这项工作是由NSERC个人发现程序的NSERC / CIHR协作的健康研究计划（CHRP）和NSERC的CREATE加拿大天体培训计划（CATP）。

1。量子溶这是一个对于CdTe的适当方法。类似的方法可以用来与其他类型的量子点，如磷化铟/硫化锌20和硒化镉/硫化锌21。 <ol><li>准备在15μM（光密度= 2.5在第一激子峰）的甲苯CdTe量子点的解决方案。 </li><li> 200μL此股票转移到玻璃小瓶。不要使用塑料！ </li><li>加入800μL的甲苯，1 mL的200 mM硼酸盐缓冲液（pH值9）和11.5米巯基丙酸（MPA）2μL。 </li><li>例小瓶和大力摇晃30-60秒。 </li><li>水和有机溶剂相分离油然而生。水相，将成为量子点的颜色。为了避免甲苯层，删除它，抛弃它。然后转移到一个干净的小瓶水相。 </li><li>四次使用500微升，10000分子量截止离心机的过滤清洗/过滤净化溶解量子点。过滤器应在13分钟3000 XG纺。至少在过去两年清洗，用理想的最终缓冲区。 </li><li>最后一次洗涤后，暂停50 mM硼酸盐缓冲液洗过的量子点所需的pH值并储存在4°C他们将是稳定为1-2周。 </li><li>吸收和发射光谱测量来估计基于公布的公式22的浓度。 </li></ol> 代表结果:图1 CdTe量子点的图像显示紫外灯照射下，发射光谱，溶水前后帽交换微不足道的变化。大小的值是通过电子显微镜的核心直径测量。 2。量子点共轭以抗生素本协议的一部分，是适用于任何带负电荷的水溶解的纳米粒子，包括最商业的量子点，金属颗粒，和更多的19。 <ol><li>任何分子将工作，eith呃自组装到带负电荷的溶解量子点，或具有活性基团，如伯胺，可以结合。在这个例子中，我们使用的是多粘菌素B（港航局），这是带正电荷和自组装，而不需要对共轭试剂。 60微米，溶解在H 2 O的港口及航运局。 （如果选择的抗生素是不溶于H 2 O，溶于DMSO或乙醇浓度在0.1-1毫米）。这将是10倍的解决方案（H2O）或100倍溶液（溶剂）。 </li><li>计算需要多少量子点的解决方案，你会为细菌的毒性试验。为0.3毫升，每口井在一式四份的96孔板，需要0.5毫升的200 nm的共轭。准备两管含100μL1微米量子点在50毫米的硼酸盐缓冲。 </li><li>如果耦合小学胺，准备了19毫克/毫升（100毫米），1 -乙基-3 - [3 -二甲氨基]碳二亚胺盐酸溶液中H 2 O（东区区议会） EDC是在溶液中不太稳定;使用立即抛出的休息带走。 </li><li>加入50μL10抗生素溶液（H 2 O）或5μL的100倍溶液（溶剂）的共轭管。为港口及航运局:碲化镉共轭摩尔比30:1，加入50μL60μM的港口及航运局。 H 2 O或溶剂的QD只控制管。 </li><li>如果需要，添加共轭管EDC原液1μL。 </li><li>带来了相应的缓冲区（硼酸盐缓冲或PBS，如Tris，不含有胺缓冲区，因为它们会抑制的EDC）共轭量0.5毫升。 </li><li>盾构管光铝箔，并在1小时nutator或摇杆的地方。如果发生聚集，重复共轭用低浓度的抗生素。滴定的浓度上升，直到颗粒不总额。 </li><li>通过适当的过滤器的清洗标记。 10000截留分子量对大多数抗生素，但不适用于蛋白质或抗体。 </li&gt; <li>根据分子，不同的方法可以用来估计每个量子点抗生素分子吸收或荧光光谱，凝胶电泳，或傅立叶变换红外光谱（FTIR）。 </li></ol> 代表性的结果。在这个例子中，港口及航运局共轭的特点是由量子点发射光谱的变化。 图2显示了与港口及航运局除了CdTe量子点的光谱。 3。 96屏幕的制备细菌抗生素的IC 50的测定; 这是适用于几乎所有的菌株生长在适当的培养基18。确切的时间记录应继续长度取决于细菌的增长速度。在我们的例子中，我们使用大肠杆菌溶源性肉汤（LB）培养基中生长。 <ol><li>为了选择适当的QD共轭浓度，重要的是要知道IC的抗生素50是共轭，如果量子点本身是有毒的。这应开始前2天，共轭实验。在晚上，从一个全新的殖民地种子10毫升细菌生长的培养基，使用正确的消毒和生物安全技术。 </li><li>第二天，实验前1-2小时，填补近最大金额的生长介质板一个明确的底部96井。使用多道移液器，种子，每口井的细菌培养与1-50μL（浓度将取决于特定菌株的生长速度有多快，将需要您的实验室校准）。 </li><li>放进酶标仪板，将它设置为读2小时，每隔10分钟，在一个确定的波长（通常是600纳米）。监控板，使细菌不生长过于密集，太快。 </li><li>当细胞达到外径= 0.1-0.15，从酶标仪板和停止录音。 </li><li>单独添加药物和量子点独自一人不同浓度至少一式三份。使用板的一面，作为一个"黑暗"的控制和一半被照亮。 图3给出了一个建议布局。浓度应跨越一个足够宽的范围内包括的浓度，抑制细菌很少，并杀死他们。 </li><li>屏蔽铝箔板的一侧的"黑暗"。揭露对方在所需波长的灯。我们使用一个自定义的96孔，440纳米灯从2.4兆瓦的发光二极体（ 图4）照射30-60分钟。有明确的底部的黑色板，帮助未曝光的细菌免受杂散光。暴露和未暴露双方之间也可以使用一个空列。 </li><li>光线照射后，放入酶标仪和纪录外径600取决于增长率为5-12小时，每10分钟的板块。保持温度&lt;32℃，如果可能的话，以避免干燥的文化。 </li><li>绘制生长曲线在选定的时间点与日志[浓度]和确定的IC 50对抗生素的使用希尔方程: </li></ol><img alt="式（1）" src="/files/ftp_upload/3969/3969eq1.jpg" /> 其中 H为Hill系数，Y 最大的增长（位于高原）的最高点，并Ÿ 分是零点，在高原也理想。这是不可能单独量子点表现出很大的毒性，在使用浓度的细胞，因此将无法确定一个值。 有代表性的成果。在录音期间结束时，明确井将显示完整的细胞死亡，细胞密度梯度应该一起出现的药物浓度增加。细菌应该显示出S形的生长曲线（ 图5）;高原最大的位置将有很大的差别应变应变，也依赖于温度。可以是一个给定的时间点为代表的选择和值绘制与日志[抗生素]给的IC 50（ 图5）。来评估量子点的毒性，生存与日志[量子点也可以被绘制，但与量子点实现显着的细菌杀死，仅仅是罕见（ 图5）。 4。细菌与抗生素/量子点的屏幕为96的制备<ol><li>一天前的试验，种子10毫升到适当的营养丰富的培养基从一个全新的殖民地文化，用正确的消毒和生物安全技术。 QD抗生素共轭应准备在这一天，除非它是非常不稳定的。 </li><li>实验前1-2小时，填补近最大金额的生长介质板一个明确的底部96井。 1-50细菌培养液使用多道移液器，种子，每口井。 </li><li>放进酶标仪板，将它设置为2小时，每10分钟在相同的波长，在第3部分读。</li><li>当细胞达到外径= 0.1-0.15，从酶标仪板和停止录音。 </li><li>至少一式四份中添加不同浓度的量子点标记。使用板的一面，作为一个"黑暗"的控制和一半被照亮。 图6给出了一个建议布局。一个细菌只控制条应始终包括在文化，温度的情况下问题，影响生长条件。一种药物只单一带和QD只也应列入，以确保它们符合以前完成控制盘。其余的井都致力于标记，以便为良好的统计。 </li><li>屏蔽铝箔板的一侧的"黑暗"。揭露对方在所需波长的灯。 </li><li>光线照射后，放入酶标仪和600记录外径为5-12小时，每10分钟的板块。保持温度&lt;32°C，如果可能，以避免博士英文化。 </li></ol> 有代表性的成果 。量子点和抗生素的结合，可能是抗生素的毒性小于单独同样有毒或毒性更大。这是可以量化的生长曲线和IC 50测量。 图7显示了一个例子，同样作为抗生素的毒性标记，更有毒的标记，例如。 5。平皿计数<ol><li>选择一个或多个井治疗的96孔板，可用于连续稀释和菌落计数。 </li><li>使每个PBS或生理盐水溶液（0.9％氯化钠）与选定的细菌样本的连续稀释。转移20μL的细菌溶液和180μL盐水稀释，改变枪头，混合和转移20μL180μL盐水稀释细菌的解决方案。重复6次。 </li><li> 10μL，在适当采取从每个稀释和平板<html>固体介质板。一个样品的全部8浓度可能被镀上了10厘米的圆形培养皿，虽然矩形菜是首选，因为它是更容易办事。 </li><li>让它干上15分钟的替补。然后在适当的温度为您的细菌孵化16个小时。 </li><li>计数菌落，计算菌落形成单位（CFU）根据（＃殖民地x稀释倍数）/体积镀CFU /毫升。 </li></ol> 图8显示了一个例子菌落形成单位板。 6。代表结果<img alt="图1" src="/files/ftp_upload/3969/3969fig1.jpg" /> 图1。CdTe量子点。 （一）八CdTe量子点的准备照亮紫外棒（365纳米）。 （二）五大小选定前，后水溶解吸收和发射光谱。虚线是在甲苯中的光谱，实线是水。 files/ftp_upload/3969/3969fig2.jpg"/&gt; 图2。QD-港航结合物的光谱和凝胶分析。橙色发光CdTe量子点用于这个例子，对其他类型的量子点的影响将需要为每个实验评估。 （一）典型的吸光度（灰线）和发射光谱（黑线）的量子点之前，港口及航运局和发射后的160个港口及航运局的摩尔等值除了（虚线）的共轭。 （二）港航比和量子点发光强度（正方形）和峰值波长位置（三角形）之间的关系。 <img alt="图3" src="/files/ftp_upload/3969/3969fig3.jpg" /> 图3。建议板控制生长板的布局。代表一个广泛的港口及航运局和QD浓度。辐照板的二分之一（高亮蓝色），相同的一半是由轻保护。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3969/3969fig3large.jpg" target="_blank">点击这里</a>查看大图。 <img alt="图4" src="/files/ftp_upload/3969/3969fig4.jpg" /> 图4。自96 LED灯板照射均匀，显示和外观关闭。一个典型的手持式紫外灯也可能被使用，但不会覆盖整个板均匀。 <img alt="图5" src="/files/ftp_upload/3969/3969fig5.jpg" /> 图5。控制生长板的例子结果。 （一）代表不同的药物浓度与细菌生长曲线，从0到完成细胞死亡。开放的符号是港口及航运局只用浓度定;坚实的符号是没有辐射的CdTe，港口及航运局和半满的符号与辐照碲化镉，港口及航运局。照射没有港航局只样品的效果，因此这些曲线，为清晰起见，省略。所有港口及航运局的CdTe标记是30:1港口及航运局:量子点比率。 （二）符合地块生长曲线值在200分钟与日志[港航]和式。 （1）。到CONTROL光线的影响，曲线是抗生素，只用灯光照射30分钟。 （三）在200分钟与CdTe量子点，量子点浓度的细菌生存。一定的毒性被曝光，但过于不大确定的IC 50值。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3969/3969fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/3969/3969fig6.jpg" /> 图6。建议共轭测试板的布局。蓝色突出显示一半的板块应暴露在光线下，保护的unhighlighted一半。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3969/3969fig6large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图7" src="/files/ftp_upload/3969/3969fig7.jpg" /> 图7例共轭测试板的结果。在200分钟的生长曲线值分别为PLotted和适合式。 （1）。 （一），碲化镉，港口及航运局的标记显示增加对港航单独的毒性。 （二）黄金纳米金港航局共轭不超过港航仅增加毒性。 <img alt="图8" src="/files/ftp_upload/3969/3969fig8.jpg" /> 图8。的菌落形成单位板块为例。E.在96孔板接种大肠杆菌与QD-港航治疗或未经照射30分钟。然后在32°C孵育4小时。每个细菌样品连续稀释用生理盐水溶液，10μL100×10 7 X稀释琼脂平板上接种。培养板在37°C和殖民地后16小时计算。板显示沿指示行的稀释;列是:（一）港口及航运局2纳米CdTe的0.06微米，（二）0.12微米的PMB + 4 nm的碲化镉（三）0.2μM的港口及航运局+ 6.7纳米CdTe的，（四） 0.06微米港口及航运局+ 2 nm的碲化镉照射，（五）0.12微米港航+ 4 nm的碲化镉照射（F）0.2μM的港口及航运局+ 6.7纳米CdTe的照射diated。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+be+inferred+from+bacterium-nanoparticle+interactions+about+the+potential+consequences+of+environmental+exposure+to+nanoparticles.">What can be inferred from bacterium-nanoparticle interactions about the potential consequences of environmental exposure to nanoparticles.</a> Ecotoxicology. 17, 362-371 (2008).</li><li>Kovochich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+C60+aggregates+toward+mammalian+cells:+role+of+tetrahydrofuran+(THF)+decomposition.">Comparative toxicity of C60 aggregates toward mammalian cells: role of tetrahydrofuran (THF) decomposition.</a> Environ. Sci. Technol. 43, 6378-6384 (2009).</li><li>Hoshino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+properties+and+cellular+toxicity+of+nanocrystal+quantum+dots+depend+on+their+surface+modification.">Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification.</a> Nano Letters. 4, 2163-2169 (2004).</li><li>Dumas, E. M., Ozenne, V., Mielke, R. E., Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+CdTe+Quantum+Dots+in+Bacterial+Strains.">Toxicity of CdTe Quantum Dots in Bacterial Strains.</a> IEEE Trans. NanoBiosci. 8, 58-64 (2009).</li><li>Park, S., Chibli, H., Wong, J., Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+and+cellular+toxicity+of+nanoparticle-polymyxin+B+conjugates.">Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates.</a> Nanotechnology. 22, 185101 (2011).</li><li>Cooper, D. R., Dimitrijevic, N. M., Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photosensitization+of+CdSe/ZnS+QDs+and+reliability+of+assays+for+reactive+oxygen+species+production.">Photosensitization of CdSe/ZnS QDs and reliability of assays for reactive oxygen species production.</a> Nanoscale. 2, 114-121 (2010).</li><li>Pong, B. K., Trout, B. L., Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+ligand-exchange+for+efficient+solubilization+of+CdSe/ZnS+quantum+dots+in+water:+A+procedure+guided+by+computational+studies.">Modified ligand-exchange for efficient solubilization of CdSe/ZnS quantum dots in water: A procedure guided by computational studies.</a> Langmuir. 24, 5270-5276 (2008).</li><li>Narayanaswamy, A., Feiner, L. F., Meijerink, A., Zaag, P. J. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+and+dot+size+on+the+spectral+properties+of+colloidal+InP/ZnS+core-shell+quantum+dots.">The effect of temperature and dot size on the spectral properties of colloidal InP/ZnS core-shell quantum dots.</a> Acs Nano. 3, 2539-2546 (2009).</li></ol>

没有利益冲突的声明。

纳米粒子代表一个充满希望的方式来创造新的抗微生物药物。生长曲线分析是一种方法来监测细菌细胞密度区分积极从生长抑制细胞生长的细胞。加上平板计数时，它可以让一个共轭抗生素潜力的深入分析。 96格式允许浓度相对高通量变化和其他条件，如光线照射;后者是光活化剂，如量子点是至关重要的。这个基本的方法上的许多变化是可能的，使它成为一个纳米毒理学的通用的方法。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>硼酸盐缓冲组件＃1 </td><td>费舍尔</td><td>硼酸-74-1 </td><td></td></tr><tr><td>硼酸盐缓冲组件＃2 </td><td> Sigma-Aldrich公司</td><td>四硼酸钠B9876 </td><td></td></tr><tr><td> MPA的</td><td> Sigma-Aldrich公司</td><td> M5801 </td><td></td></tr><tr><td> vivaspin 500 </td><td> GE医疗集团</td><td> 28-9322 </td><td>可以各种截留分子量</td></tr><tr><td>玻璃瓶</td><td>费舍尔</td><td> 03-338C </td><td></td></tr><tr><td>东区区议会</td><td> Sigma-Aldrich公司</td><td> E6383 </td><td></td></tr><tr><td>多粘菌素B </td><td> Sigma-Aldrich公司</td><td> P1004 </td><td></td></tr><tr><td>细菌GR零增长培养基（LB）组件＃1 </td><td>费舍尔</td><td>氯化钠S271 </td><td></td></tr><tr><td>细菌生长的培养基（LB）组件＃2 </td><td>屋宇署</td><td>蛋白胨211705 </td><td></td></tr><tr><td>细菌生长的培养基（LB）组件＃3 </td><td>屋宇署</td><td>酵母提取物211929 </td><td></td></tr><tr><td>光线照射灯</td><td>习俗</td><td></td><td></td></tr><tr><td>清除底的96孔板</td><td>费舍尔</td><td> 07-200-567或07-200-730 </td><td></td></tr><tr><td>荧光光谱仪</td><td>分子器件</td><td></td><td></td></tr><tr><td>吸收板读卡器</td><td>分子器件</td><td></td><td></td></tr><tr><td>用于固体介质BactoAgar </td><td> bioshop </td><td> AGR001.1 </td><td></td></tr><tr><td>培养皿中圆</td><td>费舍尔</td><td> 08-75-12 </td><td></td></tr><tr><td>培养皿中的矩形</td><td>费舍尔</td><td> 08-757-11A </td><td></td></tr></tbody></table>

solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

量子点（量子点）的大小取决于发射光谱荧光半导体纳米可以通过广泛的波长选择兴奋。量子点成像，诊断和治疗，由于其明亮，稳定的荧光1,2 3,4,5吸引了很多人的兴趣。量子点可以结合多种生物活性分子结合到细菌和哺乳动物细胞6。 量子点也被广泛的调查，有针对性的杀死细菌的细胞毒性药物。乘耐药菌株的出现，正在迅速成为一个公共健康危机，特别是在7革兰阴性菌的情况下。由于众所周知的某些纳米材料，特别是银的抗菌效果，有数以百计的研究研究纳米粒子的毒性细菌8。与其他类型的半导体纳米粒子，以及进行细菌研究，ESPECially纳米TiO 2 9,10-11，而且氧化锌12和其他包括CuO的13。在这些研究中一些比较菌株已执行，通常比较，用革兰氏阳性的革兰阴性应变。所有这些粒子毒性的机制是由于氧化作用:无论是光学生成的活性氧（ROS）的颗粒或直接释放的金属离子可以引起氧化毒性。即使这些材料，不同的研究结果差异很大。在一些研究中的革兰氏阳性的测试应变据说超过革兰氏阴性10敏感;有些是相反的14。这些研究已审查15。 在所有纳米研究，颗粒组成，大小，表面化学，样品老化/击穿，和波长，功率，光线照射的时间都可以显着影响的结果。此外，甲醇合成小号的副产品和溶剂必须考虑16 17。高通量筛选技术需要能够制定有效的新的纳米药物。 CdTe量子点有18单独或与抗生素联合抗微生物作用。在先前的研究中，我们发现，抗生素的耦合，以碲化镉可以增加对细菌的毒性，但下降到哺乳动物细胞的毒性，由于减少活性氧的生产共轭19。虽然这是不太可能将批准供人类使用含镉化合物，这类制剂可用于表面或水的杀菌消毒。 在这个协议中，我们给出一个简单的方法，以巯基丙酸（MPA）的溶解与CdTe量子点。量子点是准备在一个小时之内使用。然后，我们证明耦合抗菌剂。 该协议的第二部分演示了96抑菌试验用结合和游离量子点。读了许多小时的光密度，允许QD加法和光线照射后恢复期间，以及立即进行评估的影响。我们也说明了量化细菌生存的殖民地计数。

Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

抗微生物或抗癌的应用，如半导体量子点（量子点）的纳米粒子可以被用于创建photoactivatable代理。此技术演示如何水溶解碲化镉（碲化镉）量子点，他们共轭抗生素，并执行基于生长曲线和平板计数的细菌抑制法。

量子点的生长曲线和平皿计数的细菌毒性检测的增溶作用和生物共轭

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

solubilization-bio-conjugation-quantum-dots-bacterial-toxicity-assays

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.2K 次观看.  McGill University, Montreal, QC Canada. 抗微生物或抗癌的应用，如半导体量子点（量子点）的纳米粒子可以被用于创建photoactivatable代理。此技术演示如何水溶解碲化镉（碲化镉）量子点，他们共轭抗生素，并执行基于生长曲线和平板计数的细菌抑制法。

视频: Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长:神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

科研

生物工程

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ，1 ps Yb:YAG薄盘再生放大器

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

乳腺辐照有机体的生长与表征

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...