJ/cm2剂量为365 nm（我行）。 </li><li>晶圆放在一个烤盘和坡道从室温至95 ° C。 </li><li>曝光后烘烤25分钟的光致抗蚀剂95 ° C。 </li></ol>开发 材料： <ul><li> 100毫米玻璃餐具（5） </li><li>丙二醇甲醚醋酸酯（PGMEA） </li><li>异丙醇（IPA） </li><li>氮气</li></ul>方法： <ol><li>在化学油烟机，填补五菜PGMEA（2），“近期行动计划”，和己烷（2）。 </li><li>在1日PGMEA举行，持续10秒的晶圆</li><li>搅拌晶圆为50秒</li><li>晶圆转移到第二PGMEA PGMEA半月板。 </li><li>鼓动5秒。 </li><li>晶圆转移到“近期行动计划”与PGMEA半月板。 </li><li>鼓动20秒。 </li><li>转移晶圆第一“近期行动计划”。 </li><li>鼓动5秒。 </li><li>晶圆转移到第二国际音标。 </li><li>鼓动5秒。 </li><li>快速删除从第二届“近期行动计划”与“近期行动计划”半月板的晶圆和干燥，用N2 </li><li>检查模式。 </li><li> Hardbake晶圆在120 ° C为14-20小时，以交联SU - 8。 </li><li>骰子与钻石切割的晶圆</li><li>安装用环氧玻璃幻灯片的晶片</li><li> Fluorosilanize晶圆（见复制成型的指示） </li></ol>复制成型的mPADS 负模具 材料： <ul><li>一次性铝称量船</li><li>聚二甲基硅氧烷（PDMS）基和固化剂（道琼斯指数，美联，心肌梗死） </li><li> Razorblade </li><li>硅烷：（tridecafluoro - 1 ,1,2,2 - tetrahyrooctyl）- 1 -三氯硅烷</li><li>载玻片</li><li>玻璃巴斯德吸管</li></ul>方法： <ol><li>混合重量和德加10:1的PDMS在真空状态下1小时，以消除泡沫。 </li><li>将硅母模铝船。 </li><li>倒入50 - 60克以上掌握在铝船模的PDMS。 </li><li>德加下真空30分钟，以消除泡沫。 </li><li>吹掉快爆炸的N2在PDMS表面气泡。 </li><li>对流烤箱放置在船在110 ° C下15分钟，直到PDMS的是坚定的。 </li><li>切去铝船。 </li><li>随着razorblade，修剪了硅掌握下面的PDMS。 </li><li>皮尔的PDMS慢慢远离硅掌握和不断运动的一个方向，以避免破坏主。 </li><li>检查是否有损坏的硅主。 </li><li>切成4件负模具。 </li><li>重复步骤1-10为负模具一大批。 </li><li>氧等离子体处理1.5分钟激活表面负模具，或</li><li>臭氧处理10分钟，以激活表面负模具。 </li><li>放置在与mPAD暴露功能dessicator的负面模具。 </li><li>传播μL硅烷在玻璃幻灯片dessicator中心250-500 </li><li>广场150-200μL硅烷内dessicator提示牧场玻璃吸管。 </li><li>真空室，14小时蒸发硅烷和大衣的负面模具。 </li></ol> mPADS 材料： <ul><li>聚二甲基硅氧烷（PDMS）基和固化剂（道琼斯指数，美联，心肌梗死） </li><li>广口一次性转让移液器</li><li> 2“× 3”玻片</li><li>玻璃抄写</li><li>氮气</li></ul>方法： <ol><li>混合重量和德加10:1的PDMS在真空状态下1小时，以消除泡沫。 </li><li>花边硅烷负模具到“跨越” </li><li>移液器PDMS进入负模具使功能完全覆盖到厚度为1mm。 </li><li>等待30分钟，在PDMS任何气泡上升到表面。&lt;/ LI&gt; <li>在30&#39;的停机时间，灰尘22x22mm（第2号）的氮气流盖玻片。 </li><li>氧等离子体处理玻片半清洁和激活玻璃为1.5分钟</li><li>吹掉快爆炸的N2在PDMS表面气泡。 </li><li>玻璃盖玻片的边缘轻轻地躺在PDMS等离子清洗面临的PDMS方。慢慢地降低载玻片使PDMS的充分接触整个玻璃表面，并避免气泡载玻片下面。 </li><li>广场组装模具对流烘箱，在110 ° C 20小时完全固化。 </li><li>从烤箱中取出模具，让他们冷静到RT。 </li><li>握住一只手慢慢剥离负，顶部和底部的负面模具释放mPAD玻片。 </li><li>倒塌职位显微镜下检查mPAD。 </li><li>重新silanize通过血浆和硅烷每四个铸件模具。好复制的负面模具寿命大约是6-20蒙上。 </li></ol>到mPADS种子细胞 冲压 材料： <ul><li>为UCP的PDMS邮票（见田，J和陈，CS​​，在组织工程方法，2001年，第113-120）。 </li><li>乙醇（100％和70％） </li><li>灭菌去离子水</li><li> 50微克/ ml的无菌去离子水的人纤维连接蛋白（BD公司） </li><li> 5微克/毫升DII溶液过滤0.2微米的膜（D - 3886，分子探针，或） </li><li> 2％Pluronics F127（巴斯夫，橄榄山，新泽西州） </li><li> 1X的磷酸盐缓冲液。 </li><li> 100毫米× 25毫米培养皿（4） </li><li> 150毫米× 25毫米的培养皿（2） </li><li>铝箔</li><li>氮气</li><li>镊子</li><li>无菌遮光罩</li></ul>方法： <ol><li>修剪多余的PDMS从mPADs </li><li>如果PDMS邮票以前使用了100％的乙醇，超声5分钟擦洗污染蛋白的邮票。 </li><li>在无菌罩，保持新鲜的100％的乙醇和无菌水浸泡的邮票后，在其两侧的邮票镊子和干燥，用N2 </li><li>地点在150毫米的有工作的一面向上的Petri菜。 </li><li>每一枚邮票，分装滴纤维连接蛋白的解决方案。开始每个邮票上的每个角落，然后边，和最后的内陆地区。最初邮票的PDMS表面是疏水性，但变成亲水性蛋白质的吸附。 “外内”转变，使接触角量较低的纤维连接蛋白的解决方案是需要充分大衣邮票的PDMS。 </li><li>让我们坐1个小时，在蛋白质的吸附罩。 </li><li> DI水清洗浇菜，让水位上升在邮票的邮票。 </li><li>在100毫米培养皿充满DI水菜洗净邮票。 </li><li>取出干燥氮气。 </li><li>臭氧治疗mPADS 7分钟激活冲压，表面。 </li><li>在引擎盖下，印花税mPADS mPAD邮票的边缘，轻轻地降低，使充分接触。轻轻敲击邮票镊子顶部，以确保所有领域有良好的接触，但要小心，不崩溃的职位。 </li><li>用镊子小心取出邮票。 </li><li>淹没在100毫米的100％〜15秒的乙醇的Petri菜mPADS。转让过程中保持菜之间传输迅速从去湿微针。 </li><li>淹没在100毫米的70％〜15秒的乙醇的Petri菜mPADS。 </li><li>淹没在100毫米的培养皿〜15秒的DI水菜mPADS。 </li><li>淹没在第二个100毫米培养皿〜15秒的DI水盘mPADS。 </li><li>淹没在150毫米的DI水的Petri菜mPADS。 </li><li>淹没在150毫米的100毫升DII解决方案的Petri菜mPADS。 </li><li>盖用铝箔，浸泡1小时。 </li><li>淹没在100毫米的培养皿〜15秒的DI水菜mPADS。 </li><li>淹没在150毫米培养皿用10 mL 2％Pluronics和0.2％Pluronics，总浓度为90毫升的PBS菜mPADS。 </li><li>盖上盖子，浸泡1小时。 </li><li>淹没在100毫米的培养皿〜15秒的DI水菜mPADS。 </li><li>淹没在第二个100毫米培养皿〜15秒的DI水盘mPADS。 </li><li>淹没在第三个100毫米培养皿用PBS菜〜15秒mPADS。 </li></ol>播种 材料： <ul><li>选择单元格线</li><li>胰蛋白酶- EDTA（0.25％胰蛋白酶与EDTA•4Na，GIBCO猫号25200-056） </li><li>生长介质</li><li> 1X的磷酸盐缓冲液。 </li></ul>方法： <ol><li>在无菌罩，在100毫米的培养皿地方mPADS和填补与17毫升培养基。 </li><li>孵化器置于培养皿中菜预热至37 ° C </li><li>暂停通过胰蛋白酶的细胞</li><li>吸多次打破细胞团。 </li><li>吸取到与mPADS培养皿中的细胞悬液。加入，他们将仍然是单细胞分析时（无细胞 - 细胞接触）的细胞。细胞补充的确切数额是细胞类型的具体取决于细胞的扩散面积和增长率。 </li><li>轻轻缓慢pippetting分散在培养皿中的细胞。 </li><li>在孵化器放置2小时，让细胞来解决，并坚持到微针。 </li><li>检查mPADS如果细胞已经扩散。外观扁平细胞将跨越6个或更多的职位。 </li><li>如果细胞的扩散，转移mPADS新的100毫米培养皿用21毫升的新鲜培养基。 </li><li> mPADS准备为您的实验。 </li></ol>染色和安装mPADS 定影 材料： <ul><li>去离子水</li><li> Paraformeldahyde（16％）小瓶</li><li> 10X磷酸盐缓冲液。 </li><li> 1X的磷酸盐缓冲液。 </li></ul>方法： <ol><li>在50毫升离心管中，加入30毫升的DI，paraformeldahyde 10毫升（整个小瓶），和10倍PBS 4.5毫升的定影液。 </li><li>新增〜25毫升的定影液100毫米培养皿。 </li><li>轻轻淹没mPAD与细胞固定菜，让孵育20分钟。 </li><li>淹没在100毫米菜用1X PBS三次mPADS。 </li><li>免疫和安装准备就绪。 </li></ol>分析mPADS蜂窝力量 共焦成像 材料： <ul><li>共聚焦显微镜</li><li> 63X目的</li><li>二次抗体的筛选多维数据集</li><li>绿色过滤立方体（若丹明）DII成像微针。 </li></ul>方法： <ol><li>舞台上的地点mPADs，并找到300毫秒的曝光，没有分级的单细胞。 </li><li>微针顶端图像形象上衣。 </li><li>利用Z -控制读数，捕捉底部图像微针的图像。 </li><li>图片你有污渍（赫斯特，鬼笔环肽等）的任何其他渠道</li></ol>图像分析 材料： <ul><li> MATLAB图像处理工具箱</li></ul>方法： <ol><li>旋转和顶部和底部的图像</li><li>测量重心的底部（undeflected）和顶部（偏转） </li><li>计算的位移</li><li>使用的职位（已知，取决于后几何）弹簧常数，计算出每个职位的力矢量</li><li>误差分析的基础上，以解决部队的能力，取决于图像分辨率和质心的检测分辨率</li><li>排列的数据，为您的定制需求 - 跨不同的条件等作为一个时间过程，例如，力的大小和/或方向，许多测量</li></ol>

<ol>
	<li>Tan, J. L., Tien, J., Pirone, D., Gray, D. S., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+lying+on+a+bed+of+microneedles:+An+approach+to+isolate+mechanical+force.">Cells lying on a bed of microneedles: An approach to isolate mechanical force.</a> Proc Nat Acad Sci USA. 100, 1484-1489 (2003).</li><li>Sniadecki, N. J., Chen, C. S., Wang, Y. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Silicone+Elastomeric+Post+Arrays+for+Measuring+Traction+Forces+of+Adherent+Cells.">Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells.</a> Methods in Cell Biology - Cell Mechanics. 83, 313-328 (2007).</li><li>Lemmon, C. A., Sniadecki, N. J., Ruiz, S. A., Tan, J. T., Romer, L. H., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+Force+at+the+Cell-Matrix+Interface:+Enhanced+Analysis+For+Microfabricated+Post+Array+Detectors.">Shear Force at the Cell-Matrix Interface: Enhanced Analysis For Microfabricated Post Array Detectors.</a> Mechanics &amp; Chemistry of Biosystems. 2, 1-16 (2005).</li></ol>

microfabricated post-array-detectors (mpads): an approach to isolate mechanical forces

在这个视频中，我们将提出我们的方法来测量细胞牵引力使用微阵列的职位。通过肌球蛋白，肌动蛋白相互作用产生牵引力，并发挥着重要的生理作用。他们在发展过程中，使细胞从一个位置移动到下为了形成早期组织结构。牵引力量的帮助，在愈合过程。他们是必要的适当的伤口闭合或白细胞的迁移，并通过我们的身体爬行。癌转移或对肿瘤的血管生长的情况下，这些力量可以损害我们的健康。最常见的方法，通过它来研究细胞在体外已使用玻璃或聚苯乙烯盘。然而，承印物上的刚性，使得它无法测量细胞牵引力，并有相对较少的方法来研究牵引力。我们的实验室已开发出一种技术来克服这些限制。该方法是基于一个灵活的悬臂，刚度和大小规模，其中单个细胞遍布许多悬臂和转移的过程中垂直排列。我们使用的支柱是直径为3微米，10微米高，并定期在9微米的中心到中心间距阵列配置。但是，这些物理尺寸可以很容易地变化，以适应各种研究。我们开始用硅主，但最后的职位是由硅橡胶称为聚（二甲基硅氧烷），或硅橡胶。我们可以在显微镜下测量的挠度计算需要观察变形的牵引力量的大小和方向。我们呼吁这些基板微阵列后，探测器，或mPADs。在这里，我们将向您展示我们如何制造和使用mPADs以​​评估细胞收缩调制。

Watch this Scientific Journal Video about Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces at JoVE.com

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

在这段视频中，我们展示了如何制造和使用微加工后的阵列探测器（mPADs）评估细胞收缩的调制。

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

microfabricated-post-array-detectors-mpads-an-approach-to-isolate

Research

JoVE Journal

Biology

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

12.4K 次观看.  University of Pennsylvania. 在这段视频中，我们展示了如何制造和使用微加工后的阵列探测器（mPADs）评估细胞收缩的调制。

视频: Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

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

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

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

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

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

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...