这项工作是由美国国立卫生研究院R01 GM47607，CA163296和NSF 1334493至CET支持。我们感谢特纳实验室成员的手稿批判性阅读。本报告中显示的所有数据均来自Wormer 等2014 11转载许可。

1.准备Durotaxis商会<ol><li>为了制备1 6-孔组织培养板，去皮用50毫升锥形管中的平衡。称取约10克，在50毫升试管中的PDMS碱溶液中（该溶液是非常粘稠）。 </li><li>对于90:1的衬底（符合性〜1千帕），通过90分在管与PDMS碱溶液的测定重量来确定的交联剂溶液所需的正确的金额。与PDMS交联剂溶液的计算量添加到同一管中。 例如:10克/ 90 = 0.11克。添加0.11克PDMS交联剂溶液与PDMS碱溶液。 注意:基地和交联剂溶液在试剂盒的PDMS都提供。 </li><li>大力5分钟在RT使用小刮勺混合PDMS基/交联剂混合物。在这个阶段，混合物将含有大量气泡。 </li><li>离心机在台式离心机5分钟的PDMS衬底在50×g离心在RT以除去泡泡糖BLES。在5分钟，离心后重新如果仍有气泡。 </li><li>吸管1毫升90:1的PDMS衬底进入组织培养的每孔处理的6孔板中。存在于PDMS任何残留的气泡可以在本阶段通过使用21g的针弹出它们被消除。允许PDMS散布在井30分钟。 </li><li>煮沸12个毫米的玻璃＃1盖玻片在蒸馏水中5分钟。重复两次，并存储在蒸馏水盖玻片。 </li><li>放置一个，干燥盖玻片进入组织培养板的各孔中，轻轻触摸盖玻片一侧插入的PDMS溶液然后删除盖玻片上的PDMS。作为盖玻片沉降，与PDMS将开始侵占过盖玻片边缘，但不会完全覆盖它。这将产生固化后的PDMS和玻璃之间的界面（见图1A 中的B）。 </li><li>孵育板在70℃的烘箱中16小时以固化（硬化）的PDMS。放置高原E在细胞培养罩和紫外线消毒10分钟。 注意:最好是在使用了几天做出板。然而，板可以包在石蜡膜而没有任何缓冲，并储存在4℃达2周没有任何质量明显下降。 </li></ol> 2.细胞电镀注意:如果学习siRNA-介导击倒的效果，执行使用制造商的说明或所选择的细胞类型的优化的协议的击倒。 <ol><li>涂层的每个durotaxis室用1毫升10微克/毫升纤连蛋白在PBS无钙和镁的1小时，在37℃。替代地，纤连蛋白可以在天在4℃下施加在分析前通过将与PDMS用10微克/毫升纤连蛋白在PBS中16小时。确保整个表面浸没在在PBS溶液中的纤连蛋白。 </li><li>制备热变性1％BSA中。称取0.5克BSA，溶于50毫升的PBS。过滤消毒通过0.22微米的过滤器和热的溶液，在80℃下12分钟。 注意:准备该溶液与细胞电镀和储存在4℃下之前的一天。 </li><li>吸出纤连蛋白溶液和洗涤3次，用PBS洗涤。在PBS中添加1 ml热变性BSA的每个孔中，温育30分钟，在室温。 </li><li> Trypsinize和计数选择的细胞，而durotaxis室阻挡的热变性BSA。 </li><li>板1×10 5个细胞在一体积为2ml进入durotaxis室的每个孔中，使用所需的选择的特定细胞类型的介质。使细胞附着并在培养箱散布在基板上4小时，在37℃，5％的CO 2。 注:U2OS细胞例行维持在含10％FBS补充了2mM L-谷氨酰胺，1mM丙酮酸钠，和10国际单位/毫升青霉素，10微克/毫升链霉素。 注意:在本实施例中所用的细胞数目为优化U2OS细胞。可能需要优化的其他类型的细胞。该密度使细胞足够的空间，而无需与其他细胞相互作用显著迁移。 </li></ol> 3.活细胞成像<ol><li>在倒置显微镜下进行活细胞成像，使用了10X物镜相衬。该显微镜应装有一个封闭的环境，加湿室，允许温度的期间长期成像控制，在37℃和5％的CO 2。 </li><li>在细胞已经扩散约3.5小时，组装板显微镜室。使样品（多个）在腔室中平衡30分钟。 </li><li>设置自动，多点来访显微镜上（如果可用）。聚焦90之间的界面上:1的PDMS和玻璃盖玻片并选择各地，平均每durotaxis室40点的接口点图像。图像将细胞每10分钟达16小时。 注:重感兴趣只园将显示为两行以对应于盖玻片边缘和对应于PDMS和盖玻片之间的实际接口的内线路的外线路。参见图1B。 </li></ol> 4.数据分析<ol><li>生成的电子表格，如表1。 </li><li>算的交叉事件从PDMS从生成的每个电影的玻璃表面，反之亦然数。记录在Excel电子表格相应列的交叉事件的数量。 注意:交叉事件被定义为细胞核越过PDMS和玻璃在任一方向的边界。 </li><li>量化多个道口，计数细胞越过界面的次数。该号码应记录在对应于在其上电池位于电影结束基板excel的列。重复分析每一个跨越接口在T细胞他的电影。排除出视场的成像期间迁移的细胞。 注:同样重要的是，以验证，通过细胞在实验开始时算起，该细胞能够同样附着在纤连蛋白涂覆的PDMS和玻璃盖玻片表面。 </li><li>计算细胞的从PDMS迁移到玻璃表面（即，经历durotaxis）的百分比。添加渡从软到硬且倍数交叉事件，通过交叉事件的总数结束了硬和分裂事件的数目。 </li><li>通过将多个横跨的数目由交叉的总数计算的多个交叉的百分比。 </li></ol>

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The authors have no conflicts to disclose.

在此我们描述了一个简单的实验来研究durotaxis在细胞迁移。此法的主要优势是易于使用准备PDMS中durotaxis室的。基片的刚性可以通过改变碱的PDMS溶液的比率交联剂，以允许在测定中的各种刚性的研究很容易被操纵。然而，该系统中的一个潜在的限制是，细胞仅暴露于基板的刚性的单一变化，而 ​​不是经历由更复杂的系统4提供一个刚性梯度。重要的是，不象聚丙烯酰胺基质中，ECM组分不?...

细胞外基质（ECM）是由结构和交联蛋白，包括胶原蛋白，纤连蛋白和层粘连蛋白的一系列复杂的。虽然它是公认的对ECM提供了细胞组织重要的结构支承，有越来越多的证据表明，细胞积极应对物理变化在其细胞外基质环境来调节多种细胞过程，包括细胞存活，分化和细胞迁移。例如，在ECM的刚性差异能驱动间充质干细胞向不同谱系，具有柔软基材（〜1千帕）促进神经谱系而僵硬（〜25千帕）衬底促进成骨分化1。同样地，增加间质基质刚性已经显示出促进乳腺上皮细胞肿瘤的发生和侵袭到周围组织2,3。 这mechanosign的一个特别有趣的方面阿玲活性导致称为durotaxis，其中细胞优先迁移朝着更刚性基板4,5的处理。细胞不断通过整联蛋白受体感知它们的胞外环境的物理特性结合到ECM。这，反过来，促使许多结构性和信号蛋白的积累到其胞质结构域，以驱动被称为粘着斑或局灶性触点6,7-粘合剂结构的形成。 自整联没有固有的酶活性，信号是从ECM中继通过这些辅助蛋白来协调细胞的反应其变化的环境8。因此，参与调节mechanosignaling和durotaxis关键蛋白质的鉴定和表征是调查的一个重要领域。 各种模型系统已被开发，研究durotaxis 体外 ，但最有利用胶原包被的聚丙烯酰胺基质4。然而，聚丙烯酰胺基片的制备中可以是技术上具有挑战性并在这些试验中使用的胶原必须是化学交联到基底9。聚二甲基硅氧烷（PDMS）基片已被证明表现出的聚丙烯酰胺基片10可比机械性能。然而，PDMS衬底被通过简单地混合碱的比率来制备交联剂和这些基片可以涂有ECM蛋白无需化学交联，从而使PDMS一个更简单的工具，研究刚性对细胞行为的影响。在此，我们介绍如何编写一个简单durotaxis室，其中软PDMS基板集成了硬质玻璃盖玻片。 该法，如下所述，为研究durotaxis一种快速，简便的方法。在这项研究中，我们使用人类U2OS骨肉瘤细胞结合的siRNA介导cdGAP击倒研究在durotaxis 11本黏着蛋白的作用。重要的是，该协议可以很容易地适应个人的要求。其它细胞类型可被取代的U2OS细胞和任何蛋白质可以被击倒或过表达，以确定在durotaxis对细胞行为的影响。此外，该协议可以适于掺入荧光标记的蛋白来分析他们的动态和用FRAP行为或FRET方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

细胞外基质的成分和机械性能是组织类型之间高度可变。此结缔组织基质多样性大大影响细胞的行为，以调节正常和病理过程，包括细胞增殖，分化，粘附信令和定向迁移。在这方面，某些细胞类型的固有能力，以移向称为durotaxis较硬的，或更少的标准矩阵衬底。这种现象扮演胚胎发育，伤口修复和肿瘤细胞入侵期间起重要作用。这里，我们描述一个简单的试验来研究durotaxis， 在体外，用聚二甲基硅氧烷（PDMS）的基板。的描述durotaxis腔室准备创建相对软的PDMS凝胶和刚性玻璃盖玻片之间的刚性的接口。在提供的例子中，我们已经使用了这些durotaxis室展示为CDC42作用/ Rac1的GTP酶激活prote在，cdGAP，在mechanosensing和人类U2OS骨肉瘤细胞durotaxis调节。该测定是易于适应其它类型的细胞和/或利益的其他蛋白质的敲低，探讨在mechanosignaling和durotaxis各自的作用。

所述durotaxis室的示意图示于图1A。软PDMS衬底（90:1的混合物的PDMS碱与交联剂溶液）被铺在6孔皿，玻璃盖玻片放置在PDMS，然后部分地覆盖盖玻片的上表面的顶部上，从而产生一个接口不同的顺应的两个基板之间。软PDMS基板的刚性是〜1千帕，这与脑组织的典型合规性，而玻璃的刚性是大约1-2 GPA 1。所述PDMS和玻璃之间的界面的相衬图像显示由白色箭头在图1B。星号表?...

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A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.1K 次观看.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

视频: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

马赛克斑马鱼转基因增强子序列，以评估

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.