CG是由来自法新社法国国家（ANR-13-JSV1-0008），来自欧盟第七框架计划（居里夫人职业集成n˚8304162），由根据欧盟的地平线欧洲研究委员会（ERC）资助项目2020年研究和创新计划（ERC启动格兰特n˚639300）。

1.配体偶联磁珠注意:使用超顺磁性甲苯磺酰基活化的珠用直径2.8微米，进行配体缀合（原液浓度10 8珠/毫升，30毫克珠粒/毫升）。以下协议是基于约2×10 5个细胞，这对应于在一个60mm组织培养板上生长至80％汇合MRC-5细胞的样品。相应地，如果使用不同大小或细胞的板在不同汇流调整的珠粒和试剂的体积。使用顺序的超顺磁珠的量具有每单元2珠。因此，需要用于60毫米板4×10 5珠。 <ol><li>通过涡旋至少30秒彻底重新悬浮在原始小瓶的珠子。等分试样40微升的再悬浮超顺磁珠（相应于4×10 6珠）至1.5毫升离心管中。 </li><li> 放置在管上的磁性separati上支架以将磁珠从溶液中分离出来。弃去上清液，从磁性分离底座上卸下管和在1mL的0.1M磷酸钠pH 7.4的重悬珠子。 <ol><li>用1mL的0.1M磷酸钠pH 7.4的重复洗涤步骤两次。 </li></ol></li><li>最后一次洗涤后，重悬珠子于1mL的0.1M磷酸钠pH 7.4的。 </li><li>结合100微克牛纤连蛋白（FN）或任何其他细胞粘附受体配体以含有小珠的1毫升的0.1M磷酸钠pH 7.4的并通过移液混合。 </li><li>通过更换FN或用BSA，聚-D-赖氨酸或脱辅基转铁蛋白为阴性对照的任何其他特异性配体重复前面的步骤。 </li><li>任选地，用于凝胶分析，采取的配位体溶液中的10μL的等分试样对的交联效率分析，用浓的Laemmli样品缓冲液的适当体积混合。 </li><li>孵育为12-24小时，在37℃上的转子的配位体含有溶液珠。 </li><li>如果小珠的聚集体出现的反应发生，声处理（连续39功率W）不超过10-20之后一下。 </li><li>隔离用磁选支架上的珠子，吸出剩余的溶液，并添加1毫升的PBS / 0.2％BSA的pH 7.6的溶液。孵育在37℃下于转子1小时。 </li><li>用1毫升的PBS / 0.2％BSA的pH值7.6洗使用磁体珠两次。悬浮珠粒于1mL的PBS / 0.2％BSA的pH值7.6。 </li><li>超声处理珠子如果出现聚集不超过20-30秒。 </li><li>或者，删除10微升等分来分析免疫印迹耦合效率（用浓Laemmli样品缓冲液适当体积混合）。 </li><li>继续在4℃下对细胞测定或商店珠粒长达1个月。 </li><li>任选地，运行SDS-PAGE凝胶和染色用考马斯蓝给FN交联分析到磁性珠粒。 </li></ol>的张力对绑定到粘附受体在背配体涂层珠2.应用细胞表面<ol><li>在适当的生长培养基60mm组织培养皿培养贴壁细胞（通常的DMEM为4.5g / l D同时葡萄糖补充有FCS的10％），直至达到80％汇合。 </li><li>制备非变性非离子裂解缓冲液（20mM的Tris盐酸pH值7.6，150 mM氯化钠，2mM的MgCl 2的，0.1％的NP-40）和冷却的离心管中。 </li><li>从颗粒部分1.10，重悬涂配体珠1毫升的PBS / 0.2％BSA pH值7.6。添加涂布配体珠粒溶液至5毫升温暖的培养基和旋涡的100微升。 </li><li>至60mm培养皿中吸并加入5毫升辅以珠温暖的生长介质。 </li><li>孵育细胞培养条件下20分钟（37℃和5％的CO 2），以允许珠粒沉淀并附着到细胞中。关键的是要不超过20分钟，因为珠可以通过吞噬作用被内化。 </li><li>或者，观察使用10X或20X OBJE光学显微镜下珠莫如和微微颤抖的菜连接并漂珠区分检查珠附着力。 </li><li>同时保持培养皿在培养箱，交换为一个盖子正常盘盖与安装在该上表面上的圆38毫米钕磁铁。磁体代替由定位在盖的下表面两个较小13毫米钕磁铁保持。由于磁铁是非常强大的，小心操作它们。 </li><li>孵育进行在细胞培养条件张力所需的时间点（37℃和5％CO 2）的细胞。 </li><li>治疗后，置于冰上菜，小心吸从盘整个网上平台。 </li><li>添加细胞裂解缓冲液的300微升，并孵育在冰上10分钟。收集用细胞刮刀，并转移到预冷的1.5毫升微量离心管中的溶胞产物。 </li><li>沉淀用磁选支架磁珠和总细胞裂解物转移到一个新的预寒意编1.5毫升离心管中。在-20℃下用于进一步分析存储此级分。 </li><li>用1mL冰冷的裂解缓冲液洗涤珠粒3次。添加50微升Laemmli样品缓冲液至珠粒料，使用干块加热器5分钟，在95℃，通过移液和煮沸混合。这部分包含了孤立的粘连物。继续生化分析或储存在-20°C。 </li><li>从后珠粒分离（约300微升）中获得的总细胞裂解物，除去免疫印迹分析50微升等分试样。 注:左，可用于进一步的生化研究，如蛋白质 - 蛋白质相互作用的研究（免疫沉淀，GST下拉测定）或GTP酶活性实验的250微升（改变裂解缓冲液可以根据所关注的GTP酶是必要的）。 </li></ol>

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作者宣称没有竞争经济利益。

此处所描述的方法构成，以施加张力到细胞表面粘附受体，并允许其随后的纯化一个简单的方法。然而，一些步骤是进行高效粘附纯化的和潜在的优化可以做取决于目标粘附受体的关键。我们目前的用户可能会遇到下面的潜在问题。 我们使用直径2.8微米的磁性珠粒但较大的小珠可用于直径如4.5微米。然而，珠子直径应限制在2-5微米自吞噬可能在30-60分钟温育发生更快速和更大的珠具有更强的粘附性，使得胎圈的位移将根据磁场34,35受到限制。因此，培养时间限制到短周期和使用适当的珠尺寸是重要的。每个细胞培养的小珠的数量将影响f的量由单个细胞奥尔塞体验。而一个人想有效地刺激与张力的细胞，每个细胞过多珠可导致不相关信号途径的激活。我们通常孵育的每单元两个胎为FN包被的珠子的平均，但是该数量可以根据不同的细胞类型和细胞表面受体来调节[每单元1至5小珠。关于磁体，这里所描述的方法中使用的量上的2.8微米的合力已经测量（约30-40 PN磁体通过测量在未经稀释的甘油，牛顿液体与已知粘度31磁珠的位移，虽然它可以使用较大的磁铁，如果需要施加更大的力的量。要注意的是，这些磁体是非常强大和仔细操纵它们是重要的。 后12-24小时的温育与ECM配体（步骤1.7）以及后1小时的温育（步骤1.9），用PBS / 0.2％BSA的pH 7.6的缓冲液中，m个agnetic微珠可能形成聚集。它是那么关键分开珠粒尽可能尊重每单元2珠的比率。然而，它仍然是可以接受的一个5:1的比例。珠分离可以通过混合和移液，或通过超声处理来实现，虽然在短期内（20-30多个）。 各种蛋白质可以缀合到珠粒，包括整联配体（FN，胶原蛋白），重组蛋白质或抗体靶向特定的细胞表面受体。的涂覆配体磁珠于背细胞表面的附着可能是由配体的低耦合效率到珠粒被削弱。它建议检查配体通过向珠和在耦合过程结束的珠粒的等分试样之前收集稀释的配位体溶液的等分试样结合到珠子上。这些等分试样可被处理以SDS-PAGE分析和考马斯亮蓝染色。 如果没有变化信号通路或检测到预期mechanosensing过程中，必须考虑不同的可能性是非常重要的。识别问题的一种方法是调节与磁铁实验的持续时间。它公知的是细胞过程的速度取决于所用的细胞类型而变化。另一种选择是，以测试已知的紧张敏感的分子和检查免疫印迹的翻译后修饰（如磷酸化桩蛋白或FAK磷酸进行分析）。的力的量可以是也重要，可以用更厚的磁铁（相同的等级N52）或更大珠粒可以是一种选择。此外，配体/受体结合的效率可以通过分析附着力复杂的探索和寻找细胞表面受体（如钙粘蛋白或整合）。所述细胞汇合也可以影响到张力的细胞反应。因为已经观察到，细胞/细胞粘着可影响细胞的行为和cystokeletal预应力，它是important键注意，汇合可能影响到张力的细胞反应。尽管我们推荐张力基于整合素的粘连应用80％汇合，不同的条件可以优化实验系统进行测试。 虽然这种方法提供了一个有力的工具，以破译张力敏感adhesome以及相关的信号传导途径，它也有一些限制。首先，由于使用这样的小尺寸的磁性珠的主要关注的是由细胞的内化的危险，不能用于研究修改细胞命运诸如分化和增殖的长期机械敏感信令细胞应答此方法。另一个限制在于的力如何应用于细胞在培养板中的层的方式。的确，由于磁场是在磁体的周边总是较高，部队可以在培养板的表面与电池的减小梯度变化从外周延伸到中心，并有可能导致异质细胞应答。 此处描述的方法构成，允许研究机械敏感细胞途径以及调查受到张力粘附复合物的分子组成的简单的和成本有效的方法。其它方法，如拉伸适用循环应变于细胞的装置，导致紧张的应用到所有的细胞表面受体与胞外基质相互作用。此处所描述的方法具有能够与细胞表面受体的特定子集和大量的各种配位体的力的刺激的优点，可以使用如整合素配体或抗体的靶向细胞表面受体，从而使多个不同机械敏感系统的研究。这种方法的另一个优点是，它会导致蛋白质复合物在经历张力和对承重eleme工作纯化这是已知的NTS以机械传导36中发挥中心作用。纯化的粘附复杂，也可用于各种生化方法14，例如激酶测定法来调查响应于张力，或肌动蛋白聚合测定激酶活性。此外，本实验体系可以用磁镊子耦合到探索蜂窝机械响应，并与所识别的信号传导途径该响应相关联。有趣的是，magnetoplasmonic纳米颗粒最近已用于机械载荷的Notch和E-cadherin，在时间和空间37精确控制。这种新的发展可能有助于探索不同的空间，时间和机械投入机械敏感的信号通路。

在后生动物，机械张力通过细胞过程如增殖，分化和存活1,2无数的调节指导组织发育和内环境稳定。机械张力可以从细胞外基质产生，或可通过贴壁细胞，其样品通过拉动到细胞外基质，并通过张力敏感分子探测其刚性的肌动球蛋白收缩机械它们的胞外环境中产生的。为了应对紧张局势，机械敏感粘附蛋白发生触发复杂的信号级联反应的构象变化。反过来，这些信号传导途径编排一个mechanoresponse包含增殖，分化和存活是调节细胞行为到细胞外环境。这样的方法可以在短期时间周期进行结算（数秒至数分钟）快速反馈到机甲的环<html>通过修改机械敏感结构notransduction。例如，基于整联粘连加强在通过的Rho GTP酶-介导的细胞骨架重构3，4，5响应于张力。与此同时，其他的信号通路超过几小时或几天来控制的遗传程序，最终影响细胞的命运6激活。然而，许多研究强调矩阵刚度对细胞确定性和疾病发展1,2的效果，粘附介导的机械传导的精确分子机制仍然不清楚。 各种方法已被开发来研究细胞的行为的细胞产生的力或外力，包括流动系统，荧光共振能量转移（FRET）-tension传感器7的影响，小姑娘="外部参照"&gt; 8，柔性基板9，磁镊子，光学镊子10和原子力显微镜（AFM）11。在这里，我们使用超顺磁珠响应于施加到特定粘附受体的张力来表征机械传导途径提出的协议。超顺磁珠是放置在一个磁场时可逆地磁化的颗粒。一旦涂覆有对特定受体的配体，这些珠粒提供了有力的工具来研究细胞外力的应用程序的影响。此方法已被若干研究3，5，12验证- 17和呈现的优点在很大程度上促进在贴壁细胞的生化分析。使用类似的胶原包被的磁珠，随后通过生物化学分析，早期工作报告中的增加蛋白酪氨酸磷酸化和RhoA激活响应于张力5，18，19。下面描述的方法也被用于与纤连蛋白（FN）包被的珠从施加到整3张力下游表征信号通路。在这项研究中，Guilluy等。表明张力通过两个鸟嘌呤核苷酸交换因子（全环基金），LARG和全球环境基金-H1，以整合的粘附物招募激活的RhoA。因为，在其他的研究已经表明，GEF-H1被用不同的方法20，21，表现出这里描述的方法的稳健性招募到粘附物响应于细胞产生的张力。其结果是，活化的RhoA被证明促进粘附加固，通过细胞骨架重构。该系统也被用来探索张力施加吨O单元/细胞粘附受体。力到涂有诱导纽蛋白招募增加同样地整合伴生附着配合物12 E-钙粘蛋白的胞外域磁珠应用。柯林斯和他的同事观察到紧张的应用PECAM-1促进整合和RhoA的活性13。采用磁珠另一种实验方法是紧张适用于孤立的核研究。使用涂覆有对核包膜蛋白nesprin-1抗体珠，核膜复合物进行纯化，以显示它们响应于机械张力22动态地调节。这些结果支持在机械传导途径的研究该方法的有力度。此外，虽然流或牵引力系统刺激一般的细胞过程，磁珠特别通过使用受体配体靶向细胞粘附受体<sup系列="外部参照"&gt; 3或反对细胞表面受体13，15单克隆抗体。 这种方法的另一个优点是粘附复合物通过一个简单的配体的亲和纯化过程的隔离。这是众所周知的，加入的涂覆配体珠的细胞结合粘附受体和诱导几个粘附蛋白23的募集。力的涂覆配体磁珠进一步应用开启这些粘附配合成介导的各种张力依赖性信号通路4,24大分子平台。细胞裂解，随后通过使用磁铁珠浓度允许的附着平台的隔离。用于纯化粘附物的其它方法已经在贴壁细胞使用。他们结合化学交联，以节省蛋白质 - 蛋白质相互作用和细胞裂解步骤由洗涤剂和剪切流动或超声处理20，21，25，26，27，28。最后一步是含有粘合性复合物所得到的腹侧质膜的集合。不像这些方法，磁珠允许通过选择性靶向粘附受体的特定家族细胞粘附复合物的更大的纯化水平。磁珠已经用于纯化粘附络合物附着到涂覆配体的微珠29,30非贴壁细胞。下面模拟生物环境中描述的方法，其中施加短持续时间（数秒至数分钟）力。因此，它提供了一个有力的工具，调查两个纯化粘附复合物的分子组成和下游机械敏感-信号通路。 在这里，我们提出了一个详细的实验协议利用磁珠的张力适用于粘接面的蛋白质。永久钕磁铁被放置在培养皿表面的顶部。磁铁的磁极面被放置在6mm的高度，使得在单个2.8微米磁珠上的力是恒定的（约30-40 PN）31。张力刺激的持续时间是由根据兴趣和其时间尺度活化的分子上的操作者确定。将细胞最后裂解，粘附络合物通过珠粒分离利用磁铁纯化和生物化学分析被处理。这个协议包括的涂覆配体超顺磁珠的制备，和张力通过磁体的应用，随后通过生物化学分析。此外，我们还提供应用于基于整联-ADH数据表明张力的代表性样品esions诱导粘附重塑和改变蛋白质的酪氨酸磷酸化。

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the upper face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">DX88-N52</td>
			<td style="width:375px;">grade N52 dimension: 1 1/2&#34; dia. x 1/2&#34; thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the lower face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">D84PC-BLK</td>
			<td>grade N42 dimension: 1/2&#34; dia. x 1/4&#34; thick Black Plastic Coated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dynabeads M280 Tosylactivated</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">14203</td>
			<td>superparamagnetic beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DynaMag-2 Magnet</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fibronectin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">F1141-5MG</td>
			<td>Fibronectin from bovine plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Poly-D-Lysine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">P7280-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Apo-Transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">T1428-50MG</td>
			<td>Bovine Apo-Transferrin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">A7906-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">DMEM high glucose, GlutaMAX supplement, pyruvate&#160;</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:103px;">31966-021</td>
			<td>DMEM+GlutaMAX-I 500 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60*15 mm culture dish</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:103px;">353004</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

机械敏感细胞表面粘附物使细胞感测与其周围环境的机械性能。最近的研究已经确定了在固定区域既力传感分子和调节谱系特异性基因表达和驱动表型输出力依赖性转录因子。然而，信令网络转换机械张力成生化途径仍然难以捉摸。探索时施加到细胞表面受体的机械张力接合的信号传导途径，可以使用超顺磁性微珠。在这里，我们提出了一个协议利用磁珠力量应用到细胞表面粘附蛋白。使用这种方法，可以通过安装在涂覆配体珠粘附复合物的磁隔离，调查通过各种生物化学方法不仅力依赖性细胞质信号传导途径，而且粘附重塑。这个协议包括配体 - 共聚的制备ated超顺磁珠，和应用程序中定义的拉力接着生物化学分析。此外，我们提供的数据表明应用于基于整合素的粘附张力的代表性样品触发粘附重塑和改变蛋白质的酪氨酸磷酸化。

该技术的原理在图1a中示出。以下的配体缀合，磁珠与细胞孵育20分钟，然后一个永久磁铁用于施加约30-40 PN的拉伸力对各种量的时间。 图1b示出了2.8微米势必MRC5细胞粘附受体的FN包被的磁珠。 细胞裂解后的超顺磁珠洗涤步骤是关键的，并确定纯化的程度。建议三次洗涤最低。 GADPH免疫印迹与长时间曝光可以测试粘附复合物的纯度（图2a）是有用的。 FN包被的珠子被用来研究响应于张力的粘附物随时间发生的机械传导过程。的粘合复合馏分的磁性分离后，将裂解物和粘合复合馏分被分析免疫印迹。正如预期的那样，我们甚至在不存在机械性刺激（图2b）的观察到的密合性复合馏分踝蛋白，纽蛋白和桩，但不是GAPDH。与以前的报告一致的32，33，紧张引发纽招聘粘连物。而张力没有影响桩蛋白招募粘附配合物，其对酪氨酸31磷酸化响应于两者中的总细胞裂解物，并在粘附复杂馏分机械张力增强。 <img alt="图1" src="/files/ftp_upload/55330/55330fig1.jpg" /> 图1的方法的说明。 （ 一 ）技术的示意图。细胞在培养基培养首先培养，直至达到所需的汇合。然后，超顺磁性珠加入15-20分钟。的张力约30-40 PN是然后使用不同量的时间校准磁体应用。 （B）的纤连蛋白包被的超顺磁珠结合细胞粘附受体。细胞相差在透射光镜15分钟，在培养基中加入超顺珠（白色箭头）后成像。 （上图:比例尺= 25微米，底部图像:比例尺= 100微米） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55330/55330fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/55330/55330fig2.jpg" /> 图2.机械张力引起粘连成熟。 （A）的粘合物的净化。 GAPDH的免疫印迹用作总细胞裂解物上样对照，并检查粘附复合物的纯度。硝酸纤维素膜已使用长时间曝光来证明日成像E在附着力复杂的分数没有信号。 （B）张力引起粘连成熟。装载控制（GAPDH）和已知响应机械张力被招募或粘连物磷酸候选人（纽蛋白，塔林和桩蛋白）的免疫印迹。此实验对MRC5细胞被执行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55330/55330fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

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

细胞表面粘连是在机械传导的中心，因为它们传输机械张力，并开始参与组织稳态和发展的信号通路。这里，我们提出了解剖被响应于张力激活生化途径，使用包被配体 - 磁珠和施力到粘附受体的协议。

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 ...

analyzing-cell-surface-adhesion-remodeling-response-to-mechanical

Nanyang Technological University

Research

JoVE Journal

Biology

8.4K 次观看.  Centre de recherche UGA - INSERM U1209 - CNRS UMR. 细胞表面粘连是在机械传导的中心，因为它们传输机械张力，并开始参与组织稳态和发展的信号通路。这里，我们提出了解剖被响应于张力激活生化途径，使用包被配体 - 磁珠和施力到粘附受体的协议。 

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

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用:脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

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 ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7细胞单层中细胞基质粘附面积和细胞形状分布的定量

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...