的承认通过国防部的资助，国会导演医学研究计划（＃W81XWH-10-1-0445），美国国立卫生研究院（NIH R21 HL110056），和美国心脏协会（＃11SDG4890030）。

1。缓冲器和测试代理的制备和消毒<ol><li> 150mM NaCl的缓冲液：溶解在500毫升水nanopure4.383克NaCl的晶体。 </li><li> pH缓冲液：准备在pH为5.6，6.2，6.8和7.4的磷酸盐缓冲液混合适量的一元和二元磷酸钠。如果样品是在较低的pH值（ 即pH &lt;5.6），然后一个更合适的缓冲液，如柠檬酸盐缓冲液进行测试，应该被使用。缓冲液配方都是现成的，这里提供了一个例子参考。 </li><li>消毒瓶顶真空过滤装置，并重新检查缓冲液通过上面提到的所有缓冲区。 </li><li> 20％的Triton X-100（阳性对照）：混合20毫升纯的Triton X-100在80毫升水nanopure。涡大力超声溶解。发布在使用前在室温下过夜。 </li></ol> 2。红细胞制备<ol><li>获得25毫升的血液FROM一个匿名的人的捐助，直接绘制到K-EDTA涂层Vacutainer管，以防血液凝结。 </li></ol> 注意：所有程序必须事先批准的适当的机构审查委员会（IRB），静脉穿刺采血必须由经过培训的抽血以捐赠者的风险降到最低。标准放血程序已在其他地方发表。 <ol start="2"><li>离心血在500×g离心5分钟，和标记的血细胞比容水平（红色，下层）和血浆（微黄，上层）上管。 </li><li> 通过微量移液器轻轻吸出血浆，添加漂白剂，生物危险性废物丢弃到。 </li><li>用150mM NaCl溶液填充的血细胞比容管，标记线（原件的血浆水平）。盖上盖子，颠倒几次，轻轻拌匀。 500×g离心5分钟离心。 </li><li>重复步骤2.3-2.4再次洗血细胞。然后吸超自然蚂蚁和更换PBS在pH值7.4。反相，混合。 </li><li>斯普利特血液均匀地进入四根管，对应于将被测试的每个pH。标签的管，根据每个以进行测试（5.6，6.2，6.8，7.4）的pH值。 </li><li>在500×g离心5分钟，离心血液试管。马克的水平上管，然后吸出上清液。 </li><li>填充适当的缓冲液的pH值（2.6）每管标线。 </li><li>出版商四个50毫升锥形管（每一个pH值至测试），以及移液管49毫升PBS适当的pH值到每个锥形管。 </li><li>成相应的管中加入1毫升的红细胞（相同的pH值）为1:50的稀释。目视检查稀释血液，应浑浊，解决不受干扰。如果没有颗粒的形式，细胞裂解。 </li></ol> 3。裂解含量96孔板安装和定量分析<ol><li>储备液的配制所有的实验给药制剂，在20倍所需的最终浓度（含量进行测试将10μl的药物输送剂+ 190μl稀释的红血细胞，导致原来的药物输送剂的1/20稀释到最终测试混合物中）。建议为20，100，和800微克/毫升的股票，从而导致在最终测试浓度的1，5，和40微克/毫升，分别。 </li><li>移液管将10μl成V形底96孔板的每个原液。为了达到最佳效果，加载每个样品一式三份或四份。 </li></ol> 注意：为便于，建议为每个pH值来进行测试，用每个样品（在每一浓度）加载在n = 3-4每板一个单独的96孔板中，应制备。 <ol start="3"><li>阳性对照孔，加10微升20％的Triton X-100。 </li><li>对于阴性对照孔，加入10μl磷酸缓冲液。使用相同的pH值的缓冲液，以进行测试。 </li><li>吸取190μL稀释的红细胞（2.10），每孔，确保细胞原液保持同质传输过程中。提示：使用多道移液器简化这一任务。 </li><li>在37℃下温育平板一小时（可选：使用轨道摇床或摇臂）。 </li><li>离心5分钟的板在500 XG颗粒完好红细胞的。注意：当从离心机除去板输送到下一步骤时，小心处理，某些不破坏细胞沉淀。 </li><li>使用多通道移液管，从每个孔中加入100μl的上清液转移到一个清晰的，平底96孔板中。注：如果将细胞沉淀的任何样品（）被意外干扰，一个可以重新离心板，然后继续进行，上清液转移。 </li><li>用酶标仪测量上清液的吸光度。需要注意的是，可以使用一定范围的波长（400 - 541纳米）。 </li></ol> 注意：不同的板块读者可能有不同的饱和点和敏感，因此在选择的波长为我asurements取决于是否从实验样品的溶血数据可以可靠地对最大溶血归洗涤剂处理所诱导的。这就要求精确测量的阳性对照样品的吸光率值。 <ol start="10"><li>使用Microsoft Excel或类似的数据分析软件，找到从每个pH值（步骤3.4）的阴性对照样品的背景吸光度读数的平均值。这样的背景下吸光度值减去所有其它样品进行测量，pH值。 </li><li>背景减法之后，发现阳性对照的平均吸光度，洗涤剂处理的样品（步骤3.3）。然后正常化所有的实验数据点，这意味着吸光度值，它应该代表100％溶血。最后，乘以每口井的100％计算％溶血发生在每一个人的洗涤剂控制。 </li></ol>

<ol>
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IRB的批准程序已经批准了涉及人类受试者的范德比尔特大学机构审查委员会（IRB议定书＃111251）。没有利益冲突的声明。

的pH响应性聚合物或其他代理设计用于endosomolytic功能可以迅速和有效地筛选的基础上裂解过程中遇到的内体，在pH值的红血细胞（ 图1，pH为6.8 -早 ​​期内体，pH值6.2 -晚期内含体，pH为5.6 -溶酶体）14-17 pH依赖性溶血已用于筛选进行调解内体释放的生物大分子的治疗药物（ 例如肽，的siRNA，反义寡核苷酸，蛋白质）的载流子的能力，并且该测定的结果，可以预测的性能，作为细胞内的药物递送载体3,4,8,18,19因此，该测定表示一个有效的屏幕上的能力来衡量聚合物药物载体介导细胞内药物传递基于它们的pH依赖性的膜破坏。 正如在程序中，被检测到，通过分光光度法测量的红血细胞的上清液与实验治疗溶血代理商。因此，在除了血红蛋白，它是可能含有其它的红细胞源性胞浆组件，包括蛋白质和碳水化合物。虽然这些其它组件可贡献一个小的信号量的分光光度测量，100％的溶血校准从治疗与Triton X-100所导致的红细胞溶胞产物。假设所有来自同一供体的红细胞中含有血红蛋白和其他生物大分子的水平相似的，我们可以有把握地得出结论的&#39;污染&#39;的组件将不会带来任何文物的溶血测量，特别是在同样的血液样本用于所有测试。然而，这凸显了这样一种可能性，对于任何给定的献血者中，有可能是小天的日常血液成分和血细胞比容的变化，因此，内部控制样品（的步骤3.3-3.4，3.10-3.11）应所有实验分析。 人们还应该总是仔细检查原料ABSorbance数据。虽然它不能中的归一化的例子在图2中所示的数据可以理解，洗涤剂如Triton X-100的有效地破坏红细胞膜的稳定的pH值无关，和一个应该期望看到很少的样品，在阳性对照样本变异。的Triton X-100的吸收光谱不包括此测定法推荐的在400-600 nm范围内的峰值，因此，不应该干扰实验数据的归一化20此外，对于阴性对照样品，应该没有观察到显着的在任何使用的缓冲器（ 即 ，即使是最酸性缓冲液通常不会在此时间范围内产生溶血）中孵育1小时后溶血。阴性对照样品的背景读数应密切近似的背景上新鲜的缓冲液的吸光度读数。 理想情况下，研究人员将采用互补的战略来描述他们的经验rimental药物输送系统。为的初始endosomolytic剂筛选上天然存在的生物膜的溶血试验是有利的，但应被视为仅用于测试胞质投递代理的药物输送研究者的医疗设备中的许多工具之一。例如，溶血测定的一个潜在的缺点是它利用的红血细胞的膜，核内体膜的生物模型。然而，细胞型化妆和核内体膜的脂质含量变化，可能不能准确地由血细胞膜扼要重述。1有多种其他的，互补的检测已经发展到模仿体膜的组合物和行为21， 22的一种替代方法是利用脂质体的荧光共振能量转移（FRET）淬火成为非淬火后，不稳定的脂质体和释放到周围介质的荧光团的荧光团，。在研究日采用这种方法时，定量的非淬火荧光团已发现与车辆的能力介导其有效载荷的内体逃逸21,23显微镜为基础的测量可以提供更健壮的，但较低的吞吐量的方法，该方法是互补溶血试验。例如，它是常见的与染料标记的溶酶体（ 例如 LysoTracker由Life Technologies），以评估共存的载体或药物本身，或贩卖通路可以使用pH值敏感的染料的共轭的载体或药物（ 如 pHrodo通过其特征在于Life Technologies公司）24-26 一旦一个endosomolytic的输送系统已被证实胞浆货物交付，溶血试验，也可以在核内体逃逸的发生机制，通过提供信息。例如，基因运载工具的聚乙烯亚胺（PEI）的基础上缺乏的固有能力，扰乱磷脂膜在中性或酸性pH值11,27相反，PEI实现胞质基因传递通过的“质子海绵”效应。 PEI内部化后，缓冲区内吞体“吸收”整个体膜质子泵酸化这些车厢。最终，这将导致过量的质子和其反离子内的核内体的积聚。这将导致在一个上升的渗透压，水侵，囊泡肿胀和endosomolysis。因此，成功的质子海绵效应需要的积累的临界浓度的PEI成的内体27，送货车辆通过“质子海绵”的效果，实现内体中断不会物理破坏红血细胞或脂质体，以及它们的功效在实现细胞内药物传递，必须通过渗透压计算评估，或在体外显微镜，或功能的研究。 总之，溶血试验描述在这里是一个可靠的药物筛选模型设计的生物药物的细胞内输送。本试剂盒提供了高吞吐量的药物输送装置，车辆检查，使与细胞内的目标，提供生物制剂的制剂的快速发展。

虽然有许多潜在的高影响力的治疗靶点在细胞内，细胞内输送的代理商带来了显着的挑战。通常，药物，尤其是生物制剂，被细胞内化，贩卖到囊泡，要么导致它们的内容通过内溶酶体途径降解，或穿梭出细胞通过胞吐作用。1在后者的方法中，内部的pH值囊泡酸化至约5-6，这是活性的酶的最佳pH值，在此室的功能，如溶菌酶2 最近，一些材料经过特别设计，以充分利用酸化胞浆内吞体，以方便他们的货物交付。这种方法的一个示例中使用合成的聚合物胶束的纳米粒子，其核心是在生理pH值（ 即 7.4），两性离子和电荷中性。然而，在pH 6.0- 6.5，聚合物成为质子化并购入的净正电荷，破坏了胶束的核心，和暴露的聚合物链段相互作用和破坏核内体膜。这项活动已经表明，以促进核内体逃逸的多肽和核酸为基础的疗法，让他们来访问他们的胞浆目标。3,4调解胞内逃生，破坏黏膜屏障的方法的例子包括“膜融合肽或介导膜融合或瞬态孔形成的蛋白质，可以在磷脂双分子层中。5均聚物的阴离子的烷基丙烯酸，如聚（丙基丙烯酸酸）是另一种被充分研究的方法，并且在这些聚合物中，羧酸侧基的质子化状态的决定过渡为疏水性，膜中断状态内溶酶体的pH值范围6,7。 一个有用的模型系统，为筛选endosomolytic行为是电子x体内pH-依赖的溶血试验。8在该模型系统中，红细胞膜的脂双层膜的替代品，包围内溶酶体囊泡作为。此一般化模型已被用于别人，到评估的endosomolytic的行为的细胞穿透肽和其它聚合物的基因传递系统。8-11在本实验中，在模仿在定义的pH值的缓冲液共孵育的人的红血细胞和试验材料胞外（7.4），早期内体（6.8），和晚期内溶酶体（&lt;6.8）的环境中。潜伏期过程中释放的血红蛋白的量进行定量的红血细胞溶解，其被归一化到在阳性对照样品用洗涤剂裂解释放血红蛋白量作为衡量。 从一个小型图书馆的潜在的endosomolytic测试材料筛选，可以推断出，不产生溶血的样品，在pH值7.4，但显着升高下摆olysis在pH &lt;6.5时，将是最有效的和细胞相容性的候选人胞浆药物递送。预计将保持惰性的，而不是不分青红皂白地破坏脂质双分子层膜（ 即可能导致细胞毒性），直到被暴露后的pH值下降到溶酶体内舱的内符合这些条件的材料。 在这个协议中，红细胞从人供体中分离，并在pH为5.6，6.2，6.8或7.4与实验endosomolytic药物输送剂共同孵育。完整的红细胞沉淀，上清液（含血红蛋白释放溶解红细胞） 经由板读数器（ 图1）为特征的血红蛋白的吸光度分析。

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> BD真空采血管 - K2EDTA Vacutainer管</td><td> Fisher Scientific则</td><td> 22-253-145 </td><td>对于血液采集</td></tr><tr><td> BD真空采血管采血针，20.5号</td><td> Fisher Scientific则</td><td> 02-665-31 </td><td>对于血液采集</td></tr><tr><td> BD真空管座/针适配器</td><td> Fisher Scientific则</td><td> 22-289-953 </td><td>对于血液采集</td></tr><tr><td> BD品牌异丙基酒精棉签</td><td> Fisher Scientific则</td><td> 13-680-63 </td><td>对于血液采集</td></tr><tr><td> BD真空采无乳胶止血带</td><td> Fisher Scientific则</td><td> 02-657-6 </td><td>对于血液采集</TD&gt; </tr><tr><td>盐酸（浓） </td><td> Fisher Scientific则</td><td> A144-500 </td><td>为了调整pH值的D-PBS。 </td></tr><tr><td>的Triton X-100的</td><td> Sigma-Aldrich公司</td><td> T8787 </td><td>阳性对照</td></tr><tr><td>的Dulbecco PBS </td><td> Invitrogen公司</td><td> 14190 </td><td></td></tr><tr><td> NALGENE MF75一次性使用无菌瓶顶过滤单元与SFCA膜</td><td> Fisher Scientific则</td><td> 09-740-44A </td><td></td></tr><tr><td> BD 96孔板，平底，组织培养处理的聚苯乙烯</td><td> Fisher Scientific则</td><td> 08-772-2C </td><td>对于板读取的测定结束时。 </td></tr><tr><td> BD 96孔板，圆底，组织培养处理的聚苯乙烯</td><td> Fisher Scientific则</td><td> 08-772-17 </td><td>孵化的红血细胞与实验药物。 </td></tr></表&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

内溶酶体膜泡的磷脂双分子层构成，可以构成一道屏障，细胞内的目标提供生物药物。为了克服这个障碍，已设计了一些合成的药物载体积极破坏核内体膜，并提供货物进入细胞质。这里，我们描述的溶血测定，这可以用作快速，高通量筛选的细胞相容性和endosomolytic活性的细胞内的药物输送系统。 溶血试验，人的红血细胞和试验材料共同模仿细胞外的，早期内体，和晚期溶酶体内环境的定义的pH值的缓冲液中温育。颗粒完整的红血细胞的离心步骤之后，释放到培养基中的血红蛋白量的分光光度测定（405nm的为最佳的动态范围）。 ％的红血细胞破碎，然后相对于阳性对照SAMP量化LES用洗涤剂溶解。在该模型系统中，在红细胞膜的脂双层膜的替代品，包围内溶酶体囊泡作为。期望的结果是可以忽略不计的在生理pH（7.4）和pH约为5-6.8的溶酶体内pH值范围从鲁棒溶血溶血。

<html>通常情况下，表现出理想的pH-依赖的溶血行为的代理具有胞质交付药物，核酸，或其它生物活性分子的最高电位。描绘在图2中，在pH = 7.4，显示出最小的溶血，这是举例说明由Agent＃1，但在核内体内的pH值范围（&lt;6.5）的溶血行为急剧增加。有些剂可以表现出溶血行为的显着水平的在生理pH值范围（代理＃2（40微克/毫升）， 图2），这表明，这些药物可能不是血液相容性，并有可能在这些浓度是细胞毒素。 在大多数情况下，溶血也是剂量依赖性的，增加浓度的试验材料与溶血的较高水平，尤其是在较低的pH范围内测试（5.6 - 6.2）。 “ALT =”图1“佛：内容宽度=”4英寸“的FO：src =”/ files/ftp_upload/50166/50166fig1highres.jpg“/&gt; 图1。红细胞的溶血含量的示意图。人类红细胞了一系列的缓冲液的pH值范围从模拟生理（7.4）晚内涵体/的溶酶体（5.6），与实验endosomolytic的给药制剂的分离和培养。最佳给药制剂，不会破坏红细胞，在生理pH值，但更多的酸性条件下，会表现出强大的溶血。要评估％溶血，释放到周围介质中的血红蛋白通过平板读取器上的吸光度测定。 <img src="/files/ftp_upload/50166/50166fig2.jpg" alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50166/50166fig2highres.jpg" /> 图2。代表的溶血试验结果证明行为的两种实验Endosomolytic代理。首先，平均A 450的车辆控制（PBS）中减去在该pH值从测试的其他样本。随后，实验样品进行归一到A 450的Triton X-100处理的红细胞样品，并乘以100％。基于这些对照样品，实验转染剂溶解红细胞的能力可以被计算。通常情况下，理想的endosomolytic剂表现出剂量依赖性和pH-依赖的溶血行为。理想的代理（如Agent＃1）表现出最小的溶血，在pH值7.4，但在胞内pH值范围内（&lt;6.5）溶血行为急剧增加。有些剂表现出大量的溶血行为在生理PH范围（代理＃2 40微克/毫升），这表明，这些药物可能是细胞毒素在这些浓度。误差条表示4个独立测量的标准偏差。

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

溶血试验可作为一种快速，高通量筛选，药物传递系统的细胞相容性和内货物配送endosomolytic活动的。该试验测量作为环境pH值的函数的红细胞膜的破坏。

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

34.3K 次观看.  Vanderbilt University. 溶血试验可作为一种快速，高通量筛选，药物传递系统的细胞相容性和内货物配送endosomolytic活动的。该试验测量作为环境pH值的函数的红细胞膜的破坏。

视频: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

使用碘电磷诱导红血球中的红血球

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

免疫与感染

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

神经科学

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

用于药物发现的人视网膜类器官毒性筛选

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

生物工程

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

医学

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

生物学

Hemolysis Assay: An In Vitro Method to Measure Calcium Ionophore-Induced Lysis in Human Erythrocytes

Source: Bigdelou, P. et al., <a href="https://www.jove.com/v/60659">Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore.</a> J. Vis. Exp. (2020).
This video describes an in vitro assay to determine calcium-ionophore-induced hemolysis or lysis of red blood cells in a human sample. The assay measures the percentage of hemolysis by quantifying hemoglobin released from ruptured erythrocytes.

溶血试验:一种测量钙离子载体诱导的人红细胞裂解的体外方法

Source: Bigdelou, P. et al., Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore. J. Vis. Exp. (2020).
This video describes an in vitro ...

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

摘要

探索更多视频

此视频中的章节

可逆二硫键交联胶束生产的简便和有效途径

使用碘电磷诱导红血球中的红血球

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

在脑血管内皮细胞生物能量功能的评价

用于药物发现的人视网膜类器官毒性筛选

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性

使用高含量显微镜的细胞氧化还原分析

溶血试验:一种测量钙离子载体诱导的人红细胞裂解的体外方法

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

摘要

探索更多视频

此视频中的章节

可逆二硫键交联胶束生产的简便和有效途径

使用碘电磷诱导红血球中的红血球

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

在脑血管内皮细胞生物能量功能的评价

用于药物发现的人视网膜类器官毒性筛选

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

炎症性半月酶诱导的人单核细胞来源巨噬细胞邻近性的可视化

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

使用高含量显微镜的细胞氧化还原分析

溶血试验:一种测量钙离子载体诱导的人红细胞裂解的体外方法

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性