在此发表研究报告由美国国家癌症研究所的国家机构的健康奖号码R01CA174385下的支持。的内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1。试剂准备<ol><li>准备通过混合500毫升的RPMI-1640和5毫升的RPMI-PLGH每个青霉素 - 链霉素，L-谷氨酰胺，和HEPES溶液。 </li><li>在混合用10％胎牛血清（FBS）的RPMI-PLGH准备R10溶液。 FBS是于56℃下30分钟，加入之前热灭活。 </li><li>预保温在37℃下50毫升的RPMI-PLGH的，15毫升PBS中，和15ml无菌锥形管中的R10。 </li><li>在PDMS:硅主阵列的nanowells采用光刻基本上如前所述，10的制造。调匀Sylgard的184弹性体套件碱剂和固化剂按10:1的重量比在一个一次性杯使用塑料刀。脱气的混合物1小时，在真空室中。到纳为阵列，倒入密封载玻片上，让它坐在30分钟。的组件转移到设置为80℃下进行2小时的烘箱中，并在室温下搅拌1小时，放凉。弹性体电流E在此期间，将债券载玻片上。含有PDMS纳为阵列的载玻片仔细升空硅主站，覆盖着透明胶带和保存，直到将来使用。 </li></ol> 2。靶细胞（T）标签<ol><li>使用血细胞计数器和颗粒细胞计数5000000从细胞培养的靶细胞在无菌的15毫升锥形管，通过离心分离，在340×g离心5分钟。被分裂的细胞培养的前一天，在5毫升的总体积（密度= 1万个/ ml），并培养在T-25烧瓶（VWR）。已经描述了用于细胞计数使用血细胞计数仪的协议在前面已经详细11 </li><li>吸出多余的介质，留下了约50微升的媒体。 </li><li>准备一个有效的解决方案的细胞追踪红（CTR）的混合0.5微升CTR股票（1毫米），150μLRPMI-PLGH（终浓度为2.5μM）。到靶细胞的工作溶液，并加入150μl调匀使用P200的吸移管。PKH 26（最终浓度为2μM）或者，可以使用的标签的靶细胞，按照制造商的说明。 </li><li>在37℃/ 5％CO 2中孵育20分钟。 </li></ol> 3。效应（E）细胞标记<ol><li>计数5000000效应细胞在无菌的15毫升锥形管使用血细胞计数器和颗粒细胞在340×g离心5分钟，通过离心从细胞培养。 </li><li>吸出多余的介质</li><li>准备Vybrant DyeCycleViolet染色溶液的工作溶液的1微升的5mM Vybrant紫罗兰库存通过添加1毫升的R10培养基中（终浓度为5μM）。添加到细胞中的工作溶液，并重新悬浮使用P200的吸移管。 PKH 67（最终浓度为2μM）或者，可以使用的标签的靶细胞，按照制造商的说明。如果使用PKH 67，然后SYTOX格林不应使用，因为它们是在相同的荧光信道。凋亡的目标是只使用一个枚举在这种情况下，nnexinV染色。 注:如果执行的时间推移，而不是端点测量，应避免UV染料类似Vybrant紫罗兰。 </li><li>在37℃/ 5％CO 2中孵育20分钟。 </li></ol> 4。使用密度梯度活的和死细胞的分离<ol><li>加入6.8毫升和6.0毫升RPMI-PLGH的目标和效应细胞，分别由上下吹打轻轻混合。 </li><li>层的FICOLL帕克加上每个锥形管的底部包含目标和效应细胞的3.5毫升。层FICOLL慢慢地确保形成层之间FICOLL和媒体。 </li><li>在340×g离心30分钟，离心两管。没有制动和加速。 </li><li>在离心过程中，转移到4 - 孔板7毫升预先温热的PBS。准备纳为阵列（PDMS中制作）:清洁底部的载玻片上，用乙醇和氧化PDMS在高RF设置使用1分钟的等离子体氧化。这一步将STErilize纳为阵列中，同时也使PDMS的亲水性。立即将纳为阵列到板PBS。 </li><li>板，生物安全柜，吸去多余的PBS。 10毫升1％高尚的琼脂100毫克琼脂粉加入10毫升去离子水，然后微波30-45秒。应用温暖1％贵金属琼脂上的顶部和底部边缘的载玻片，包含纳为阵列固定阵列，等待10分钟的琼脂固化。加入3毫升PBS和吸出多余的琼脂。 </li><li>加入3毫升R10纳为阵列的顶部上，并让它〜10分钟的平衡。 </li><li> FICOLL离心分离之后，从每个管顶端层吸5毫升媒体。要小心，不要吸出含有细胞层。 </li><li>收获细胞（白层）通过回收从与P1，000吸移管和移动到新的无菌锥形管FICOLL和介质层之间的1-2毫升。重复此为套细胞，同时确保你的格言IZE恢复细胞。 </li><li>加入3毫升预热的RPMI-PLGH每个管中，通过移液混合和颗粒通过离心分离在340×g离心5分钟，充分的加速度和制动。 </li><li>吸出多余的介质，加入5毫升预热的RPMI-PLGH，每个试管和，重复离心340×g离心5分钟。 </li><li>吸出多余的介质和悬浮细胞沉淀在500μl的R10。 </li><li>使用台盼蓝排除方法血球计数器的计数活细胞。 </li></ol> 5。手机装上纳为阵列<ol><li>吸纳为阵列的多余的介质，并加入2毫升的新鲜R10在阵列中。吸再次而确保纳为阵列的顶部是不是太湿/干的，因为它会影响细胞分布。 </li><li>存款1×10 5效应细胞纳为阵列表面在200微升R10到（密度= 5×10 5细胞/ ml）。 </li><li>让细胞沉降5分钟，并使用标准的组织培养显微镜检查为en确定细胞的分布优选。如果需要添加更多的细胞。 </li><li>存款1×10 5靶细胞在200μlR10纳为阵列表面上（密度= 5×10 5细胞/ ml）。 </li><li>让细胞5分钟，并使用标准的组织培养显微镜检查，以确保细胞的分布优选定居。如果需要添加更多的细胞。 </li><li>仔细冲洗纳为阵列，用2毫升R10和吸出多余的介质。 </li><li>准备在15毫升无菌锥形管3毫升预先温热的R10。加入3μl的0.5mM的SYTOX格林核酸着色漆（最终浓度为0.5μM）和60μl的膜联蛋白V-的Alexa Fluor 647准备工作溶液。仔细，同时确保细胞不从井到4孔板中添加有效的解决方案。 </li><li>在37℃/ 5％CO 2中孵育15分钟。 </li></ol> 6。成像1 <ol><li>采集图像的纳为阵列使用荧光MICR的Oscope:在这个例子中，我们使用ZEISS观察，Z-1显微镜配备了一个机动阶段和Lambda DG4。 </li><li>初始化的软件和显微镜检查的信号强度上的透射光，和其他荧光通道。总体而言，将有五道相对应的透射光，膜联蛋白V-647（Cy5的过滤器），CTR（红色荧光蛋白过滤器），SYTOX绿色（FITC过滤器），并Vybrant的紫（DAPI过滤器）。确保显微镜的设置，以确保最大的信号噪音，同时避免饱和。 </li><li>设置为纳为阵列的X和Y位置。零位置设置和初始化焦点上的中心的7×7纳为阵列块上的纳为阵列邮票左上角开始过程。设置的x，y的位置，以配合每个块的中心的z值，以准确地反映在每个块上的焦点。 </li><li>一旦位置和焦点都设置，确保线性模式设置图像采集和采集图像。 <s豪华型&gt;注意:如果需要打开孵化器和CO 2在显微镜实验前至少30分钟。 </li></ol> 7。成像后转移到6小时的孵化器（37℃/ 5％CO 2）的4 -孔板含有纳为阵列。 8。成像2 采集图像在所述第二时间点，在步骤6中所概述。 9。图像分析<ol><li>细胞内的nanowells占用确定通过使用适当的图像分割程序（ 例如 ImageJ的， <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ）基本上如前面所述的9,12。 </li><li>的显微镜图像（t = 0时小时; 吨0）的初始集合的分析是用来生成一个数据库含有一个活的靶细胞（确定个别nanowells[的CellTracker红色，红色），无死细胞（Annexin V染色鉴定，绿色）[T1T 0 nanowells。 </li><li>显微镜图像的第二组（ 吨 = 6小时;吨6）分析，用于独立地确定一个第二数据库的nanowells含有1靶细胞（红色）[T1T 6 nanowells]。实现的完整性校验（数据库查询），以确保在t = 0的所有活靶在t 6的目标相匹配，以产生最终的集的匹配目标（作为具有T1T 0 = 6 T1T指定为T1match的nanowells实现） </li><li>在nanowells效应细胞的数目被确定使用Vybrant紫染色在 t 0 [E1 nanowells] </li><li>实现数据库查询确定含有单效应的nanowells共同培养的单一目标（E1和T1的比赛作为nanowells，指定为E1T1的定义） </li><li>由我打进效应诱导细胞凋亡<html> dentifying的目标是在t = 0和Annexin V阳性T 4（E1T1与Annexin V的目标染色在t 6），膜联蛋白V负。 </li></ol> 10。可选的延时成像的杀戮使用尼康BioStation IM <ol><li>盖玻片底培养皿中切出一小片的纳为阵列芯片（步骤5）编制的盖玻片上，将完全适合。请确保该芯片切割时，保持湿润。 </li><li>混合料1×10 6个目标和1×10 6的T细胞在100μl培养基，移液到盖玻片。 </li><li>允许约15-30分钟，让细胞定居。 </li><li>快速反转的小纳为阵列芯片，并把它放在盖玻片。 </li><li>为了确保芯片将无法移动的摄像过程中，放置芯片的顶部上的一个或两个20毫米x 20mm的盖玻片。轻轻地施加压力，推动芯片的底部的菜。 </li></ol>10吨"&gt; 注:的纳为阵列芯片的底部是过于厚的高清晰度成像，因此具有向被打开倒挂，在此期间，许多细胞被洗出。由于本可以不被容易控制，细胞数步9.2在步骤9.3中，孵育时间必须进行优化设计。

<ol>
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没有利益冲突的声明。

我们概述了协议，用于高吞吐量的单细胞的细胞溶解测定启动阵列nanowells（ 图1）中的效应和目标通过共同孵育。除了吞吐量的技术的一个主要优点是能够监视对所希望的靶细胞效应的介导的细胞毒性，而不需要为这又允许使用自体或匹配/主肿瘤细胞作为靶细胞的靶细胞工程。的空间限制允许检索并随后在结束测定9功能性T细胞克隆。检测的一个限制是需要专门的自动化阶段?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI-1640 W / O型L-谷氨酰胺</td><td> Cellgro </td><td> 15-040-CV </td><td></td></tr><tr><td>青霉素，链霉素</td><td> Cellgro </td><td> 30-002-CI </td><td> 10,000 IU青霉素10,000μg/ ml的链霉素</td></tr><tr><td> L-谷氨酰胺</td><td> Cellgro </td><td> 25-005-CI </td><td> 200毫米的解决方案</td></tr><tr><td> HEPES </td><td> Sigma Aldrich公司</td><td> H3537 </td><td> 1M </td></tr><tr><td>牛胎儿血清（FBS）的</td><td>亚特兰大生物</td><td> S11150 </td><td>地段测试</td></tr><tr><td>手机追踪器红染色</td><td> Invitrogen公司</td><td> C34552 </td><td> 50微克</td></tr><tr><td> Vybrant DyeCycle紫染色</td><td> Invitrogen公司</td><td> V35003 </td><td> 5毫米</td></tr><tr><td> SYTOX绿色核酸染料</td><td> Invitrogen公司</td><td> S7020 </td><td> 5毫米</td></tr><tr><td>膜联蛋白V-的Alexa Fluor 647 </td><td> Invitrogen公司</td><td> A23204 </td><td> 500μL </td></tr><tr><td>的Dulbecco PBS </td><td> Cellgro </td><td> 21-031-CV </td><td> 500毫升</td></tr><tr><td>高贵的琼脂</td><td> DIFCO </td><td> 2M220 </td><td> 100克</td></tr><tr><td>台盼蓝</td><td> Sigma Aldrich公司</td><td> T8154 </td><td> 0.4％液，无菌过滤</td></tr><tr><td>血球计数仪</td><td>豪塞尔科学</td><td> 1492 </td><td>亮线</td></tr><tr><td> 4 - 孔板</td><td>赛默飞世尔</td><td> 167603 </td><td></td></tr><tr><td> Harrick旅游等离子清洗机</td><td> Harrick旅游等离子</td><td> PDC-32G </td><td>基本的等离子清洗机</td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><tD&gt; </td><td>荧光显微镜（与下面的三个部分） </td></tr><tr><td>拉姆达10-3 </td><td>萨特仪器</td><td></td><td>过滤器控制器</td></tr><tr><td>拉姆达DG-4 </td><td>萨特仪器</td><td></td><td>超高速波长转换器</td></tr><tr><td>滨松EM-CCD相机</td><td>滨松</td><td> C9100-13 </td><td> CCD显微镜摄像头</td></tr><tr><td> 15毫升锥形管</td><td> BD猎鹰</td><td> 352097 </td><td></td></tr><tr><td> 50毫升锥形管</td><td> VWR </td><td> 3282-345-300 </td><td></td></tr><tr><td>尼康Biostation </td><td>尼康仪器公司</td><td> Biostation IM </td><td></td></tr><tr><td>玻璃底培养皿</td><td> MatTek公司</td><td> P35G-0 </td><td> 35毫米培养皿中，10毫米微孔</td></tr></tbody></table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

肿瘤免疫治疗可以利用的目标和消灭肿瘤的特异性免疫反应。继细胞治疗（ACT）T细胞过继的转基因表达的嵌合抗原受体（CAR）的基础上，已表现出相当大的希望在临床试验中1-4。有几个好处使用CAR + T细胞治疗癌症的能力为目标，非MHC限制性抗原和功能化的T细胞以达到最佳的生存，归巢内的主机和持久性，并最终诱导细胞凋亡的CAR + T细胞在宿主毒性5的事件。 划定的最佳功能的CAR +与临床受益相关的T细胞是必不可少的设计的临床试验的下一代。多光子显微镜等活的动物成像的最新进展，彻底改变了我的免疫细胞功能的研究体内6,7。虽然这些研究已经推进了我们理解，T细胞在体内的功能，基于T-细胞的ACT在临床试验中需要需要链接分子和功能特性的T-细胞的准备预浸液与临床疗效输注后，通过利用在体外实验监测T细胞的功能，如，细胞毒性和细胞因子分泌。已经开发了确定在单细胞水平的T细胞数量的整体运作，但这些是不适合用于监测共轭形成和寿命或相同的小区的能力，杀多个目标8标准基于流式细胞仪检测。 微型阵列设计生物相容性的聚合物，如聚二甲基硅氧烷（PDMS）是一个特别有吸引力的方法，在空间上限制小体积效应和目标9。结合时间的推移自动荧光显微镜，寿usands效靶相互作用可同时监控纳为阵列的成像个别井。在这里，我们提出一种高通量的方法，用于监测在单细胞水平，可以广泛地应用于研究T细胞的细胞溶解功能的T细胞介导的​​细胞毒性。

在图2中的应用程序的一个示例的高通量的细胞溶解测定表明。简要地说，标记的CD19-特异性CAR + T细胞的共同培养与标记的小鼠EL4靶细胞中的纳为阵列的各个井（第1-5）。最初的图像被记录在自动荧光显微镜来识别占用阵列（第6章）（效应和/或目标）的每一个纳为。用于图像处理，找出所有包含一个单一的目标和单效应和nanowells以排除nanowells含有死的目标（第9条）。被转移?...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

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

我们描述了一种单细胞的高通量的检测，以测量与肿瘤靶细胞的培养时的细胞毒性T细胞。此方法采用致密的，弹性体子纳升井（〜100,000井/数组）的数组，在空间上局限在T细胞和靶细胞，在规定的比例，和被耦合到的荧光显微镜来监测效靶共轭和随后的细胞凋亡。

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 ...

quantitative-high-throughput-single-cell-cytotoxicity-assay-for-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.9K 次观看.  University of Houston. 我们描述了一种单细胞的高通量的检测，以测量与肿瘤靶细胞的培养时的细胞毒性T细胞。此方法采用致密的，弹性体子纳升井（〜100,000井/数组）的数组，在空间上局限在T细胞和靶细胞，在规定的比例，和被耦合到的荧光显微镜来监测效靶共轭和随后的细胞凋亡。

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

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

科研

免疫与感染

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

癌症研究

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

医学

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

定量高通量单细胞的细胞毒性T细胞的测定

摘要

探索更多视频

此视频中的章节

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

基于细胞的流式细胞仪检测，以测量细胞毒活性

使用患者源性肿瘤类器官的高通量体外检测

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

实时的细胞毒性分析在人全血

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

基于液滴的细胞毒性测定，用于在单细胞水平评估嵌合抗原受体 T 细胞

定量高通量单细胞的细胞毒性T细胞的测定

摘要

探索更多视频

此视频中的章节

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

基于细胞的流式细胞仪检测，以测量细胞毒活性

使用患者源性肿瘤类器官的高通量 体外 检测

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

实时的细胞毒性分析在人全血

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

一种使用 mRNA 生成瞬时嵌合抗原受体 T 细胞用于癌症免疫治疗的非病毒方法

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