1。跑步机在小鼠模型揭示肌肉损伤的程度上运行<ol><li>建立运行协议期间使用的跑步机表面的角度。一般来说，平级或7 °和15 °度之间的角度下坡或上坡。一些跑步机有不可分割的仪器来调整倾斜，而有些则需要通过其他方式升高的跑步机。 </li><li>前放置一个托盘下面的跑步机或蓝色实验室垫在跑步机上的动物在运行协议的任何废物收集动物。 </li><li>运行小鼠前应驯化跑步机的环境。这涉及到电网和皮带驱动电机，但带不动（即定为0米/秒的速度），放置15分钟，在跑步机上的动物。 </li><li>打开上的动机电网。通常可以使用脉冲的强度和频率上跑步机控制，但变化从一个制造商。一般来说，最大强度是不需要激励小鼠运行，高频率（至少每两秒一次），同时将提高遵守。 </li><li>激活的跑步机带开始运行的协议。一个约5米/分的初始的速度应该用于为小鼠的热身期。可以加速跑步的速度缓慢，通常加入1-2米/分钟，每分钟在跑步机上开始。 </li><li>动物在大多数协议的合规性，可以提高运行实验开始前，在温暖的速度为3至10天进行一系列的短期运行（5分钟）。 </li><li>急性衰竭运动通常会涉及增加跑步机的速度随着时间的推移，直至最高速度（一般&lt;30 M / M），是实现和保持，直到小鼠显示疲惫的迹象。用尽判断标准各有不同，但一般包括鼠标支出电网或电网连续5秒，而无需返回到跑步机表面上超过一半的时间。一个单独的鼠标是用尽后，它可以从跑步机可以记录和运行的总时间。 </li><li>改变跑步机的角度，可用于急性运行试验，增加运动负荷（上坡角度）或诱发破坏性的离心收缩（有一个下坡的角度），肌纤维撕裂的细胞膜膜。伊文思蓝染料可以注入动物前使用作为一个组织学染料，以确定受损的肌肉纤维 2,3程序。 </li><li>运行协议的耐力训练运动负荷的动物提供了一个长时间（个月），以生产的肌肉或心血管系统的重塑。热身过程与上面相同（步骤1.5），但最高速度一般较低，运行时间可能很长（&gt; 1小时）。 </li><li>完成运行小鼠后可以返回他们的笼子。跑步机通常会变得非常脏，而老鼠运行，特别是长期协议。这是必要的，通常用70％乙醇的喷雾，每次使用后清洁跑步机。 </li></ol> 2。孤立的肌纤维膜的维修能力的体外检测，经过紫外线激光损伤<ol><li>解剖，从鼠标脚的趾短屈肌（FDB）肌肉的表面层。首先，削减中线唯一开放的皮肤，同时注意不要破坏下面的肌肉，然后进行横向削减和消除皮肤。可以看出FDB肌肉（近端肌腱）的白色平面肌腱连接到跟骨骨。说白了单独由钳肌腱割断肌腱附着部位接近，抢钳自由肌腱，轻轻把它从周围组织中分离出来的FDB同时切割保持连接的结缔组织。见状远端肌腱分支FDB可能削减这些肌腱连接到更深一层的肌肉中删除个别数字。 </li><li> FDB肌束把1毫升的胶原酶溶液预先加热至37 ° C在1.5 mL Eppendorf管，胶带管在37℃轨道摇床的水平方向和摇晃65分钟200转。孵育时间可能会有所调整，以确保有足够的消化，这是由磨损的外观和肌肉苍白色指示。 </li><li>小心地转移到1.5μL离心〜600 2.5的Ca 2 +台氏液管通过1毫升吸管与切端，有一个直径足以让消化捆绑轻松传递无中断的一角消化FDB包。轻轻翻转试管撼动从松散的纤维束。 </li><li>剪下30μL枪头的diameter仅略高于肌束小（15至 - 直径在20微米）使用移液器吸管轻轻划到捆绑，然后推入解决方案。重复此过程，直到大部分的纤维脱离捆绑。 </li><li>充分混匀，轻轻转动管脱钩纤维，玻璃底三角洲T盘上采取所需的金额和下降。隔离是有用的，需要在一个特定的协议盘纤维的数量效率的基础上，加载量将有所不同。剩下的纤维可以储存在4 ° C约6小时额外的研究，在管。 </li><li>允许的菜，坐了5分钟不受干扰。这使得纤维坚持以玻璃底部的菜。 </li><li>将配备的紫外激光共聚焦显微镜上的玻璃见底菜。 &gt; 100X倍率相差显微镜下观察纤维检查存在一个正常的条纹图案和一条直线状杆。 </li><li> FM1 - 43或4 FM4 - 64 styrylpyridinium染料添加到终浓度为2.5微米5解决方案。 </li><li>为了诱导损伤纤维的位置，它在成像窗口，使纤维为导向，在一个45 °角，从右下角的视野（图1）左上方。照射5x5的像素使用紫外激光（80兆瓦或更大，364分之351纳米）设置为5秒的最大功率的等离子体膜的面积（约0.9μmx0.9μm）。照射区域应分开质膜;就是5x5的方块的一半应纤维内部，而其余的应该是外​​纤维（图1）。 </li><li>捕捉到每5秒的时间间隔纤维的染料进入长达5分钟的图像。 </li><li>重复步骤菜不超过3％纤维2.8，荧光染料，最终将被吞噬成纤维和复杂化的任何数据分析。 3纤维后，应准备一个新菜，从步骤2.5。 </li><li>分析，通过计算每个捕获帧之间的荧光强度的变化（ΔF/ F 0）测量量的染料进入的数据。这需要测量的一个面积约200微米的平均荧光 2使用分析软件，如公开ImageJ（http://rsbweb.nih.gov/ij/ ）。纤维之间的比较，下面的计算，应为每个帧：ΔF/ F 0，其中f 0是感兴趣的区域的平均荧光，在第一个捕获帧（T = 0;之前受伤），和ΔF在其后每帧的荧光变化（FF） 。 </li></ol> 3。活细胞共聚焦成像监视器细胞膜修复的动态过程<ol><li>从PYREX毛细血管微量移液器。毛细管被放置在一个微量拉马，由预先设定的程序（热= 695;拉= 50;的Vel .= 55;时间= 250）拉。 </li><li>微管连接到一个3轴（XYZ）显微。 </li><li>细胞转染质粒DNA使用正常的方法在玻璃底部三角洲T菜。媒体应切换到2.5MM的Ca 2 +台氏缓冲在实验开始前。 </li><li>玻璃菜含有转染细胞分别置于激光扫描共聚焦显微镜40X 1.3NA油浸物镜。 </li><li>活细胞急性损伤是由微管插入细胞膜，然后迅速收回的微量细胞3,6。连续活细胞图像获得间隔为1.54秒/帧。 </li><li>皂甙诱导的膜破坏试验，在2.5mm 的钙皂甙0.005％（Sigma公司）+台氏缓冲液灌注重力流定制组装灌注系统 7 。灌注率约1毫升/分钟和灌注提示应直接放在上述成像细胞。 </li></ol> 4。代表性的成果： 一个鼠标的跑步机上跑步的代表电影，可在拍摄过程中。这部影片将说明的降低运行能力的mg53 - / -小鼠由于骨骼肌损害。 上面详细的协议紫外激光损伤造成的损害程度取决于分析纤维膜的修复能力。这种能力是影响纤维分离的鼠标的基因型，外条件，和蛋白表达（转染或感染）的任何改变。一个正常的野生型纤维将允许轻度至中度染料项目（图2a）。肌肉纤维派生从mg53 - / -小鼠膜修复能力受损，会显示更重要的染料进入纤维（图2b） 。 典型的结果显示电极损伤易位MG53含vesiCLES到损伤部位。图3a和3b显示GFP - MG53转染C2C12肌管插入到细胞膜上的微量损坏。在微管损坏前的细胞，GFP - MG53是位于细胞膜和细胞质 3 。微量渗透，GFP - MG53含有囊泡移向损伤位点（箭头表示）。图3c所示的GFP - MG53转染C2C12成肌细胞治疗与0.005％的皂素，清洁剂，可以通透质膜。皂甙治疗后，GFP - MG53从细胞质易位到细胞膜。 <img src="/files/ftp_upload/2717/2717fig1.jpg" alt="图1"/> 魏斯勒德，等。图1。光纤对准和损伤区域定义。所示的伤害地区的观点和定义领域内的孤立的肌纤维的正确方向。 <img src="/files/ftp_upload/2717/2717fig2.jpg" alt="图2"/> 魏斯勒德，等。图2。紫外激光损伤的FDB光纤图像显示纤维损伤（0秒，左）和200秒伤后（右）。从野生型小鼠（一）的纤维显示轻伤。膜修复受损的小鼠的纤维（二）让更多的染料进入。 <img src="/files/ftp_upload/2717/2717fig3.jpg" alt="图3"/> 魏斯勒德，等。图3。微电极和皂甙培养细胞的损害 。图像显示C2C12成肌管（A，B）和一个C2C12成肌细胞的损伤（以上）和伤后（下）（三）前。用微电极（A，B）受伤的细胞显示MG53移位到损伤部位（箭头）。皂素（C）处理的细胞显示MG53易位到细胞膜。

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跑步机上跑步是一种有用的方法治疗的动物提供一个运动负荷。作为测量肌肉的功能或续航能力方法，它是出了名的嘈杂的方法，是难以执行， 一贯重现时尚8。在一般情况下，消耗体力的时间可以被用来作为终点，以解决重大的实验组之间的差异，如之间的一些基因敲除小鼠的线条和野生型小鼠9。产生更微妙的差异，如一些药品试验，实验操作，可能不会解决上述这种技术带来的噪音的差异。为了最大限度地发挥这些技术的重复性和灵敏度，重要的是保持在会话的条件非常密切。一天的时间行使的动物，以及作为热身期间使用条件，都是重要的因素，从审判审判应保持一致。 应变效应可以跑步机协议的设计上有重大影响，跑步机比其他品系小鼠的某些菌株可以运行更多的时间和不同的反应耐力运动10。作为一般指引，许多小鼠将能够运行在跑步机的速度不断的增加（5 M / M 1 M / M的初始速度为每分钟后开始添加）在用尽研究200-300平方米。对于耐力运动的老鼠可以运行9日至12日之间的M / M 2或30分钟，每周3次。然而，个别的研究，通常会需要一些协议，以配合个别的实验需要优化。 紫外激光损伤过程已被证明是一个有效的方法用于测量骨骼肌纤维膜3,6,11修复能力。这些实验的成功的一个重要组成部分，是只使用肌纤维显示一条直线，杆形美观的条纹的规律和平稳，不会出现皱纹肌膜。如果选择其他的肌肉纤维膜破坏，可能已经在这些纤维和从这些实验结果的解释可能是复杂的。同样重要的是，在图1中定义的纤维方向和照射区域的定义。这提供了一个重复性大小的损伤，从而使纤维之间的比较。如果损伤定义的区域内完全在于纤维，内伤将被创建，而不是破坏肌膜。此外，ΔF/ F 0的计算值是非常敏感的地区平均荧光测量选定的利益。面积大约为200μm2，和包括损伤部位附近，是适当的。受伤定义的区域不同，这方面应该完全躺在内的纤维。如果包括纤维以外的空间，由此产生的空白区域将增加小染料进入纤维 ΔF/ F 0，同时降低更严重的表型纤维ΔF / F 0。这降低了检测的灵敏度，从而使纤维间的比较困难。 对于微量的损伤检测，之间的微管和玻璃底菜的角度应约45度，为了有效地破坏细胞膜。被选为细胞微管损坏，应紧紧地贴盘底部和圆润的细胞不应该，因为最有可能，他们将分离后微量戳。皂素permeabilzation实验技术要求高，相对更快地执行比较微量渗透检测。不过，也有一些局限性皂素的损害，包括只有一个细胞，每皿可用于以来皂素会扩散并破坏其他细胞，菜。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>鼠类跑步机</td><td>哥伦布仪器公司行使3 / 6或同等</td><td></td></tr><tr><td> 70％的乙醇</td><td> ISC的Bioexpress </td><td></td></tr><tr><td>解剖工具，包括精尖钳，弹簧剪刀</td><td>世界精密仪器</td><td></td></tr><tr><td> 2 mg / mL胶原酶I型</td><td>西格玛</td><td></td></tr><tr><td> 0的Ca 2 +台氏缓冲液（140毫米氯化钠，氯化钾5毫米，10毫米的HEPES，2毫米氯化镁 2，pH值7.2，290 mosm ） </td><td>西格玛</td><td></td></tr><tr><td> 2.5MM的Ca 2 +台氏缓冲液（氯化钠140毫米，2.5 毫米氯化钙，氯化钾5毫米，10毫米的HEPES，2毫米氯化镁 2，pH值7.2，290 mosm ） </td><td>西格玛</td><td></td></tr><tr><td>温度可控的轨道摇床</td><td>新不伦瑞克省或同等学历</td><td></td></tr><tr><td>三角洲赤芍菜</td><td> Fisher Scientific则</td><td> 1207133 </td></tr><tr><td> LSM 510企业80兆瓦的紫外线激光或有同等作用的共聚焦显微镜共聚焦显微镜</td><td>蔡司</td><td></td></tr><tr><td> FM1 -​​ 43或FM4 - 64染料</td><td> Invitrogen公司</td><td></td></tr><tr><td>高硼硅毛细管尺寸0.8-1.0 × 100毫米</td><td> PYREX </td><td>零件编号9530-2 </td></tr><tr><td>微管拉拔</td><td>萨特仪器</td><td>模型P - 97 </td></tr><tr><td> 3轴显微</td><td> Narishige </td><td> MHW - 3 </td></tr><tr><td>光辉2100激光扫描共聚焦显微镜或相当于显微镜</td><td> BioRad公司</td><td> MHW - 3 </td></tr><tr><td>皂素</td><td>西格玛</td><td> 47036 </td></tr></tbody></table>

visualization of mg53-mediated cell membrane repair using in vivo and in vitro systems

急性损伤细胞膜的修复是一个正常的细胞生理学的元素的过程中，已与许多退化的人类疾病和有缺陷的膜修复。最近发现的膜再加盖印章机器的重要组成部分MG53允许以一个更好的组织修复的基础生物学，以及在再生医学的潜力翻译应用分子了解。 ：我们在体内的MG53的功能，在使用跑步机上锻炼的小鼠模型协议，用于测试测量到孤立的肌纤维的染料进入体内膜的修复能力，肌肉损伤的修复，探索和监控的动态过程详细的实验协议MG53 -介导的囊泡贩运和使用活细胞共聚焦显微镜培养细胞的细胞膜修复。

Watch this Scientific Journal Video about Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems at JoVE.com

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

这里描述的是MG53介导的细胞在整个动物的膜修复，并在细胞水平上的动态过程可视化的协议。这些方法可以适用于调查的质膜再加盖印章和再生医学细胞生物学。

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

visualization-mg53-mediated-cell-membrane-repair-using-vivo-vitro

Research

JoVE Journal

Biology

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

12.3K 次观看.  Robert Wood Johnson Medical School. 这里描述的是MG53介导的细胞在整个动物的膜修复，并在细胞水平上的动态过程可视化的协议。这些方法可以适用于调查的质膜再加盖印章和再生医学细胞生物学。

视频: Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

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

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

前体mRNA底物的3&#39;末端切割：用无细胞体系中的RNA加工反应分析<em&gt;在体外</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.