Name: a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules
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Description: Watch this Scientific Journal Video about A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules at JoVE.com

这项工作是由蒙特利尔大学提供的默克Frosst启动资金支持。我们愿在免疫与癌症研究所研究从高通量平台，感谢博士让Duchaine和Dominic Salois他们的讨论，发表意见和反馈。 Moutih Rafei持有全宗德拉RECHERCHE EN桑特魁北克少年1奖。

所有动物方案都被蒙特利尔大学的动物管理委员会批准。小鼠通过CO 2的渐进吸入安乐死，直到没有生命体征观察颈椎脱位。该程序是由经过认证的人员进行，以确保动物以人道的方式实施安乐死，并根据加拿大议会关于动物保护的建议。 1.脾细胞培养基和流式细胞仪缓冲液的制备<ol><li>执行70％的乙醇，生物清洗引擎盖下的所有步骤。 </li><li>取出70毫升预热罗斯威?...

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数读出的方法已被开发用于灵敏和可靠的HTS测定法的发展。这些措施包括色度，发光或荧光方法。虽然比色法是简单的设置，它们需要的化学品的多次加入，其可干扰或中断所测试的细胞。 23此外，它们不作为药理学作用是在特定端点评估允许生物应答的动态评估。此外，如果使用自动的HTS此方法可能需要昂贵的机器人手臂。这些因素使色度读奏与HTS的目标相容性较差，由于其?...

高通量筛选（HTS）是一个久经考验的战略新的治疗分子的鉴定或为新的医学指征FDA批准的药物重新定位广泛采用。 1到目前为止，取得HTS成功可以通过的先前发现的药物过多测定。例如，用于乳腺癌，西他列汀的治疗酪氨酸激酶抑制剂拉帕替尼;一个二肽基肽-4（DPP-4）抑制剂用作抗高血糖药物，以及用于治疗慢性髓细胞性白血病的治疗口服的Bcr-Abl的酪氨酸激酶抑制剂达沙替代表批准的药物最初由发现的一长列的几个例子HTS。 2虽然制药业的生产力近来从缺乏的新化学实体的发现遭受的，成功的药物发现的可能性可以通过增加在临床前念珠菌的数量提高TES显示调节生物学/生物化学特性。因此，适于表型筛选新HTS测定法的发展可以提供为新药命中的发现提供了重要的药理学工具的潜力。 3，4，5，6此外，高温超导现在可以以更快的速度，由于近年来显著技术改造包括定制设计的灵活的机器人装置，新颖读出的技术和大量的小型化进行。 2,7在有助于在使用表型筛选（又名向前药理学）的日益增长的兴趣的因素是人们认为着眼于功能效应，而不是关于分子靶（基于目标的筛选/生化反应）过于简化的还原假设是更有可能以翔<html>瓦特临床疗效。因此，表型筛选持有的承诺，发现新的潜在的治疗化合物和目前无法治疗的疾病的分子途径。 2 适当地识别一个给定的分子靶标或细胞功能抑制剂或活化剂，高度敏感的，可靠的检测是必需的，以便真正命中和误报之间进行区分。那么，是什么使一个良好的试验？一个给定的测定法的质量必须由信噪比（通过为Z因子反射的）首先判断。 8其次，有针对性的效果或屏幕的目标应明确规定。例如，功能性的基于细胞的方法可以用于受体的筛选提供显著优点，而不是专门设计来评估配体 - 受体结合的测定法。这样做的原因是，在后一种方法不能激动剂和拮抗剂配体之间区分。SS =“外部参照”&gt; 9相反，基于细胞的方法很可能是作为受体功能可以在一个生物的表型被直接评估更为有效（增殖，细胞周期停滞，细胞凋亡，和/或分化）。然而，必须指出的是，生物化学测定可在表型测定中提供显著优点，因为它们违反在一个特定的细胞内靶标。一个精心优化的生化检测通常会有比表型筛选数据散射少，同时简化后的相关行动的药物分子机制的研究。然而，目标为基础的或生物化学分析的主要缺点是扩增在生物系统（最初在生物化学测定法研究了特异性的损失）测试时，可能会影响非特定目标的假阳性命中率的机会。 10虽然阴性和阳性之间命中一个完善的分界点可以最大限度地减少误报的数量我n中的初步筛选，使用了生理学相关的系统模拟天然细胞环境如完整细胞，完整的组织或整个动物仍然测定设计钟摆的核心。因此，表型筛选使铅发现与对于没有标识的药物靶标疾病期望生物/表型效应，而无需化合物的活性或作用模式的先验知识。 11 在本文中研究涉及基于两个重要部件的优化和可再现的表型筛选的开发和测试：市售小鼠模型和化学化合物的聚集亚家族。对于动物模型，测定法依赖于在其中绿色荧光蛋白 （GFP）的表达是由第驱动用来自小鼠品系（Nur77 GFP）带有含一个盒的细菌人工染色体衍生的淋巴细胞ËNur77子。 12这种刺激的标志是基于这样的事实，Nur77是立即早期基因上调下列T细胞受体（TCR）或B细胞受体（BCR）刺激。 12对于筛选方法本身，使用的方法，以帮助避免琐碎类似物的筛选，同时最小化，以评估一个大化学文库（&gt; 10 5的化合物）所需的时间。这样做，通过使用虚拟筛选工具药物化学家选择的化学化合物的数据库利用来识别使用已知活性种子的结构作为参考拓扑类似的化合物。这个方法允许我们筛选较136000化学实体的整体库4398的化合物。

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:211px;">Nur77GFP mice</td>
			<td style="width:157px;">The Jackson Laboratory</td>
			<td style="width:135px;">Mouse strain No. 016617</td>
			<td style="width:224px;">An in house colony was established at our animal facility&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">96 wells-U culture plates, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">10062-902</td>
			<td style="width:224px;">T-cell activation using the magnetic beads</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">70&#181;m cell strainer, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352350</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:211px;">5 ml polystyrene round bottom tubes, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352058</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:211px;">50 ml polypropylene conical bottom tubes, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">89039-656&#160;</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">10 ml syringe without needle, sterile</td>
			<td style="width:157px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305482</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">T-25 culture flask</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">690 175</td>
			<td style="width:224px;">To incudabte B cells during activation</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">5 ml cell culture dish, sterile</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">627 160</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Penicillin- Streptomycin (10,000 U/mL)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">450-200-EL&#160;&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">RPMI 1600 with sodium bicarbonate and L- glutamine</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">350-002-CL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:211px;">MEM non-essential amino acids</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">321-010-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HEPES free acid 1 M</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">330-050-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Sodium pyruvate solution (100mM)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">600-110-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:211px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">080-910&#160;</td>
			<td style="width:224px;">Component of the splenocyte media and flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Phosphate buffered saline (PBS)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">311-010-CL</td>
			<td style="width:224px;">Component of flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">2-Mercaptoethanol (55mM)</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">21985-023</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:211px;">T- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19851</td>
			<td style="width:224px;">To isolate T cells</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:211px;">B- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19854</td>
			<td style="width:224px;">To isolate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Mouse T-Activator CD3/CD28 superparamagnetic beads</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11452D</td>
			<td style="width:224px;">To activate T cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Cell isolation magnet</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">18000</td>
			<td style="width:224px;">To isolate T cells and remove the magnetic beads&#160;</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG + IgM (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:135px;">115-006-068</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Recombinant Murine IL-7</td>
			<td style="width:157px;">Peprotech</td>
			<td style="width:135px;">217-17&#160;</td>
			<td style="width:224px;">To support T-cell survival during activation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Recombinant CD40L</td>
			<td style="width:157px;">R&#38;D Systems</td>
			<td style="width:135px;">8230-CL/CF</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD3 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">561799</td>
			<td style="width:224px;">To stain T cells for flow-cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD19 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">553786</td>
			<td style="width:224px;">To stain B cells for flow-cytometry</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:211px;">Biomed FXp</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">A31842</td>
			<td style="width:224px;">To re-suspend cells after 24 hours incubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Opera Phenix High Content Screening System</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">HH14000000</td>
			<td style="width:224px;">To analyze GFP/Hoechst signal</td>
		</tr>
	</tbody>
</table>

a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules

高通量筛选（HTS）是目前能够调节的生化反应，或细胞过程的化学实体的标识支柱。随着生物技术的进步和小分子的高潜力转化，一批新药研发创新方法的演变，这也解释了在使用HTS的复苏兴趣。肿瘤学领域是目前药物筛选中最活跃的研究领域，与靶向移植相关并发症或自身免疫性疾病的新免疫调节化合物的鉴定没有取得重大突破。在这里，我们提出体外鼠基于荧光检测淋巴细胞一种新型容易地适应新的免疫调节化合物的鉴定。这种测定法使用从转基因小鼠，其中该Nur77启动子后的T细胞或B细胞受体刺激驱动GFP的表达衍生的T或B细胞。由于GFP强度反映了靶细胞的活化/转录活性，我们的测定法定义了一种新的工具来研究对细胞/生物反应给出化合物（S）的作用。例如，使用在不存在“目标假说”，它导致了160显示免疫调节活性的潜在的命中鉴定4398化合物进行初筛。因此，使用该测定的是适合用于药物开发的程序探索进一步在体外 /体内验证研究之前大化学文库。

高温超导实验的设计设计此荧光分析时有两个重要的因素考虑在内。首先，我们需要复制在其中的T或B细胞激活将代表一个疾病（ 例如移植物抗宿主病）的生理状态。第二，应该使用灵敏和定量的方法来进行细胞活化的评估。荧光是时下高温超导读数的主要选择之一，因为它符合这些需求。 ...

Watch this Scientific Journal Video about A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules at JoVE.com

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

我们提出在目前的研究使用从转基因小鼠衍生的淋巴细胞的新颖的基于荧光的测定法。该测定是适于赋予的任抑制或促进淋巴细胞的活化的能力的小分子的高通量筛选（HTS）。

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 ...

Research

JoVE Journal

Immunology and Infection

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

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 ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...