Name: a flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled cxc chemokine ligand 12 to cxc chemokine receptor 4
Uploaded: 2018-03-10T13:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4 at JoVE.c

作者感谢埃里克. 芳廷承袭提供了出色的技术援助。这项工作得到了库鲁汶 (格兰特) 的支持。PF/10/018), 全宗与客厅里 Wetenschappelijk Onderzoek (FWO, 授予 no。G. 485.08) 和基金会 Dormeur 瓦杜兹。

1. 细胞培养的维护
注: 1 和2所述的所有步骤均在层流柜的无菌条件下进行。
<ol><li>在37摄氏度和 5% CO2的 T75 培养瓶中生长细胞在一个湿润的孵化器。 注: 在本试验中, 使用 Jurkat 细胞 (即,内在表达 CXCR417) 的人白血病 T 淋巴细胞细胞。CXCR4 在细胞表面的表达应通过流式细胞仪在细胞培养过程中进行评价。但是, 对流式细胞术和试剂的描述, 以确...

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与其他类型的结合分析 (即,饱和结合和动力学结合实验) 相比, 竞争结合检测最适合于筛选目的。事实上, 它们允许对大量未标记化合物 (例如小分子) 进行评估, 通过评分它们的能力来干扰固定数量的标记受体配体的绑定。与标记配体绑定到其他受体部位的化合物在化验中可能仍未被发现。虽然本文所描述的竞争约束实验尤其侧重于趋化因子受体 CXCR4, 但它也可能成为其他受体竞争结合分析?...

G 蛋白耦合受体 (受体) 是细胞表面蛋白, 可由胞外配体 (例如,肽, 蛋白质激素, 胺) 激活, 从而调节许多生理和发育过程1。当激动剂占据其 GPCR 结合袋, 诱导构象变化的受体蛋白促进与细胞内受体相关的 heterotrimeric g 蛋白的结合, 由 gα-GDP 和 gβγ亚基组成。随后, gα亚基的 GDP 交换 GTP 导致 g 蛋白亚基 (gα-GTP 和 gβγ) 的离解, 进而将进一步启动下游信号通路23。当 gα-GTP 水解后, gα-GDP 和 Gβγ亚基的重新组合将 g 蛋白转换回休眠状态3,4。不同类型的 g 蛋白存在 (gs, g i/o,gq, g12/13), 这些分类基于序列相似性与 Gα亚基5。所有这些 G 蛋白诱导定义的细胞内信号通路的基础上的生物反应的受体活化。受体活化后, GPCR 激酶 (GRKs) phosphorylate 受体的细胞内尾部, 从而促进与β arrestins 的相互作用。这一过程导致 G 蛋白信号的终止, 受体脱敏和内部化6。β-arrestins 也是多分子复合体的一部分, 触发信号叶栅独立于 G 蛋白信号7。
受体是治疗干预的最有效的分子靶点之一, 因为解除 GPCR 介导的信号, 例如由于受体基因或受体过度表达的功能增益突变, 导致许多人类疾病8。因此, 受体代表了制药行业调查的最重要的药物靶类之一8,9,10。临床相关 GPCR 的一个显著例子是 CXC 趋化因子受体 4 (CXCR4), 它可以由唯一的天然配体激活, CXC 趋化因子配体 12 (CXCL12)11。由于其作为人体免疫机能丧失病毒 1 (HIV-1) 进入和感染的主要联合受体在分化 4 (CD4) 阳性 T 淋巴细胞12中已确立的作用, CXCR4 首先被调查为抗病毒药物靶。CXCL12-CXCR4 在骨髓中的相互作用进一步调节茎和祖细胞的保留和归巢13。此外, 考虑到它参与癌症生物学的许多方面 (例如,肿瘤细胞存活, 转移, 肿瘤相关血管生成)14和其他一些人类疾病 (例如,炎症疾病)15, CXCR4作为药物发现的一个有希望的目标, 引起了极大的兴趣。AMD3100, 一个专门针对 CXCR4 的小分子, 最初被发现为抗 HIV 药物候选16 , 仍然是迄今为止17所描述的最有效的 CXCR4 拮抗剂之一。然而, 它作为抗病毒药物的发展已停止了18。目前该分子作为干细胞动员剂在治疗多发性骨髓瘤和淋巴瘤患者中的应用18。其他一些化学无关的小分子和生物制剂, 抑制 CXCR4 功能的不同效力被描述为19。
受体结合方法是药理学中有价值的工具, 可以识别与 GPCR 直接作用的化合物 (例如,小分子)。为了进行具有约束力的研究, 不需要事先了解一个给定 GPCR 的胞内信号特性或功能。虽然这可以被认为是一个优势, 它意味着可以证明受体结合的化合物需要进一步的特点, 评估其潜在的竞赛或拮抗活性。这项活动可以用药理学或生物化验方法来评估, GPCR 正在研究中。依赖于它们的活动剖面, 受体结合分子可能会进化成为新的铅化合物, 用于研究前临床和临床研究。分子, 专门绑定到一个高亲和力的受体也可以作为支架, 以产生治疗或诊断工具, 例如通过 radiolabeling 他们为无创性的在体内成像肿瘤细胞20, 或作为潜在的有针对性地提供治疗方法的车辆21。在 CXCR4 的情况下, 肿瘤细胞的体内成像已经用老鼠模型证明了, 其中标记了 CXCR4-targeting 分子允许人类癌症的可视化20,22,23.
在本报告中, 我们描述了一项竞争结合试验的详细协议, 它能够识别直接干扰激动剂 (CXCL12) 与 CXCR4 结合的小分子和生物制剂。该检测的基本原理是在固定量的荧光标记配体之间的竞争 (CXCL12AF647, 参见材料和试剂表) 和未标记的化合物绑定到受体蛋白17,24. 然后用流式细胞仪分析标记配体与单细胞表达 CXCR4 的特异荧光信号。当未标记的小分子破坏 CXCL12AF647和 CXCR4 之间的交互时, 此荧光信号将会减少。该化验使用非操作的活细胞内在表达 CXCR4 (即, Jurkat 细胞)。因此, 不需要细胞膜的准备, 这使得化验方便, 快速, 并与增加的吞吐量兼容。由于使用了荧光标记配体, 放射性被避免。
由于 CXCL12 是 CXCR4 的自然激动剂, 因此干扰 CXCL12AF647绑定的小分子化合物很可能与 orthosteric 受体结合站点 (即,由自然激动剂)。如果它们不影响 CXCL12 的束缚, 那么与 orthosteric 结合部位地形不同的受体结合部位的分子将保持不被察觉。例如, 积极和消极的构调制器, 一个重要的和新兴的类别的 GPCR 靶向分子作用于构结合点25, 将有可能不会被拿起这个化验。此外, 是否有这种结合的检测功能的化合物, 作为受体拮抗剂或兴奋剂不能获得。因此, 需要对所鉴定的化合物进行其他药理学或功能受体相关化验。这些化验可能包括 (组合) 细胞荧光或基于发光的检测第二信使 (例如, Ca2 +, 循环腺苷磷酸 (阵营)), 表型或生物测定和β arrestin招聘分析的选择取决于研究中 GPCR 的具体信号特性。因此, 本文所描述的竞争性结合试验主要是一种初步筛选化验, 需要辅以其他细胞化验, 以使化合物具有受体结合效力的深入描述。

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		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSCanto II</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td style="width:304px;">Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSDIVA Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td>Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray System Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphpad Prism</td>
			<td style="width:247px;">Graphpad</td>
			<td style="width:281px;"></td>
			<td>software package used for nonlinear regression analysis in Figure 2 and Figure 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:387px;">FlowJo</td>
			<td style="width:247px;">FlowJo is now a wholly owned subsidiary of BD.</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Vi-CELL</td>
			<td style="width:247px;">Beckman Coulter</td>
			<td style="width:281px;">Not applicable</td>
			<td>cell viability analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sigma 3-18 KS</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">Not applicable</td>
			<td>centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">AMD3100</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A5602-5mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Maraviroc</td>
			<td style="width:247px;">Pfizer</td>
			<td style="width:281px;"></td>
			<td>antiretroviral drug, CCR5 antagonist, available for research at Selleckchem (cat#S2003), Sigma (cat#PZ0002)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">h-SDF1a (AF647)</td>
			<td style="width:247px;">ALMAC</td>
			<td style="width:281px;">CAF-11-B-01</td>
			<td>fluorescently labeled CXCL12, CXCL12AF647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A1933-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HBSS (10x), calcium, magnesium, no phenol red</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14065-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HEPES (1M)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">15630-056</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Jurkat cells</td>
			<td style="width:247px;">ATCC</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Reagent reservoir PP</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">BR703411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid flow filter: 0.2 &#181;m aPES</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sterilin microtiter plate, 96-well, U bottom, clear</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td style="width:281px;">611U96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Falcon tubes, 50ml</td>
			<td style="width:247px;">Greiner Bio-One</td>
			<td style="width:281px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Tissue culture flask (T75)</td>
			<td style="width:247px;">Corning</td>
			<td style="width:281px;">353024</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled cxc chemokine ligand 12 to cxc chemokine receptor 4

药物靶向 G 蛋白偶联受体 (受体) 是非常重要的人类健康, 因为功能失调的 GPCR 介导信号有助于许多疾病的进展。配体/受体对 CXC 趋化因子配体 12 (CXCL12)/CXC 趋化因子受体 4 (CXCR4) 引起了重要的临床兴趣, 例如作为治疗癌症和炎症性疾病的潜在目标。因此, 小分子以及专门针对 CXCR4 和抑制受体功能的治疗性抗体被认为是宝贵的药理工具。本文介绍了一种基于流式细胞仪的细胞检测方法, 它可以识别出将 CXCL12 绑定到 CXCR4 的化合物 (例如,小分子)。从根本上说, 该检测依赖于固定量的荧光标记为 CXCL12, CXCR4 的自然趋化因子激动剂和未标记化合物之间的受体结合的竞争。因此, 本试验避免了放射性标记探针的不良使用。此外, 活细胞被用作受体 (CXCR4) 的来源, 而不是细胞膜的准备。这样就可以方便地将检测方法与平板格式进行调整, 从而提高了吞吐量。这种检测已被证明是一种有价值的通用药物发现检测, 以确定 CXCR4-targeting 化合物。该协议可能会适应其他受体, 至少如果荧光标记配体可用或可以生成。对于在激活这些受体时诱导的细胞内信号通路的先验知识是不需要的。

绑定分析的一般工作流显示在图 1A中。在图 1B中描述了在标准实验中为不同样本类型获得的流式细胞仪数据的类型 (即负控制、正控制和实验样本), 以及可能的版面布局来执行96井板格式的化验在图 1C中给出。Jurkat 细胞的孵化, 内在表达趋化因子受体 CXCR4 (即,这一检测中的兴趣 GPCR), 随着荧光?...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4 at JoVE.c

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

本文介绍了一种基于流式细胞术的细胞结合检测方法, 主要用作筛选工具, 用于识别抑制荧光标记为 CXC 趋化因子配体 12 (CXCL12) 的化合物对 CXC 趋化因子受体 4 (CXCR4) 的束缚。

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Biochemical Reconstitution of Steroid ReceptorÃƒÂ¢Ã¢â€šÂ¬Ã‚Â¢Hsp90 Protein  Complexes and Reactivation of Ligand Binding

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...