工作是我们的实验室是由美国国立卫生研究院的赞助支持。

制备<ul><li>盖玻片应高压灭菌。 </li><li>应使用无菌蒸馏水或去离子水，准备解决方案和洗涤盖玻片。 </li><li> AC（40％W / V）和双AC（1％W / V）的解决方案是0.2微米的过滤消毒。不久之前使用无菌过滤，准备10％过硫酸铵（APS;100μg/ml水）。每月更换的APS解决方案。 </li><li>不能进行高压灭菌，如3 APTMS，氯仿，戊二醛，NHS的，并SurfaSil化学试剂均保存在分配瓶仅用于水凝胶的制备。 </li><li>凝胶为了达到最佳效果，应利用适当的ECM蛋白孵育过夜后一两天内。 </li><li>在氯仿（10毫升通常足以供20个顶级盖玻片）的10％SurfaSil解决方案之前准备在50毫升聚丙烯猎鹰管的水凝胶的浇筑。 （我们的实验室通常siliconizes在0.5％戊二醛孵化步骤盖玻片）。添加盖玻片猎鹰管和岩石至少10分钟。倒出SurfaSil解决方案和空气干燥凝胶将准备在生物安全柜的盖玻片上Kimwipes。 </li><li>准备热灭活的BSA溶液如下：PBS中的自由脂肪酸的牛血清白蛋白20毫克/毫升的解决方案是在一个68℃水浴30分钟孵育。解决的办法是无菌过滤，并保存在4 ° C </li></ul>程序<ol><li>广场上的一个150毫米的培养皿底部的半层的封口膜。 </li><li>广场9 25毫米的封口膜顶部盖玻片覆盖1毫升0.1 M的氢氧化钠。孵育3分钟，然后吸真空线。 </li><li>在化学罩，放置0.5毫升3 APTMS每个盖玻片工作。孵育3分钟，然后吸APTMS。如果等待时间过长，会形成泡沫。 </li><li>在同一盘菜用20 ml去离子水冲洗盖玻片一次。菜用弯钳取出盖玻片和转移，其治疗面朝上，一个新的150毫米盘。盖玻片，摇杆去离子水清洗三次，每次洗10分钟。如果你不能消除所有的APTMS，它会在下一步与戊二醛反应，留下白色阴天的沉淀物（图1）。 </li><li>解冻后的戊二醛（约10分钟），使用前。 </li><li>用弯钳，转移到一个干净的菜用封口膜分层的盖玻片和吸任何剩余的液体。剩余的水，需要使用真空线或Kimwipe印迹。 </li><li>完全相互覆盖盖玻片，用0.5毫升0.5％戊二醛灭菌去离子水孵育30分钟在化学罩。吸戊二醛。冲洗和洗的盖玻片，在第4步。完全干的盖玻片，[你可以停在这里，在干燥的地方离家数周的盖玻片 ] 。 </li><li>当你准备准备的水凝胶，用弯钳，盖玻片，无朝上，转移到一个表的封口膜生物安全柜的表面，一直到录音。确保在盖玻片上的封口膜表面的平坦。 </li><li>准备在甲苯饱和NHS的解决方案。 （在特定的实验足够甲苯溶解少量的NHS的不断增加位NHS位直到NHS的就不再溶解。饱和溶液通常是阴天和粉红色。） </li><li>下一步，准备丙烯酰胺，双丙烯酰胺，水和APS，以达到所需的丙烯酰胺的百分比。在离心管中添加如下所述总量的0.8毫升试剂。 </li><li>然后，一次一个等份，加入NHS和TEMED 0.8毫升交流解决方案，涡简要立即倒入3-5凝胶在生物安全罩的使用140μL盖玻片。只要有可能，这一点从所有步骤应在生物安全柜。 [注：根据需要，可以利用不同大小的盖玻片。如果使用18毫米盖玻片，用〜33μL每盖玻片AC解决方案和一个18毫米的顶级硅化盖玻片的。] <table border="1"><tbody><tr><td></td><td colspan="4"><div align="center"> 双AC（％） </div></td></tr><tr><td></td><td> 0.3（僵硬） </td><td> 0.15 </td><td> 0.06 </td><td> 0.03（软） </td></tr><tr><td></td><td> μL </td><td> μL </td><td> μL </td><td> μL </td></tr><tr><td>水</td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td>交流</td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td>双AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS</td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS的</td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li>快速放置硅化25毫米盖玻片每个凝胶的顶部，然后才开始聚合。顶部盖玻片除应允许交流完全覆盖底部的盖玻片。在室温下孵育这种“三明治”，直到AC聚合。 （检查的离心管中残余的交流解决方案，以确定何时发生聚合。僵硬凝胶通常几分钟，一个柔软的长一点就足够了。） </li><li>小心地拿起三明治（无菌手套）和滑落，直到它出挑的聚合凝胶的顶端盖玻片。然后，您可以撬起它的凝胶。如果等待时间太长才卸下顶盖盖玻片，凝胶将RIP盖玻片被删除。 </li><li>丢弃顶部盖玻片。放入2毫升PBS / 6孔板底部凝胶盖玻片（以下简称水凝胶）。 PBS可以添加之前或之后凝胶盖玻片每口井。 PBS洗3次冲洗水凝胶，摇杆，每洗5分钟。 </li><li>重复步骤11-14，已准备，直到所需数量的水凝胶。 </li><li>覆盖每一个纤维连接蛋白的解决方案（3微克/毫升的PBS）或其他ECM蛋白（Klein等详细信息，13。）2毫升水凝胶。 ECM蛋白成为在4 ° C共价键结合在孵育过夜水凝胶</li><li>吸的ECM解决方案和块1mg/ml的热灭活脂肪酸免费BSA的无血清培养基与未反应的NHS的至少30分钟，在37 ° C在细胞培养孵化器。一次冲洗水凝胶，用无菌PBS或细胞培养液中。 </li><li>板细胞在适当的文化含有胎牛血清的介质。应由用户根据细胞扩散和进行实验所需的汇合程度的凝胶种子细胞的数量。约10 5个细胞通常是足够的免疫印迹和定量PCR分析。 </li><li>潜伏期后，细胞蛋白或mRNA可以被提取。小心取出的盖玻片的井，用弯钳和100μL裂解液已间距沿实验室的长椅上的封口膜表（2 - 3cm的）的液滴上放置（细胞面朝下）。 （我们使用标准的SDS样品缓冲液和TRIZOL，分别准备样本免疫印迹和RNA的定量PCR隔离。）用了整整一分钟的裂解液培养的细胞。取出盖玻片和裂解液转移到离心管。或者仅适用于RNA的提取，用户可以转移到一个新的6孔板每个凝胶，并添加TRIZOL /1毫升。孵育3分钟，并删除存储在离心管TRIZOL解决方案。 </li></ol>如免疫程序的附加信息，BrdU染色，染等，对水凝胶的种子细胞是在克莱因等。 2007 13。 代表性的成果彻底清洗的盖玻片APTMS除了产生“反应”的盖玻片的重要一步。如果一个人不能完全消除的APTMS，它会在下面的步骤与戊二醛反应和产生的白色阴天沉淀物，如在图1A所示。图1B显示正确的洗涤，干燥，盖玻片。如果沉淀物的发展，整个过程必须从头开始重新盖玻片不再可用。 水凝胶的形成及与ECM蛋白一夜之间涂层，种子细胞可翌日。如图2所示，硬与软凝胶的细胞扩散之间有一个明显的区别。可以看出，在小鼠胚胎成纤维细胞（MEFs）由鬼笔环肽染色细胞的扩散就僵在更大程度上较软凝胶。事实上，大多数细胞附着的软凝胶会保持紧凑，重视效率较低。 虽然只有MEF的形态，如图2所示，在细胞扩散的差异是一致的其他几个细胞株测试 11-12,14 。 成功的秘诀<ol><li>一旦程序启动时，它是非常重要的，保持与“功”的一面的盖玻片，并记住哪一方被涂。 </li><li>手套应戴在任何时间，在手术过程中提供的工作环境，也就是尽可能无菌。 </li><li>大多数的最初步骤执行之间的化学通风柜，实验台。所有步骤，随后从盖玻片删除多余的戊二醛应执行下一个生物安全柜。 </li><li>由于细胞中的差异蔓延（图2），我们建议僵硬的水凝胶相比，播种软凝胶的细胞数量的两倍。 </li><li>交流聚合过程是非常快。对于首次用户，可以减少量的APS或TEMED的解决方案，以延长聚合过程中。不要尝试一次准备多几个盖玻片。 </li><li>在G0同步需要在细胞周期研究中，我们通常血清饿死在塑料培养皿中的细胞，trypsinize细胞和补种不同符合水凝胶中存在的有丝分裂原（FBS和/或生长因子）。这个过程可以确保人口的G0同步细胞开始在所有样品相同。然而，对于许多信号转导的研究，所需要的有丝分裂原刺激期间可能没有足够长的时间，让细胞附着和传播。在这种情况下，我们血清饿死上的水凝胶的细胞，然后直接与有丝分裂原刺激。 </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="图1a" /> 图1A。不当洗涤盖玻片。APTMS此外，盖玻片洗涤1-2分钟，除了之前的戊二醛溶液。沉淀形式尚未洗净根据在程序中所述的步骤盖玻片。 <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="图1b" /> 图1b。洗净的盖玻片 。APTMS以下此外，盖玻片前三次，每次10分钟另外的戊二醛溶液冲洗。无沉淀形成的盖玻片上。 <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="图2" /> 图2。不同刚度。凝胶细胞形态成立MEFs 9个小时的高或低刚度的纤维连接蛋白涂层凝胶种子。潜伏期后，细胞固定，透和F -肌动蛋白结合的FITC -鬼笔环肽染色。高刚度凝胶MEFs表现出应力纤维相比，低刚度凝胶种子的传播。比例尺为50μm。 <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="图3" /> 图3。代表鼠标cyclin D1的mRNA水平的定量PCR结果血清饥饿小鼠胚胎成纤维细胞接种到高（3％丙烯酰胺）或低（0.3％丙烯酰胺）刚度凝胶和10％的FBS为9小时刺激。实时定量PCR分析RNA的提取，cyclin D1的mRNA水平（标准化18S核糖核酸）。 G0代表从静止细胞的细胞周期蛋白D1的表达。 Cyclin D1的mRNA水平显着增加，僵硬的水凝胶，但不软凝胶。数据以均数+ / - SD重复的PCR反应。

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	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. 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凝胶聚合过程的一个关键因素是，以避免气泡的形成，这将使细胞绑定到ECM涂层凝胶本身，而不是玻璃盖玻片。这是可以预防的仔细吹打后震荡和可视化，确保无气泡已成为被困在凝胶的聚合解决方案。我们始终建议准备额外的“反应”盖玻片和凝胶，以确保有足够的实验。 凝胶用PBS冲洗时，应特别注意支付。吸痰过程中必须避免与水凝胶本身，因为它可能被抓住，撕裂的?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

组织刚度，是一个重要的细胞功能因素，并组织刚度的变化通常与纤维化，癌症和心血管疾病的1-11的关联。传统的细胞研究细胞功能的生物方法涉及一个刚性基质上培养细胞（塑料餐具或盖玻片），它可以不考虑弹性ECM或流脑组织之间的刚度变化的影响。 在体内组织遵守的条件，在体外模型，我们和其他人使用ECM涂水凝胶。在我们的实验室，是基于水凝胶聚丙烯酰胺可以模仿生物12见过的组织符合。除了3 APTMS孵化用NaOH产生“反应”的盖玻片。戊二醛是用于交叉连接的3 APTMS和聚丙烯酰胺凝胶。用于水凝胶的聚合丙烯酰胺（AC），双丙烯酰胺（BIS - AC），过硫酸铵溶液。 N -羟基琥珀酰亚胺（NHS）是纳入交联的ECM蛋白凝胶交流解决方案。聚合凝胶，凝胶表面涂有流脑的首选，如纤维连接蛋白，vitronectin，胶原蛋白等蛋白质水凝胶的硬度，可以由流变或原子力显微镜（AFM）和由不同的解决方案中的12的交流和/或双AC百分比调整。在这种方式下，下层刚度可以匹配生物组织的刚度，也可使用流变或AFM量化。细胞可以接种这些水凝胶和培养根据所需的实验条件。成像的细胞和分子生物学分析他们的恢复很简单。在这篇文章中，我们定义为那些有&lt;3000帕斯卡与E和僵硬的组织下层/&gt; 20,000帕斯卡的弹性模量（E）软下层。

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

可以仿照下层刚度对细胞功能的影响<em&gt;在体外</em&gt;使用不同的符合标准的聚丙烯酰胺凝胶。

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

studying-effects-matrix-stiffness-on-cellular-function-using

Research

JoVE Journal

Biology

21.9K 次观看.  University of Pennsylvania. 可以仿照下层刚度对细胞功能的影响在体外使用不同的符合标准的聚丙烯酰胺凝胶。

视频: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

科研

生物学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

研究蛋白质和微RNA调制对脂肪细胞功能的影响的脂肪细胞培养模型

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...