这项工作是由美国国家卫生研究院（AI050677，HD-051998，GM103433），马尔科姆·费斯特心血管研究奖学金，和美国心脏协会博士前的奖学金（10PRE4200007）的补助支持。

1。明胶涂层的盖玻片的制备<ol><li>放置酸洗玻璃盖玻片（15毫米直径），在无菌塑料100mm皿（晚餐）。将每道菜8-9盖玻片，并确保他们不接触对方或双方的菜.. </li></ol> 注：盖玻片，针和镊子必须是无菌的，以消除可能的污染的微生物，以及内毒素，这将影响细胞功能，包括蠕动。 <ol start="2"><li>称量明胶粉末，并重新悬浮在去离子水中的粉末，使明胶溶液的最终浓度为0.5 g/300毫升。接着，将明胶溶液需要以进行高压灭菌。 </li><li>移取2-3滴明胶（约100-150毫升），每个盖玻片上。 </li></ol> 注意：请小心不要让明胶触摸盘，否则您将无法从盘子里取出盖玻片。 <ol start="4"&gt; <li>烘烤10分钟在90℃下在烘箱中在100毫米培养皿明胶包衣的盖玻片。 </li><li>删除多余的，食用明胶温柔的移液。 </li><li> 45分钟，在100毫米的培养皿在烘箱中在70℃下干燥盖玻片。 </li><li>一旦干时，取出盖玻片100毫米的盘使用无菌，无内毒素针和镊子（针是用来轻轻上调盖玻片，使其访问的镊子轻轻取出）。 </li><li>将个人明胶包被盖玻片的24孔板到单独的井。 </li></ol> 2。制备胶体金涂层盖玻片的<ol><li>称取适量的氯金酸（四氯金酸三水合物; HAuCl 4·3H 2 O），然后重新悬浮于无菌的去离子水，以制备最终的14.5 1mM溶液（毒性）。准备1.5毫升的溶液每8-9盖玻片。 </li></ol> 警告：氯金酸为h抱吞咽，引起严重的皮肤灼伤和眼睛损伤，并可能导致皮肤过敏反应。吞食有毒。 <ol start="2"><li>称取适量的柠檬酸钠（磷酸三钠二氢2 -羟基丙烷-1，2,3 -三甲酸三乙酯;的Na 3 C 6 H 5 O 7），然后重新悬浮在去离子水中的粉末作最后的0.5％的溶液。准备1毫升的溶液每8-9盖玻片。 </li></ol> 警告：柠檬酸钠可能会引起眼睛和皮肤有刺激性。它也可能导致呼吸道和消化道的刺激。 <ol start="3"><li>在无菌的，无内毒素的烧杯中的1.5毫升的无菌14.5 mM的HAuCl 4溶液和13.5毫升的无菌去离子水相结合;结果应该得到淡黄色溶液。上述量将允许8-9胶体金盖玻片。 </li><li>在连续搅拌的同时，在热板上加热该溶液，直到它开始到b油。 </li></ol> 警告：加热该溶液应在通风橱中进行，如可以是蒸气的有害/有毒。蒸气组织的粘膜和上呼吸道有破坏性的。 <ol start="5"><li>删除从热板的烧杯中。 </li><li>在搅拌的同时，加入0.7毫升的0.5％柠檬酸钠溶液中。上述量将允许8-9胶体金盖玻片。 </li><li>保持这种组合的解决方案搅拌约2分钟（注：溶液颜色会逐渐改变，从淡淡的黄色，清除，灰色，紫色，深紫色，最后才到达红葡萄酒，最好是铁锈色） 。该产品是您的胶体金溶液。 </li><li>冷却的10毫升移液管的胶体金溶液中1-2分钟（为了保持盖玻片的明胶层上时，从熔化的胶体金溶液中加入），然后加入1.0-2.0毫升的colloida升金溶液到每个明胶包被的盖玻片放置在24孔板（ES）（您添加量的胶体金溶液的明胶包被的盖玻片上取决于如何以及在溶液中的金颗粒沉淀 - 下面的评论）。 </li></ol> 注意：因为有可能会对在金颗粒沉淀的效率的变化，它是表示第一次添加的胶体金溶液的明胶包衣盖玻片0.5-1毫升，0.5小时，然后放置在24孔培养皿中孵化器。此温育时间后，应检查盖玻片，在光学显微镜下，适当的密度的金颗粒上的盖玻片。过低和过高的浓度的金颗粒的实施例的20倍的显微镜图像，参见图1。请参阅图2为一个理想的浓度的金颗粒（至少用于与单核细胞）的40倍的显微镜图像。巴勒斯坦被占领土IMAL在幻灯片上的金纳米粒子密度应使用每种细胞类型的实验确定的，作为不同的细胞的特性将决定他们的运动的强度上的盖玻片。如果金颗粒的浓度是不足的，增加一个额外的胶体金溶液的明胶包衣盖玻片0.5-1毫升。 根据细胞的大小，适当浓度的金颗粒可以不同，从而将最终依赖于细胞的大小检查蠕动。例如，与人单核细胞是小尺寸的细胞（〜10-20微米28），需要更高浓度的金颗粒在我们的分析。黄金纳米粒子的适当分配为研究人员提供了简单而有效的区分边缘细胞的轨道。甲金颗粒浓度太高会妨碍细胞的能力，移动，因此，准确地测量他们的蠕动，而金颗粒的浓度过低限制的能力划定一个准确的跟踪蠕动。 重要注意事项：如果是有问题的黄金纳米粒子的合成，合成的纳米金是市售的尺寸从5纳米到400纳米。此外，荧光微球也被用来研究细胞运动29。但是，这些微球的使用需要分析细胞迁移的荧光显微镜。 <ol start="9"><li>与胶体金覆盖的盖玻片的培养箱中放置24孔培养皿，在37℃下孵育1小时至过夜。 </li><li>孵育后，冲洗/通过浸渍到无菌的磷酸盐缓冲盐水（PBS，pH 7.4）中的的盖玻片（3×），除去未结合的金。 </li><li>将这些胶体金涂层盖玻片，在一个干净的12孔板（ES）的PBS。 </li><li>将这些盖玻片，在4°C，直到准备（封口膜板放置在冰箱前）。 </li></ol> 重要注意：始终保持在某种类型的液体/介质的胶体金涂覆盖玻片作为金颗粒剥落从盖玻片，如果让其干燥。胶体金盖玻片，应使用之日起，他们在2-3个月内。 质量控制：在一个单一的phagokinetic轨道蠕动测定，它是由一个类似的浓度的金颗粒，其特征在于使用的盖玻片的关键。这个简单的点，将允许在蜂窝蠕动样品之间的定量差异的性质的处理，而不是胶体金涂层盖玻片的物理性质是纯粹由于。我们赞成使用只在个别的实验中在同一时间所作的盖玻片，虽然我们已经表明，只要金纳米粒子的密度是相等的，使用盖玻片从不同的制剂是适当的。 3。细胞运动分析<ol><li>将胶体金涂层盖玻片放置在一个适当的蜂窝媒体所关注的细胞。 </li><li>适当的处理后的细胞转移到胶体金涂层的盖玻片放置在24孔培养皿（晚餐） </li></ol> 注：转移到单井细胞的数目，需要确定每种细胞类型的研究。理想的情况下，细胞需要平均分布在胶体金涂层盖玻片，这反过来将允许创建的单个单元格的唯一的磁道的统计分析。如果细胞接种于浓度太高，它变得很可能发生的，将要看到的多个小区的重叠轨道。重叠的轨迹不能准确地定量，因此，它们不能被考虑在最后的分析测试细胞的移动。作为一个结果，重叠轨道参与多个细胞通常是很容易观察到，作为合并或划线的轨迹（在分析领域内的），其中两个或更多的细胞中可以观察到相同的连续清除区域。 <ol start="3"><li>孵育在37℃，C / 5％CO 2的24小时（或任何合适的时间， 例如，我们已经分析在6小时和24小时的后处理和血管内皮细胞在12小时后处理）之间的单核细胞蠕动。会随着研究的细胞类型，因此应通过实验确定了不同的细胞类型（一个很好的起点，但是，是6 - 12小时后处理）的确定和测量细胞运动的最佳时间框架。 </li></ol> 注：如果实验胶体金涂覆盖玻片需要将被存储和/或分析在稍后的时间，将细胞和金纳米粒子需要被固定在盖玻片。要做到这一点固定步骤;以下步骤3.3，先洗盖玻片仔细的2倍，1倍PBS（浸渍盖玻片是可取的，因为，从盖玻片必须避免的黄金纳米粒子的去除），然后使用标准的细胞固定方法，如3％的多聚甲醛室温孵育。孵育15分钟后，被删除的3％的多聚甲醛和盖玻片仔细洗3次，用1×PBS。可以保存在冰箱中的固定盖玻片。 <ol start="4"><li>使用光学显微镜，拍摄图像创建一个移动单元格（注：用于细胞的轨道拍照，放大倍率肯定会有所不同的细胞类型根据调查）的轨道。在图2中所示的实施例的蜂窝轨道创建的非能动和能动的细胞对胶体金涂层盖玻片。 </li></ol> 注：phagokinetic轨道蠕动测定中使用的大小的金纳米粒子已被发现是完全无毒的细胞30。如果有必要进行评估的可行性研究细胞台盼蓝染色或其他标志物检测细胞活力。人们必须考虑到需要为固定，固定型等，如果需要进行这一步。 <ol start="5"><li>使用免费的软件，如ImageJ软件（ <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ）或NIH图像（ <a href="http://rsb.info.nih.gov/nih-image/" target="_blank">http://rsb.info.nih.gov/nih-image/</a> ）的，无论是发达国家在美国国立卫生研究院（ <a href="http://ddsdx.uthscsa.edu/dig/itdesc.html" target="_blank">http://ddsdx.uthscsa.edu/dig/itdesc.html</a> ） ，或ImageTool的大学，得克萨斯大学健康科学中心圣安东尼奥，清除胶体金的平均面积（任意单位） 10-20或更多的细胞（每个样品）确定为每个实验臂从所捕获的图像。在T就可以进行统计他收集的结果。例如，结果可以绘制为意味着±的标准误差的装置（SEM）与学生的 t测试执行，和一个P值，&lt;0.05作为统计意义样品之间的措施。 图3和4展示的步骤在清除由该单元的胶体金的区域分析。 </li></ol>

<ol>
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Soc. 11, 55-75 (1951).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size+and+sol+stability+in+mental+colloids.">Particle size and sol stability in mental colloids.</a> Colloid &#38; Polymer Science. 250, 736-741 (1972).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+nucleation+for+the+regulation+of+the+particle+size+in+monodisperse+gold+suspensions.">Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions.</a> Phys. Sci. 241, 20-22 (1973).</li><li>Zetter, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+capillary+endothelial+cell+migration.">Assay of capillary endothelial cell migration.</a> Methods Enzymol. 147, 135-144 (1987).</li><li>Scott, W. N., McCool, K., Nelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+the+production+of+gold+colloid+monolayers+for+use+in+the+phagokinetic+track+assay+for+cell+motility.">Improved method for the production of gold colloid monolayers for use in the phagokinetic track assay for cell motility.</a> Anal. Biochem. 287, 343-344 (2000).</li><li>Wang, S. Y., Mak, K. L., Chen, L. Y., Chou, M. P., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+blood+monocyte:+two+subpopulations+with+different+sizes,+phenotypes+and+functions.">Heterogeneity of human blood monocyte: two subpopulations with different sizes, phenotypes and functions.</a> Immunology. 77, 298-303 (1992).</li><li>Windler-Hart, S. L., Chen, K. Y., Chenn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+behavior+screen:+identification,+sorting,+and+enrichment+of+cells+based+on+motility.">A cell behavior screen: identification, sorting, and enrichment of cells based on motility.</a> BMC Cell Biol. 6, 14 (2005).</li><li>Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-dependent+cytotoxicity+of+gold+nanoparticles.">Size-dependent cytotoxicity of gold nanoparticles.</a> Small. 3, 1941-1949 (2007).</li><li>Rodriguez, L. G., Wu, X., Guan, 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=Wound-healing+assay.">Wound-healing assay.</a> Methods Mol. Biol. 294, 23-29 (2005).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J. Exp. Med. 115, 453-466 (1962).</li><li>Brooks, D. M., Brooks, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Invasion+Assay+Using+Matrigel(R).">In Vitro Invasion Assay Using Matrigel(R).</a> Methods Mol. Med. 58, 61-70 (2001).</li><li>Kuo, J. C., Wang, W. J., Yao, C. C., Wu, P. R., Chen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+suppressor+DAPK+inhibits+cell+motility+by+blocking+the+integrin-mediated+polarity+pathway.">The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway.</a> J. Cell Biol. 172, 619-631 (2006).</li><li>Benazeraf, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+random+cell+motility+gradient+downstream+of+FGF+controls+elongation+of+an+amniote+embryo.">A random cell motility gradient downstream of FGF controls elongation of an amniote embryo.</a> Nature. 466, 248-252 (2010).</li></ol>

没有利益冲突的声明。

轨道活力的phagokinetic在这篇文章中提出的分析是定量分析细胞迁移的一个简单而非常有效的方法。由于多种细胞类型可以分析9-17，这种方法有可能跨越多个学科的广泛使用。胶体金镀膜玻璃盖玻片的使用允许测量由移动的小区的轨道区清零。该法可衡量的效果，不同的刺激（ 如生长因子，纯化的ECM配体，病毒，细菌）对细胞活力。此外，缺乏监测细胞迁移的要求，进行细胞固定，...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td>盖玻片（15毫米） </td><td> Fisher Scientific则</td><td> 12-545-83 </td><td></td></tr><tr><td>明胶300布鲁姆</td><td> Sigma-Aldrich公司</td><td> G-1890 </td><td></td></tr><tr><td>四氯金酸三水合物</td><td>费舍尔化工</td><td> G54-1 </td><td> 14.5毫米（最终工作液） </td></tr><tr><td>柠檬酸钠</td><td> Fisher Scientific则</td><td> BP327-500 </td><td> （最后工作溶液）0.5％ </td></tr><tr><td>多聚甲醛</td><td> Fisher Scientific则</td><td> O4042 </td><td> 3％（最后工作溶液） </td></tr><tr><td> 100毫米组织培养皿</td><td>萨尔斯塔特</td><td> 83.1802 </td><td></td></tr><tr><td> 12孔板</td><td> Fisher Scientific则</td><td> 08-772-29 </td><td></td></tr><tr><td> 24孔板</td><td> Fisher Scientific则</td><td> 07-200-84 </td><td></td></tr><tr><td>泰科的烤箱Hybridiser HB-1D </td><td> LabPlanet </td><td> 2040500 </td><td>标准的实验室烤箱就足够了</td></tr><tr><td> 10毫升血清学移液管</td><td>萨尔斯塔特</td><td> 86.1254.001 </td><td></td></tr><tr><td>吸取援助灌装机/饮水机</td><td>德拉蒙德</td><td> 13-681-15 </td><td></td></tr><tr><td> P200单通道手动移液器</td><td> RAININ </td><td> PR-200 </td><td></td></tr><tr><td> 200毫升障碍提示</td><td>中电</td><td> BT200 </td><td></td></tr><tr><td> ImageJ软件</td><td> <a href="http://rsb.info.nih.gov/ij/" target="_blank">HTT</a>p :/ / rsb.info.nih.gov的/ IJ / </td><td></td><td>许可证：公共领域</td></tr><tr><td>尼康的Eclipse TE300一个配光CoolSNAPfx的单色12位的CCD相机</td><td>尼康</td><td></td><td>停产;最具可比性的规格有尼康的Eclipse钛，以及较低的尼康80I将是适当的。其他品牌也提供了类似的显微镜。 </td></tr><tr><td></td><td></td><td></td><td> 注意：下面列出的试剂和设备已动用我们在我们的各种研究。其他供应品，供应商，试剂和设备都可以使用，只要他们有类似的规范。 </td></tr></tbody></table>

a quantitative evaluation of cell migration by the phagokinetic track motility assay

单细胞和多细胞生物体细胞的活力是一个重要的生物过程。它的来源的营养物或远离不适合的条件，以及在多细胞生物组织的发展，免疫监视和伤口愈合，只是提了几个角色1,2,3的单细胞生物向运动是必不可少的。撤销这个过程可能会导致严重的神经系统，心血管疾病和免疫系统疾病，以及加剧了肿瘤的形成和传播4,5。分子，肌动蛋白聚合和受体回收已被证明发挥了重要作用，在细胞的扩展名（片状伪足），即推动向前运动的细胞6,7,8。然而，许多生物细胞迁移的问题仍然没有答案。 在人类健康和疾病的细胞运动中的核心作用突出的重要性，了解具体的MEChanisms参与了这一过程，并作出准确的方法来评估细胞的运动，特别是重要。显微镜通常是用于可视化细胞的运动。然而，细胞移动相当缓慢，耗费资源的过程，需要昂贵的相机和软件创建时间的推移定量电影运动细胞的细胞迁移的定量测量。因此，进行定量测量细胞迁移，是符合成本效益的，不费力，而且采用了常见的实验室设备的能力是一个伟大的许多研究人员的需要。 phagokinetic轨道蠕动测定法利用能力的移动单元以清除金颗粒，从它的路径建立一个可测量的磁道上的胶体金涂覆的玻璃盖玻片9,10。使用的自由软件，可以评估多个音轨，每次治疗完成统计的要求。该试验可以用来评估许多类型的细胞，如癌细胞11,12，成纤维细胞9，嗜中性粒细胞13，骨骼肌细胞14，角质形成细胞15，的滋养层16，血管内皮细胞17，和单核细胞10,18-22能动性。该协议涉及创建由柠檬酸钠，氯金酸（互惠3 +）的减少所生成的金纳米粒子（互惠°）涂覆的幻灯片。这种方法的开发由Turkevich 等人于1951年23，然后在20世纪70年代的改善Frens 等。24,25。作为一个结果，该化学还原步骤中，金颗粒（10-20 nm直径）从反应混合物中沉淀，并可以应用到玻璃盖玻片，然后准备用于在细胞迁移分析9,26,27。 在一般情况下，phagokinetic轨道蠕动法是一种快速，定量和容易的细胞能动性的措施。此外，它可以用来作为一个简单的高通量的检测，使用的细胞类型，不适合时间推移的成像，以及其他用途，取决于需要的研究员。一起，定量地测量多种细胞类型的细胞运动，而不需要昂贵的显微镜和软件的能力，以及常见的实验室设备和化学品的使用，作出的phagokinetic的轨道活力测定了坚实的选择，有兴趣的科学家了解细胞蠕动。

所示出的是示出了轨道区清零由一个单一的细胞（单核细胞从我们的实验是在图2中所示）在光学显微镜下拍摄的图像的一个例子。非运动细胞创建特征的小，椭圆形或圆形大片自己周围指示低基础水平的运动为这些未刺激的细胞（ 图2A和2B）。高度运动细胞[在我们的系统中，人巨细胞病毒（HCMV）感染的细胞]与此相反，其特征在于由作为细长的轨道区?...

Watch this Scientific Journal Video about A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay at JoVE.com

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

phagokinetic蠕动轨道法是一种方法，用于评估细胞的运动。具体而言，测定测量化学增活现象（随机细胞运动性），以定量的方式随着时间的推移。该试验利用细胞的能力，以建立一个可测量其运动轨迹胶体金涂覆盖玻片。

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

a-quantitative-evaluation-cell-migration-phagokinetic-track-motility

Research

JoVE Journal

Immunology and Infection

13.8K 次观看.  Louisiana State University Health Sciences Center. phagokinetic蠕动轨道法是一种方法，用于评估细胞的运动。具体而言，测定测量化学增活现象（随机细胞运动性），以定量的方式随着时间的推移。该试验利用细胞的能力，以建立一个可测量其运动轨迹胶体金涂覆盖玻片。

视频: A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

科研

免疫与感染

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...