我们感谢博士。约翰汉考克，雷烧烤，安德鲁莫里斯和奥尔加Chumakova的支持。 FlexStation 3系统UTHSC Cytodynamic成像设施的一部分。我们也感谢博士。 Ardem Patapoutian和迈克尔Bandell来自斯克里普斯研究所和诺华研究基金会（GNF）的基因组研究所提供的HEK293 - hTRPA1稳定的细胞株。支持这项研究是由NIH（GM081658 MXZ）和德州医学中心消化内科中心（HH）和使命连接/ TIRR基金会（农户）的赠款部分。

1。在96孔板细胞的准备 HEK293细胞与人类TRPA1基因瞬时trasfected <ol><li> HEK293细胞成长为一到两天贝科的最小必要中等含有4.5 mg / ml的葡萄糖（Thermo Scientific的，SH3002201）（DMEM），辅以10％热灭活胎牛血清（Invitrogen公司，16000-044），50个单位/ ml青霉素和50μg/ mL链霉素（Invitrogen公司，15140-163）。在一天的转染，细胞密度应达到70-90％。 </li><li>大衣96孔板吹打，每孔50μL聚- L -鸟氨酸（Sigma公司P3655，20微克/毫升无菌蒸馏水）和孵化在37℃至少15分钟的彗星。每口井一次冲洗贝科磷酸盐缓冲液（DPBS）无镁2 +和Ca 2 +（Thermo Scientific的，SH3002803）。该板块可能会留在细胞培养罩了几个小时。 </li><li>对于每一个96孔板以及稀释的1.5 mL Eppendorf管0.2微克的cDNA 25μLOPTI - MEM（Invitrogen公司，31985-070）。扩大比例按井数相同的cDNA转。另外一个单独的1.5 ml Eppendorf管0.4μL脂质体在2000年25μLOPTI - MEM（Invitrogen公司，11668-019）以及每个要转稀。扩大根据的染井总数。 </li><li>简要涡稀释后的cDNA和脂质体2000，并让他们坐了约5分钟，在室温下。 </li><li>转让等体积稀释的脂质体2000包含稀释后的cDNA，涡管，让混合物在室温下坐了15分钟至2小时。在这段时间内，转让的cDNA /脂质体2000的混合物在49μL/聚- L -鸟氨酸的涂层板。 </li><li> Trypsinize HEK293细胞（步骤1.1），使用血球或伯爵夫人自动细胞计数器（Invitrogen公司，C10227）计数细胞密度，转移所需的细胞量（一般约〜125,000-150,000细胞/孔）15 ml无菌锥底离心管。在1000 × 5分钟的转速的离心机。 〜1,250,00-1,500,000细胞/毫升在10％热灭活胎牛血清的DMEM培养液补充，但不含抗生素的悬浮细胞。 </li><li>点样98μL细胞悬液，每孔含有脂质体的cDNA - 2000混合使用多pipetter或MicroFill微孔板饮水机（BIO - TEK）。让坐在板至少30分钟在引擎盖之前，加湿细胞培养孵化器。孵育于37℃，5％的CO 2为20-44小时。 </li></ol> 对于HEK293细胞中稳定表达TRPA1 <ol start="8"><li>按照步骤1.2大衣96孔板。 </li><li> A. Patapoutian（​​斯克里普斯研究所）博士，并慷慨地提供诱导的HEK293 - hTRPA1细胞株保​​持在同一介质为野生型的HEK293细胞（步骤1.1），但辅以5微克/毫升杀稻瘟（Invitrogen公司， A1113902）和50μg/ ml的潮霉素B（Invitrogen公司，10687010）。当细胞密度达到90％左右，trypsinize细胞，计数细胞密度和细胞在5分钟的转速为1000 ×的离心所需数量。在相同的文化与强力霉素（费舍尔，BP265​​35，0.25微克/毫升）培养基在〜100细胞/ ml密度悬浮细胞。 </li><li>配制细胞悬液，以聚- L -鸟氨酸涂层板在100μL/孔用多pipetter或MicroFill（BIO - TEK）。让坐在板至少30分钟在引擎盖之前，加湿细胞培养孵化器。在37℃，5％的CO 2为16 - 24小时。 </li></ol> 2。解决方案<ol><li>汉克的缓冲盐溶液（HBSS）作为细胞外液。它包含（毫米）：137氯化钠，5.4氯化钾，0.25 NA 2 HPO 4，0.44 KH 2 PO 4，1.3，1.0氯化钙硫酸镁 ，1.0 10氯化镁2，使用1N氢氧化钠调整至7.4的pH值，葡萄糖，10 HEPES） 。 </li><li>荧光4缓冲液中包含的HBSS丙磺舒2毫米。使250毫米存量为0.5 N氢氧化钠丙磺舒（Sigma公司，P8761）。在1:125比例稀释到的HBSS。重新调整pH至7.4使用1个N盐酸。 </li></ol> 3。细胞装载荧光凌晨4点<ol><li>溶解于912μL无水二甲基亚砜1毫克氟- 4 AM（Invitrogen公司，F14202），使1毫米的原液。荧光4 AM原液可在-20 ° C保存至少一个月。 </li><li>混合10微升1毫米荧光4上午10μL聚醚F - 127（Invitrogen公司，P3000MP，20％（W / V）在DMSO溶液中），然后整个内容传送到5毫升荧光4缓冲液（步骤2.2）辅以0.1％牛血清白蛋白（Sigma公司，A9418）。 </li><li>细胞生长在96孔板（步骤1.7或1.10）80μL/ 2使用洗板机（如来自Bio - TEK ELx405TM）倍的HBSS洗净。 </li><li>新增的氟-4上午装载解决方案（步骤3.2）50μL/孔用多pipetter或MicroFill（BIO - TEK）和板在37 ° C为1小时。 </li><li>洗涤细胞荧光分析缓冲液使用ELx405TM洗板机（2.2）的3倍。最后一次洗涤后，离开的荧光4缓冲液，每孔80μL。 </li></ol> 4。制备复合板<ol><li>在1小时细胞加载期间，准备了一系列激动剂的荧光4缓冲液的最终浓度的3倍（或拮抗剂）的3倍稀释。 </li><li>吸取250-300μL的稀释后的复合解决方案，以一个96孔的复合板。安排在同一列的复合板的化合物和相应的阳性和阴性对照的系列稀释，只要有可能，因为FlexStation 3在一次读取一个单个列。 </li></ol> 5。读板FlexStation 3 <ol><li>打开系统（FlexStation 3，计算机，SoftMax PRO 5.2软件） </li><li>保存一个新的文件名实验。在设置中，选择FLEX模式，并输入以下值： </li></ol> 协议＃1，受体激动剂的影响 <img src="/files/ftp_upload/3149/3149table1.jpg" alt="表1" /> 协议＃2，拮抗效应 <img src="/files/ftp_upload/3149/3149table2.jpg" alt="表2" /><ol start="3"><li>一个新的提示框，复合板和阅读板放置到相应的托盘在FlexStation 3，点击“读取”按钮。 FlexStation 3会一次读取一列，并添加化合物在预先设定的时间点，不中断阅读。这大约需要3分钟（5分钟协议＃2）读完一列的机器将继续读取下一列化合物进行补充，为用户在步骤5.2编程，直到所有的列只读。 </li></ol> 6。数据分析<ol><li>处理实验数据可直接使用SoftMax PRO 5.2软件或复制并粘贴到任何电子表格程序（如Microsoft Excel，）。使用下面的最小二乘拟合常规构造一个TRPA1受体激动剂flufenamic酸（FFA）的浓度 - 反应曲线。 <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="公式1" /> ， 其中 Y是观察到的反应，Y 最大正常化的最大响应，C为相应的药物浓度， 欧共体 50的浓度，引起了半最大反应， 和 N H是明显的Hill系数。一个公认的TRPA1拮抗剂抑制浓度曲线，一个是通过使用固定浓度的FFA（100μM），并逐步增加的化合物A的IC 50值复合浓度，一个是由装修以下最小二乘计算方程。 <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="公式（2）" /> ，其中Ÿ是在观察响应和Ÿ 最大是规范化的峰值响应，[蚂蚁]是一个的浓度的相应化合物，IC 50的拮抗剂浓度产生的一半，最大抑制游离脂肪酸，引起的反应，N H的表观山系数。 </li></ol> 7。代表性的成果： TRPA1是一个渗透性的Ca 2 +离子通道，激活钙离子内流的Ca 2 +敏感的染料，荧光4（1-2），可以通过检测。 ，一个稳定的HEK293 - hTRPA1细胞株用于研究TRPA1受体激动剂和拮抗剂的浓度 - 反应关系。细胞装有荧光凌晨4点，放置在80μL缓冲液，并阅读使用FlexStation 3。录制30秒的基线荧光后，分别加入40μL的TRPA1受体激动剂，FFA系列稀释，每3倍所需的最终浓度，通过多渠道pipetter细胞作为流体模块的一部分FlexStation 3。荧光的变化进行了监测额外的180号ΔF=（F - F 0），F和F 0的阅读兴趣和开始时的荧光值，分别表示为相对荧光4荧光每口井的变化。代表时间课程的痕迹，在一系列的FFA浓度荧光的变化如图所示。 1。注意：在过去的四个游离脂肪酸的浓度，虽然最大的响应水平都差不多，激活动力学的速度显然比在较低的游离脂肪酸浓度较高。因此，要区分这些差异，浓度 - 反应关系是通过测量后，加入不同浓度的FFA（图2）的初始利率的荧光增加（斜率）的构造。半最大效应IVE浓度的FFA（EC 50）被确定为55.4μM（n = 3时）。要测试公认的TRPA1受体拮抗剂，化合物A，我们进行了一个类似的实验，使用的协议＃2（步骤2）。在这种情况下，在30秒（3X理想的最终浓度为40μL），并分别加入系列稀释的拮抗剂FFA的增加了180秒后终浓度为100微米300微米（60μL）。化合物A剂量依赖性降低半的最大抑制浓度（IC50）为4.3μM（N = 3）（图2）为FFA的TRPA1细胞的反应。 <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="图1"/> 图1。FFA的浓度依赖性地激活人力TRPA1 。痕迹随着时间的推移增加FFA的诱发荧光（显示在第一个60秒的唯一反应是为了更好地说明FFA的诱发反应的快速动力学）。请注意，在较高浓度的游离脂肪酸诱发荧光增加一个更快的动能比，即使在低浓度的荧光峰值水平相似。 <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="图2"/> 图2浓度- FFA诱导荧光变化和复合A诱导抑制FFA人类TRPA1在HEK293细胞中稳定表达的响应的响应关系。 Δ，游离脂肪酸的浓度 - 反应关系确定后，加入不同浓度的FFA测量的初始利率正常化的荧光增加（斜率）。一个公认的TRPA1受体拮抗剂抑制游离脂肪酸诱发的钙响应的浓度 - 反应关系，澳，化合物A

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没有利益冲突的声明。

细胞内的Ca 2 +在许多生理和病理条件下，如神经递质的释放（3），心肌动作电位（4），肌肉细胞收缩（5），细胞信号转导（6-7）和兴奋性细胞死亡（，起着重要作用8）。细胞内Ca 2 +浓度的测量有助于澄清功能的变化质膜或细胞器中的Ca 2 +渗透的渠道的贡献 的Ca 2 +以上的流程（9-13） 。该检测也可以用于调查“G -蛋白偶联受体的功能，其中许多激活后...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> FlexStation 3 </td><td>分子器件</td><td> FLEX3 </td><td></td></tr><tr><td> 96孔，FlexStation吸取技巧</td><td>分子器件</td><td> 9000-0912 </td><td></td></tr><tr><td> 96孔板</td><td>格雷纳生物一</td><td> 655090 </td><td></td></tr><tr><td> 96，那么清除平底板</td><td>配角</td><td> 3595 </td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 12430-104 </td><td></td></tr><tr><td> OPTI - MEM </td><td> Invitrogen公司</td><td> 31985-070 </td><td></td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668-019 </td><td></td></tr><tr><td>荧光凌晨4点</td><td> Invitrogen公司</td><td> F14202 </td><td></td></tr><tr><td> PluronicF - 127 </td><td> Invitrogen公司</td><td> P3000MP </td><td></td></tr><tr><td>聚- L -鸟氨酸</td><td>西格玛</td><td> P36551 </td><td></td></tr><tr><td>丙磺舒</td><td>西格玛</td><td> P8761 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

“分子器件FlexStation 3是一个台式多模酶标仪能够自动在多孔板荧光测量。它是理想的学术环境中高通量屏幕。它集成了一个流体输送模块，配备了多渠道pipetter机读取一列，在一个时间来监视各种荧光试剂的荧光变化。例如，FlexStation 3已被用于研究的Ca 2 +渗透离子通道和G蛋白偶联受体胞内游离Ca 2 +浓度的变化，通过测量的功能。瞬时受体电位（TRP）通道是一个大家族的非选择性阳离子通道，在许多生理和病理的功能发挥的重要作用。 TRP通道是钙离子渗透性和诱导激活后钙离子内流。在这段视频中，我们展示FlexStation 3的应用药理学研究TRPA1通道，为许多有害刺激的分子传感器。 HEK293细胞中瞬时或稳定表达人类TRPA1通道，生长在96孔板，装入一个的Ca 2 +敏感的荧光染料，荧光4，并在这些细胞中实时荧光变化的测量之前和期间的应用一个TRPA1受体激动剂，使用弹性模式FlexStation 3。一个公认的TRPA1受体拮抗剂的作用进行了研究。 SoftMax Pro软件的数据传送构建TRPA1激活剂和抑制剂的浓度 - 反应关系。

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

此视频提供了药理学研究，人类TRPA1使用FlexStation 3的渠道的详细协议。该协议包括细胞的准备，染料装载和酶标仪，FlexStation 3操作细节。

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Research

JoVE Journal

Bioengineering

21.0K 次观看.  The University of Texas Health Science Center at Houston. 此视频提供了药理学研究，人类TRPA1使用FlexStation 3的渠道的详细协议。该协议包括细胞的准备，染料装载和酶标仪，FlexStation 3操作细节。

视频: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto：一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

中国仓鼠卵巢细胞培养中高通量自动化微生物反应器工艺优化

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...