这项研究得到国家卫生和医学研究理事会澳大利亚（项目赠款。6288480）和Scobie和克莱尔麦金农信托。

1。微观一个RT反应<ol><li>在执行与RT反应的微​​观，RT反应所需的温度平衡的微混合器。 <ol><li>将微混合器内的37 ° C（或由RT供应商建议的温度）RT反应发病前至少1小时的孵化器。 </li></ol></li><li>根据逆转录供应商设置的RT组合（例如，自Qiagen Omniscript自Promega MMTV - RT）的说明，除了使用单细胞的金额，输入的总RNA（例如0.1-1pg/μl），而不是毫微微克供应商建议的数额。 <ol><li>我们使用标准的无菌，无核酸200μL薄壁PCR管。 </li></ol></li><li>位置RT管牢固地进入微混合器尽快搅拌。 RT反应时间应保持37℃孵化器内的微混合器。 </li><li>选择适当的频率和幅度的微观混合。在这个例子中，我们使用150Hz的。 </li><li>开关的微混合器，离开它为整个工期的RT反应（60分钟）。 </li><li>开关的微混合器和采取的RT管RT反应完成后。 </li><li>放在冰RT管，并进行进一步的应用，如定量聚合酶链反应（定量PCR或实时PCR）为正常。 </li></ol> 2。代表性的成果: 在逆转录反应中的微观的好处是在使用时，其他的混合方法（如摇晃，震荡或trituration）cDNA的产量相对增加。可以看出，例如，随后的cDNA用定量聚合酶链反应（定量PCR或实时PCR）分析后。图2a给出了第5分钟的微观效应（蓝色，"micromixed5"）或整个60分钟（红色，"micromixed60"）与RT反应trituration前夕的RT孵化（黑色，"trituration相比" ）。在这种情况下，前面的RT反应体积为25μL，它包含2.5pg的总RNA。如上图所示，使用的引物设计到检测的cDNA代表Nurr - 1的mRNA的qPCR的痕迹和手段±周期，以达到在3重复这同样的实验50％的最大荧光中小企业绘制如下。星号表示差异有统计学意义（单向方差分析与杜克多重比较）。注意，前面的RT反应的前5分钟的微观生产比trituration非显着改善，而整个60分钟前RT反应的微​​观产生了显着的改善超过trituration，幅度约相当于15的qPCR周期，平均。 对微观混合的好处，也可以看出，在随后的qPCR产品获得熔解曲线分析。在图2b所示的实验，为整个60分钟前RT反应的微​​观导致在重复样本的qPCR信号（图2BA，2个红色的痕迹），而第5分钟，或只产生不一致的qPCR trituration的微观痕迹（图2BA，2个蓝色的痕迹和2个黑色的痕迹，分别）。灰色的痕迹，阴性对照组（"南德"）在其中的cDNA的qPCR反应。当从本实验的qPCR产品，通过提高他们的温度（即熔解曲线分析），它是明显的变性，适当的PCR产物（即是相同的，当浓度高得多的mRNA反转录和定量PCR扩增获得的扩增，数据未显示），目前只有以下RT，已为60分钟（图2BB，2个红色的痕迹）micromixed的反应。所有其他人（即micromixed5，trituration阴性对照）所产生的替代扩增产品（如引物二聚体）。 <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="图1"/> 图1。申请的微观标准实验室RT反应的协议的流量图。涉及的物理原理，在微混合器，以及如何建立一个内部的微混合器提供更详细的描述中引用13,14 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="图2"/> 图2代表的qPCR数据显示的微观应用到前面的逆转录反应，包括在其他标准的实验室逆转录反应的单细胞的mRNA数量的好处 。一个比方的qPCR检测的cDNA代表Nurr - 1基因的mRNA编码的痕迹。前面的RT反应在25μL体积的总RNA中包含2.5pg，并在37℃，60分钟，在一个孵化器。逆转录反应混合的R triturationT组合前潜伏期（黑的痕迹，"trituration"），micromixed RT反应（蓝色的痕迹，"micromixed5"）在第5分钟，或为整个60分钟的RT反应micromixed的（红色的痕迹，"micromixed60" ）。平均值±中小企业的数量要求达到50％，最大荧光定量PCR周期曲线所示为n = 3的重复这个实验。星号表示显着性差异（单程与杜克多重比较方差分析）。 乙另一个qPCR的实验研究Nurr - 1基因的产品，这个时候包含在一个体积25μL25pg总RNA逆转录反应。 qPCR的痕迹（一）以及熔解曲线分析定量PCR获得的产品（B）所示为不同的搅拌条件下。还包括在没有基因包含在前面的RT反应的阴性对照（灰色的痕迹，"负"）。浓度高得多的mRNA RT反应也和生产micromixed60（二）（红色痕迹）样品相同熔解曲线峰（数据未显示）。

<ol>
	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

作者们没有透露。

应用标准实验室RT反应的微​​观描述的方法可以，当然，涉及的mRNA通过任何方法（如细胞裂​​解，激光捕获显微切割）的收获。它也可以包括任何品牌或类型的RT试剂，任何温度（微混合器的材料的承受能力范围内），和任何时期。例如，我们已经看到了包括随机六聚体或寡DT引物的RT反应的cDNA产量提高。它也可能被应用到其他生化转换（例如DNA杂交15），符合以下限制。当前技术的主要制约因素是必须发生在一个小的（微升）解决方案的下降，形成一个弧形的液体空中接口（如半月板）的反应。我们经常使用卷10或25μL，200μL薄壁PCR管生产半月板分别为4.7或6.9毫米，曲率半径。这是一个在下降，从而拉长了不同的解决方案之间的接口设置一个旋涡流的要求，并允许扩散到地方在较短的时间尺度。这个约束，因此，适用于所有可能被用来在其中的微观情况。事实上，使用小卷可以是一个优势，因为往往是输入材料（如RNA，DNA）的数量是有限的，加上其他试剂可以大大节省。在特定背景下的RT反应产率提高，反应也必须包括一个mRNA的限制金额。我们已经表明的微观标准实验室RT反应，包括输入RNA的单细胞数量增加搅拌（摇晃，震荡或trituration），包括Nurr - 1说明以外的其他基因的标准方法，约10 - 100倍的cDNA产量这里14。我们还表明，这种改进降低输入RNA的浓度增加14。想必这是因为在较高的RNA浓度，增加扩散率的微观混合的情况下，确保更多的基因是转化为cDNA，它会降低之前。 微观混合的方法，将受益于高高保真通过反应釜反应液的声信号的传输。我们帮助实现小心混合覆亚克力板扬声器（图1），他们正是适合的200μL薄壁PCR管锥孔加工。这最大化之间的接触管和亚克力板，因此最大化的声信号的传输。人们也需要，以确保安全反应管内混合孔举行，因为他们可以在混合的机械振动。 我们声学microstreaming的定义是可能比一些现象学为基础的定义更广泛。然而，有一个良好莱利16的根本，而不是现象学，审查，分类这些流动。 Navier - Stokes方程的非线性项，已足够大，以便在第二为了有一个平均流量。由于这个词是一个速度倍速度梯度，我们做任何使速度变化足够小尺度（即足够大的速度梯度），可以带来一个microstreaming流。我们的声学microstreaming为基础的装置（"微混合器"）能够在音频频率（即低功耗），确保系统中有一个小半径曲率 13的液气界面声microstreaming。相比之下，"声流"往往是定义一个非线性项是由一个大的速度（即高功率）大。在我们的例子，在一个良好的小水滴是由振荡;连同其粘度，表面张力下降，可能会发挥创造了平均流量为混合 13的作用。实验还表明，大型（10 -100μm的）粒子可在 ​​振动较大的下降比我们使用的移动，但是这可能是由不同的机制， 具体到粒子17。 总之，应用的微观标准实验室RT反应，包括单细胞的RNA金额大幅增加他们的cDNA产量的有效途径。这种改进及以上的混合微升卷使用目前可用的方法（即摇晃，震荡或trituration），可以实现。虽然有很多情况下，在微流体为基础的设备，执行RT将是一个巨大的进步，在其他情况下，包括我们自己的，单独的微观提供显着的和足够的利益，而不需要作进一步的专门设备和方法。

<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>从管理单元冻结，敏锐地准备成年小鼠的脑切片中提取总RNA </td><td></td><td></td><td></td></tr><tr><td> PicoPure的RNA分离试剂盒</td><td>大角星，CA，USA </td><td> KIT0204 </td><td>现在该工具包是从美国应用生物系统公司</td></tr><tr><td>无DNA脱氧核糖核酸酶处理和去除试剂</td><td> Ambion公司</td><td> AM1906M </td><td></td></tr><tr><td>随机六聚体引物</td><td> Promega公司</td><td> C1181 </td><td></td></tr><tr><td> AMV - RT </td><td> Promega公司</td><td> M5101 </td><td></td></tr><tr><td> dNTP的设置</td><td> Promega公司</td><td> U1240 </td><td></td></tr><tr><td> RNasin 核糖核酸酶抑制因子</td><td> Promega公司</td><td> N2111 </td><td></td></tr><tr><td>无核酸酶水</td><td> Promega公司</td><td> P1193 </td><td></td></tr><tr><td> SYBR 绿色PCR扩增预混试剂</td><td>应用生物系统</td><td> 4309155 </td><td></td></tr><tr><td> HPRT前锋（20mer）:铁通TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> HPRT反向（20mer）:达GTC CCC认证总工会TGA CTG的AT </td><td></td><td></td><td></td></tr><tr><td> NURR1前锋（23mer）:CAG的反恐委员会CGA TTT铁通的AAC台泥公司</td><td></td><td></td><td></td></tr><tr><td> NURR1反向（23mer）:GGT GAG GTC公司的CAT GCT的AAA级铁通GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000分光光度计。 </td><td> Thermo Scientific的</td><td></td><td></td></tr><tr><td>科贝特转子基因RG - 6000 </td><td>科比特生命科学</td><td></td><td>科比特生命科学学院于2008年7月由QIAGEN公司收购</td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

细胞的行为及其相关基因表达的理想是做在单细胞水平。然而，这是不容易实现的，因为少量不稳定的mRNA在一个单元格（1 - 50pg总RNA的1-5％，或0.01 - 2.5pg的mRNA，每单元 1）目前大多降低，然后才可以反转录进入一个稳定的基因复制。例如，使用标准的实验室试剂和硬件，只有少数基因可以定性评估每单元 2 。一种方式来提高效率标准的实验室逆转录（RT）的反应，包括单细胞mRNA的金额（即标准试剂微升卷）将更加迅速地混合使用的试剂，使可转换的mRNA为cDNA，然后它会降低。然而，这是不平凡的，因为在微升尺度液体流动层，即目前可用混合方法（即晃动，涡旋和trituration）不能产生足够的混沌运动，有效地组合试剂。为了解决这个问题，微观尺度的混合技术也将用于 3,4 。已开发的基于微流体混合技术，成功地增加RT反应产率 5-8 。然而，微流体技术需要专门的硬件是相对昂贵，尚未广泛使用。一个更便宜，更方便的解决方案是可取的。这项研究的主要目的是演示了如何应用小说"的微观"的技术标准实验室RT反应包括单细胞的mRNA数量大大增加了他们的cDNA产量。我们发现cDNA的产量增加约10 - 100倍，从而使:（1）更大的数字分析每个细胞的基因;（2）基因表达的定量分析;（3）更好的低丰度基因检测在单细胞。的微观是基于声microstreaming 9-12，声波传播围绕一个小的障碍，创建一个障碍物附近的平均流量的现象。我们已经开发出一个关键的简化声microstreaming为基础的装置（"微混合器"）;声microstreaming可以确保系统中有一个小半径曲率 13的液气界面在音频实现。在管的解决方案微升量的半月板提供了一个适当的曲率半径小。音频频率的使用，意味着硬件可以廉价和通用的13，和不被损坏，像他们可以与标准实验室sonicators核酸和其他生化试剂。

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

我们描述了一个从单细胞的mRNA数量增加在其他标准的实验室逆转录反应的cDNA产量的新方法。求新驻留在一个微混合器的使用，它采用的声学microstreaming现象，微升尺度混合流体比摇晃，震荡或trituration更有效。

增加从单细胞的数量标准实验室逆转录反应的mRNA的cDNA产量，使用声Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.1K 次观看.  University of Melbourne. 我们描述了一个从单细胞的mRNA数量增加在其他标准的实验室逆转录反应的cDNA产量的新方法。求新驻留在一个微混合器的使用，它采用的声学microstreaming现象，微升尺度混合流体比摇晃，震荡或trituration更有效。

视频: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

科研

生物工程

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

从未引用的太平洋牡蛎生成虚拟序列的 cDNA 库的聚合策略

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

使用生物活性珊瑚基质提高解离神经细胞培养物的耐久性

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

用于木材粘合剂内聚强度发展的标准测试方法 ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

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 ...