通过了美国国家科学基金会，美国国立卫生研究院（NIH），材料技术中心（MTC）和ORDA在SIUC提供了财政支持这项工作。我们感谢美国国家科学基金会的资助（CHE-0959568）购买一个FE-SEM。我们希望，感谢马修麦卡罗尔的有益讨论。朱莉娅·雷耶斯会喜欢她的奖学金和财政支持，感谢COLCIENCIAS，哥伦比亚机构和大学Pedagogica的的ŸTecnologica哥伦比亚。

A. PDA脂质体4,5,6的合成与表征注1：保护PDA解决方案，光在所有的实验步骤，每一容器上使用的铝箔包装。 注2：两个不同的脂质体溶液（B和C）以下步骤A（PDA脂质体的合成与表征）。 1。合成N-羟基Diacteylene（NHS-PCDA） <ol><li>为了准备脂质体，PCDA-NHS的一个重要成分是必需的。我们已经合成PCDA-NHS使用下列程序： </li><li>添加10,12 - pentacosadiynoic酸（PCDA）（0.267克，0.713毫摩尔），N-羟基琥珀酰亚胺（0.0914克，​​0.786毫摩尔）和1 - （3 - （二甲基氨基）丙基）-3 - 乙基碳二亚胺盐酸盐（0.144克，0.713毫摩尔）在无水CH 2 Cl 2（20毫升）中。 </li><li>在室温下搅拌该溶液2小时。 </li><li>小心地取下溶剂的使用rotary蒸发器，得到干燥的薄膜。 </li><li>使用分液漏斗中提取残余物，用乙醚（25毫升）和水（25毫升）的三倍。 </li><li>干燥有机层，用MgSO 4（1.0克）为半小时。过滤并通过旋转蒸发除去溶剂，得到白色固体粉末（0.24克，&gt; 90％）。 </li><li>根据核磁共振（NMR）分析最终的化合物。 </li><li> 1 H NMR（300兆赫，DMSO），δ（ppm的）：0.893（吨，3H），1.268（米，26H），1.512（米，4H），1.754（米，2H），2.252（吨，4H）， 2.365（1H），2.610（米，1H），2.842（2H）。 </li></ol> 2。脂质体制备5,6,7 <ol><li>溶解PCDA：PCDA-NHS：1,2 -二肉豆蔻酰-sn-甘油-3 -磷酸胆碱（DMPC）:: 8：1：1在20毫升二氯甲烷中。 </li><li>用滤纸过滤溶液以除去团粒。 </li><li>完全蒸发溶剂，得到的薄膜的单体。 </li><li>过夜真空干燥的薄膜UUM。 </li><li>水合物的膜用去离子水（50毫升），使所需的浓度（0.65-1毫摩尔）的脂质体溶液。 </li><li>超声处理将所得的悬浮液用探针超声波仪，在76℃〜18分钟。 </li><li>小心地通过该溶液通过一个纸过滤器，以除去脂质团聚体</li><li>在4℃过夜，以促进自我组装的单体溶液冷却。最终的解决方案应该是光学透明的。 </li><li>聚合自组装的联乙炔单体（脂质体），通过照射与〜2分钟使用钢笔射线UV源（4.5毫瓦/平方厘米2）在空气中的254 nm的紫外线辐射。 </li><li>的脂质体溶液在室温下是稳定的，至少两个星期。解决的办法是更稳定的冷藏时。 </li></ol> B.罗丹明标记牛血清白蛋白（BSA-RH）修改PCDA脂质体的制备 1。到脂质体表面的结合BSA-Rh型<ol><li> BSA-RH溶解在PBS BUFFER（离子浓度为0.01M，pH 7.2）中，以使最终浓度达到1.2μM的BSA的罗丹明B溶液。 </li><li>加入2毫升（1.2μM）BSA-罗丹明A.2在步骤（见上文），在室温下制备的脂质体溶液至10毫升。 </li><li>一直沿袭古典的结合从蛋白质的赖氨酸残基的羧酸由NHS组激活的胺基反应（反应方案在图2中）。 NHS-PCDA使用NHS-胺的反应（ 图2，步骤2）的脂质体的共价结合的蛋白质分子的设计（ 图2，步骤1）。 NHS是一个极好的离去剂，在向前的方向驱动的胺羧酸反应。在适当的条件下，反应收率是定量的。 </li><li> 取消的免费BSA-RH：一个光谱浸泡在去离子瓦特/ POR生物纤维素酯（CE）膜（MW C O：100000）ATER为15分钟。该膜是用于透析的未反应的BSA-罗丹明（分子量〜66000沓）在去离子水中。 </li><li>小心地将溶液转移到透析膜。 </li><li>在透析过程中的水在2小时，8小时，14小时，24小时，和36小时变更。 </li><li>收集用铝箔覆盖的最终溶液的小瓶中。 </li></ol> C.制备的SR-二胺和生物素标记的脂质体 1。而不是使用DMPC与在步骤2.1中，我们将使用生物素标记的-（1,2 - 二油酰-sn-甘油-3 - 磷酸乙醇胺-N-（生物素基）（生物素-DOPE）。 <ol><li>所有的步骤2.2至2.9。 </li><li>相反，在步骤B中的BSA-Rh型，使用的二胺的标签Sulphorhodamine（SR-二胺）。 </li><li>在B.按照所有进一步的步骤</li><li>在此制备含有生物素的脂质体，并在其表面上的SR-二胺。随后的步骤A和B（见上文），但有一个例外：链霉亲和素的的解决方案生物素 - 链霉亲和素相互作用，通过改变荧光共振能量转移效率。 </li></ol> D.代表性的成果<img alt="标题图" src="/files/ftp_upload/3805/3805figtitle.jpg" /> 题名图。卡通反应说明和FRET在脂质体的表面上的过程中发生的（按照步骤B制备的）。 A.监测蛋白质附着脂质体使用荧光共振能量转移1 罗丹明和PDA准备步骤B中的脂质体之间的荧光共振能量转移监控 BSA-Rh型的激发和发射光谱和吸收光谱的PDA（ 图3A）。我们可以清楚地看到，发射光谱的吸收光谱PDA BSA-RH重叠。 FRET的机制，这满足谐振条件。 BSA-罗丹明标记的脂质体聚合之前和之后，分析用UV-V光谱和荧光光谱。对于隔离的施主和受主，FRET效率是高度依赖于供体-受体的距离（r）和 J 1第（j-值）。观察中的排放量的淬火（ 图3B），因为若丹明和PDA由于电子吸收光谱的蓝色的光聚合后的PDA的外观之间的FRET。在我们的例子中，FRET效率是零的未聚合的脂质体与罗丹明因为 J = 0，在可见光区域的未聚合的脂质体。 我们进行了类似的实验，只有PDA不包含NHS在其表面的脂质体。在这些情况下，未标记BSA-Rh型的脂质体的表面。在这种情况下，若丹明和PDA相关（r 平均值 ）之间的平均距离远大于福斯特半径（R 0 = 2.8纳米）。因此，我们并没有观察到荧光强度下降幅度大。这种看法。莱索托的荧光猝灭是占主导地位，当r &lt;2.8 nm的。 J和R 0的值，使用以下公式计算：[1] J（λ）=∫F D（λ）εA（λ）λ4Dλ R 0 = 0.211 K 2 N-4 Q D J（λ）] 1/6 蓝PDA的消光系数（ε）的范围是在〜2000-10000 M -1厘米-1之前，加入链霉抗生物素蛋白。蓝PDA的消光系数是依赖于光聚合条件。 [2]对于例如，ε的值将是较大的溶液聚合的时间较长。另外，自组装体的联乙炔单体也可能会影响的ε值。 在 J演算ations需要考虑这些变化，又反映了由于生物素-链霉亲和素分子间的相互作用在PDA消光系数的变化。用实验的PDA的吸收和SR-101发射数据，J值计算。 J值的变化是由于转移后，加入到该溶液中（ 图4C）的链霉抗生物素蛋白的PDA中的电子吸收光谱。 J值的单位是M -1 cm -1的纳米4。 B.监测FRET，加入链霉抗生物素蛋白的生物素标记的脂质体溶液注1：监控罗丹明​​准备在上述步骤C和PDA脂质体之间的荧光共振能量转移。 激发和发射光谱Sulphorhodamine标签二胺（SR-二胺）和吸收光谱的PDA（ 图4A）。未聚合的聚合生物素标记的脂质体进行了分析使用的UV-Vis光谱和荧光光谱。聚合（ 图4B），表明由于FRET的荧光淬灭后，若丹明（SR-101）的排放量降低45％左右。加入40μl等分试样（1μM）的链霉抗生物素蛋白溶液至2毫升脂质体溶液。链霉抗生物素蛋白与添加到该溶液中，在J值的变化被观察到（ 图4C）。图4D示出了作为生物素结合的链霉抗生物素蛋白，蓝色的PDA，脂 ​​质体（中心在图1中在〜645 nm）的峰强度的下降，而在540 nm处的吸收峰的增加，观察到（F IGURE 1S）。在FRET的变化效率。荧光共振能量转移效率下降，链霉亲和素浓度的增加，也与我们的预测一致。 记录每40微升等分此外，链霉亲和素的的SR排放后。我们观察到一个稳定增长后，在罗丹明排放增加的链霉抗生物素蛋白（ 图5）。罗丹明排放这增加是由于为sulphorhodamine生物素-链霉抗生物素蛋白相互作用后的发射光谱和PDA的吸收光谱中的J值减少。在分子水平上，生物素-链霉抗生物素蛋白的相互作用导致有效的共轭长度的微妙的变化，这将导致减少在蓝PDA的形式到热力学更稳定的红色PDA表单2的PDA。这是在J个值中的变化的基础。有趣的是，分子间的相互作用为共价键或非共价键合的生物素到PDA脂质体的微妙差异可以探测使用我们检测测定4。 我们也进行对照实验中，在相同的实验条件下的那些链霉抗生物素蛋白 - 生物素系统监测的对照样品的发射。对照实验包括：（1）脂质体的解决方案，包含生物素在他们的表面中加入​​相同的体积和浓度的缓冲溶液中，和（2）无生物素受体在其表面上的脂质体溶液中加入链霉抗生物素蛋白的相同的体积和浓度。生物素标记的脂质体溶液后，加入链霉抗生物素蛋白的强度表现出增强的强度，但对照实验的脂质体溶液中的强度（例如，请参阅图2S），另一方面，具有在发光强度下降。这是归因于该溶液稀释。这些实验清楚地表明，该解决方案的增强发射是由于特定的分子间的相互作用。 福斯特半径（R 0），若丹明和PDA对计算（式（2））为〜2.80 nm的。这意味着，孤立的PDA -罗丹明对罗丹明分子的激发态的50％将有自己的能量转移到PDA 当 r为2.80纳米。 我们OBS<html> erved，当生物素共价连接到PDA的骨干网，排放量增加的非共价键合生物素的脂质体的2-3倍大于4这些结果有力地表明，我们所提出的系统是敏感的区分中的相互作用的微妙的差异在换能器（连接器之间的生物素和脂质体双层）由于共价和非共价键连接到脂质体受体。根据分光光度计，实时监控（毫秒为单位的第二时间尺度）的蛋白质的相互作用（在UV-Vis光谱）的扫描和数据采集能力是可能的与该感测系统。 <img alt="图1" src="/files/ftp_upload/3805/3805fig1.jpg" /> 图1的吸收光谱的蓝色和红色的PDA解决方案。 （插图）光学显微照片拍摄的数码相机。 G“/&gt; 图2为PCDA-NHS的合成（步骤1）的反应式。 PCDA-NHS反应的胺取代基的蛋白质（第2步）。第2步的基础上的赖氨酸残基的蛋白质的PCDA的羧酸单体的结合。 <a href="http://www.jove.com/files/ftp_upload/3805/3805fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3A" src="/files/ftp_upload/3805/3805fig3A.jpg" /> 图3A。变动观察到FRET效率是由于在PDA中的吸收光谱的变化。聚合之前有BSA-Rh型发射和PDA的吸收，但在聚合后与BSA-Rh型发射PDA吸收重叠，这是要求对FRET之间没有重叠。罗丹明是施主（红色）和聚合的PDA脂质体作为受体（蓝色）。 <img alt="图3B" src="/files/ftp_upload/3805/3805fig3B.jpg" /> 图3B。BSA-Rh型标记的脂质体之前（蓝色）和后：PCDA聚合（红色）的荧光光谱。一个大的减少在罗丹明排放的观察由于FRET罗丹明和PDA之间。 <img alt="图4A" src="/files/ftp_upload/3805/3805fig4A.jpg" /> 图4A。光谱重叠（J）为PDA（蓝色或红色）吸收光谱和Sulphorhodamine的发光光谱（橙色）的变化。 图4A是极端条件下的代表性的频谱，即，当被加入到溶液中过量的链霉抗生物素蛋白。图4A示出了几乎完全的蓝色到红色的PDA变换后，添加链霉抗生物素蛋白的过量。可以清楚地看出，J（光谱重叠）增加与PDA吸收光谱的蓝移<img alt="图4B" src="/files/ftp_upload/3805/3805fig4B.jpg" /> 图4B。前荧光光谱（蓝色）和后相关（rED）脂质体的聚合。 <img alt="图4C" src="/files/ftp_upload/3805/3805fig4C.jpg" /> 图4C。对FRET状态：J之间的变化给体（sulphorhodamine）和受体（PDA）与链霉抗生物素蛋白的另外的脂质体溶液。 <img alt="图4D" src="/files/ftp_upload/3805/3805fig4D.jpg" /> 图4D。FRET效率的变化之间的给体（sulphorhodamine）和受体（PDA）与链霉抗生物素蛋白的另外的脂质体溶液。 <img alt="图5" src="/files/ftp_upload/3805/3805fig5.jpg" /> 图5。罗丹明到PDA的脂质体溶液后，添加链霉抗生物素蛋白的等分试样的发光光谱。插图显示了一个更大的排放SR-101光谱的变化。

<ol>
	<li>Lakowicz, J. 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> Principles of Fluorescence Spectroscopy. , (1999).</li><li>Charych, D. H., Nagy, J. O., Spevak, W., Bednarski, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Colorimetric+Detection+of+a+Receptor-Ligand+Interaction+by+a+Polymerized+Bilayer+Assembly.">Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly.</a> Science. 261, 585-588 (1993).</li><li>Yoon, B., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Conceptual+and+Technological+Advances+in+Polydiacetylene-based+Supramolecular+Chemosensors.">Recent Conceptual and Technological Advances in Polydiacetylene-based Supramolecular Chemosensors.</a> Chem. Soc. Rev. 38, 1958-1968 (2009).</li><li>Xuelian, L. i., Kohli, P. u. n. i. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Molecular+Interactions+in+Biosensors+Based+on+Fluorescence+Resonance+Energy+Transfer.">Investigating Molecular Interactions in Biosensors Based on Fluorescence Resonance Energy Transfer.</a> J. Phys. Chem. C. 114, 6255-6264 (2010).</li><li>Li, X., Matthews, S., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Resonance+Energy+Transfer+in+Polydiacetylene+Liposomes.">Fluorescence Resonance Energy Transfer in Polydiacetylene Liposomes.</a> J. Phys. Chem. B. 112, 13263-13272 (2008).</li><li>Li, X., McCarroll, M., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+Fluorescence+Resonance+Energy+Transfer+in+Conjugated+Liposomes.">Modulating Fluorescence Resonance Energy Transfer in Conjugated Liposomes.</a> Langmuir. 22, (2006).</li><li>New, R. R. C., New, R. R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liposomes: A Practical Approach. , 33-104 (1990).</li><li>Chen, X., Lee, J., Jou, M. J., Kim, J. -. M., Yoon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+and+Fluorometric+Detection+of+Cationic+Surfactants+Based+on+Conjugated+Polydiacetylene+Supramolecules.">Colorimetric and Fluorometric Detection of Cationic Surfactants Based on Conjugated Polydiacetylene Supramolecules.</a> Chem. Commun. , 3434-3436 (2009).</li><li>Yarimaga, O., Im, M., Choi, Y. -. K., Kim, T. W., Jung, Y. K., Park, H. G., Lee, S., Kim, J. -. 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+Color+Display+System+Based+on+Thermochromic+Conjugated+Polydiacetylene.">A Color Display System Based on Thermochromic Conjugated Polydiacetylene.</a> Macromolecular Research. 18, 404-407 (2010).</li></ol>

没有利益冲突的声明。

我们已经进行了选择性结合脂质体的表面上的蛋白质的赖氨酸残基的使用NHS-胺反应。 FRET为基础的方法是能够进行实时监控，生物素 - 链霉亲和素结合蛋白（BSA）结合到脂质体表面。类似的过程可以被应用到与他们的选择性受体的各种蛋白质相互作用的结合动力学研究。有灵活性是在选择的荧光基团，将提供的荧光的光谱特性，根据J值的变化。 PDA是一个普遍的接受。因此，随着多个荧光?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的10,12 pentacosadiynoic酸（PCDA） </td><td>政府飞行服务队的化学品</td><td> 3261 </td><td>光敏感</td></tr><tr><td> N-羟基琥珀酰亚胺（NHS） </td><td> Acros Organics公司</td><td> 157270250 </td><td>湿度敏感</td></tr><tr><td> 1 - （3 - （二甲基氨基）丙基）-3 - 乙基碳二亚胺盐酸盐（EDC） </td><td> CHEM-IMPEX国际</td><td> 00050 </td><td></td></tr><tr><td> 1,2 -二肉豆蔻酰-SN-甘油-3 -磷酸胆碱（DMPC） </td><td>阿凡提极性脂质</td><td> 850345P </td><td></td></tr><tr><td>罗丹明标记牛丝氨酸的嗯白蛋白（BSA-RH） </td><td> Sigma Aldrich公司</td><td> A4537 </td><td></td></tr><tr><td> （1,2 -二油酰-sn-甘油-3 -磷酸乙醇胺-N-（生物素基）（生物素-DOPE） </td><td>阿凡提极性脂质</td><td> 870282 </td><td></td></tr></tbody></table>

real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer

荧光共振能量转移是一个过程，非辐射能量是从激发供体分子转移到基态的受体分子通过远程偶极-偶极相互作用1。我们在本传感检测，利用PDA的一个有趣的特性：蓝移后的UV-Vis的PDA的电子吸收光谱（ 图1）中的分析物相互作用与受体连接到PDA 2,3,4,7。这种在PDA中的吸收光谱的移位提供了在光谱重叠（J），PDA（受体）和若丹明（供体）之间的变化导致在FRET效率的变化。因此，分析物（配体）和受体之间的相互作用是通过FRET检测到供体荧光和PDA之间。特别是，我们显示了一个的模型蛋白质分子链霉亲和素的检测。我们还证明了牛血清白蛋白（BSA）的脂质体的表面用共价键结合的FRET机制。这些t之间的相互作用他双分子层脂质体和蛋白质分子可以在实时感测到。所提出的方法是用于感测小的化学和大型生物化学分子的一般方法。由于荧光本质上是更敏感比比色法，测定的检出限，可以在子纳摩尔范围或低8。另外，PDA可以作为一个通用的受体，在FRET，这意味着，多个传感器可以开发PDA（受体）官能化与捐助者和PDA的脂质体的表面上附着的不同的受体。

Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

我们表明FRET之间的共轭聚合物的聚二乙炔（PDA）和荧光基团连接到PDA脂质体的生物分子的传感表面。 PDA脂质体也包含在其表面上的生物分子的受体分子可以用作探针。配体 - 受体相互作用导致这是传感机构的基础的荧光团和PDA之间的FRET效率的变化。

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

real-time-monitoring-ligand-receptor-interactions-with-fluorescence

Research

JoVE Journal

Biology

14.3K 次观看.  Southern Illinois University. 我们表明FRET之间的共轭聚合物的聚二乙炔（PDA）和荧光基团连接到PDA脂质体的生物分子的传感表面。 PDA脂质体也包含在其表面上的生物分子的受体分子可以用作探针。配体 - 受体相互作用导致这是传感机构的基础的荧光团和PDA之间的FRET效率的变化。

视频: Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

科研

生物学

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量：受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...