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, 以确定细胞表面的受体表达水平, 但不属于本协议的范围, 但以前已经描述过17。</li>
	<li>让 Jurkat 细胞在悬浮中生长, 直到它们达到 80-85% 融合。在将细胞传代到新的烧瓶之前, 允许所有试剂达到室温 (RT)。</li>
	<li>添加20毫升新鲜完整的生长培养基 (RPMI-1640 培养基, 10% 胎牛血清 (血清), 2 毫米谷氨酰胺) 到一个新的 T75 培养瓶。</li>
	<li>添加5毫升的 Jurkat 细胞悬浮从原来的 T75 瓶 (包含25毫升细胞悬浮) 到新的 T75 培养瓶。孵育在37°c 和 5% CO2在一个湿润的孵化器。</li>
</ol>2. 制备 CXCL12、化验缓冲器和 Jurkat 细胞进行竞争结合试验。
<ol><li>准备 CXCL12AF647 (20 µg/毫升) 的库存解决方案; 请参阅材料和试剂表) 溶解冻干试剂 (存储在-80 °c, 在黑暗中) 在超纯水补充 0.01% (容量/体积) 吐温20。存储单使用整除数从这个库存解决方案在-80 摄氏度, 保护免受光。</li>
	<li>通过添加40毫升 HEPES (1 米, 20 毫米最终浓度) 到200毫升汉克的平衡盐溶液 (HBSS, 10x, 不含酚红色和不含碳酸氢钠, 1x 最终浓度) 制备化验缓冲器。添加超纯水以获得 2 l 的最终体积, 添加4克 (0.2% 重量/体积) 牛血清白蛋白 (bsa), 并通过磁性搅拌溶解 bsa。最后, 调整 pH 值为 7.4 (使用氢氧化钠) 和过滤溶液通过0.2 µm 毛孔 (参见材料表和试剂) 使用真空歧管。 注: 此检测缓冲器将用于该议定书的所有进一步步骤。</li>
	<li>计算单元格的数量和生存能力。为此, 取一个细胞悬浮的样品和稀释它在磷酸盐缓冲盐水 (PBS)。 注意: 我们经常使用自动细胞生存能力分析仪 (参见材料和试剂表), 能够在不同浓度下计数细胞悬浮液。对于细胞计数, 稀释0.5 毫升细胞悬浮在1.5 毫升 pbs (其他稀释,例如, 0.1 毫升在1.9 毫升 pbs, 也是可能的)。此方法的使用是基于台盼蓝染料排除方法, 以前已经描述了26。还有一些其他的设备在商业上可用于计数细胞数和生存能力, 应该同样良好的工作。</li>
	<li>收集所需的单元格数 (即, ~ 24 x 106在无菌50毫升管中使用一个完整的 96-井板来运行化验结果) 离心 (参见材料和试剂的为离心机的类型) 在 400 x g 为5分钟在rt.</li>
	<li>在不干扰细胞颗粒的情况下, 轻轻地倒上清液。添加新鲜化验缓冲区 (例如, 20 毫升), 并通过轻轻吹打向上和向下并用重悬细胞。</li>
	<li>再次离心细胞在 400 x g 5 分钟的 RT。</li>
	<li>再次倒入上清液, 并在新鲜的化验缓冲器中并用重悬细胞颗粒, 以获得 5 x10 6 细胞/毫升的密度。</li>
</ol>3. 竞争结合试验
注: 实际的竞争结合试验是在 RT 进行的, 可以在无菌条件下进行。
<ol><li>稀释检测缓冲物中所研究的化合物 (见 2.2) 以获得所需浓度。要么准备一个固定浓度的化合物初始筛选 (例如, 10 µM 最终浓度), 或者, 或者, 一个系列稀释系列的浓度, 以更详细的描述化合物 (例如, 1/3, 1/4 或 1/5稀释系列从1µM 开始, 最后集中)。请记住, 复合溶液最终将成为2x 稀释的化验;因此, 准备一个2x 集中解决方案。</li>
	<li>将100µL 的复合溶液 (2x 浓缩) 成一个清晰的96井圆底板 (参见材料目录和试剂表), 按照预定义的实验布局 (例如, 图 1C)。 注: 在这个阶段, 阴性和阳性对照样品包括在化验中。在负控制样本中, 将100µL 的检测缓冲器添加到96井板的井中, 而不是复合物。对于正则控件示例, 在此步骤中还添加了检测缓冲区。另请参见图 1C中的典型实验布局, 其中对几种化合物的稀释系列进行了测试。</li>
	<li>添加50µL 的细胞悬浮 (见 2.7; 0.25 x 106细胞) 从试剂库到96井板使用多通道吸管。在黑暗中在 RT 中孵化出15分钟的盘子。</li>
	<li>添加50µL 的荧光标记为 CXCL12 (即, 100 ng/毫升 CXCL12AF647在化验缓冲区, 4x 浓缩, 25 ng/毫升最终浓度) 从一个类似的试剂库到96井板井。在黑暗中在 RT 孵化30分钟。 注意: 对于负控件示例, 请改用检测缓冲区。因此, 在负控制样本中检测到的荧光信号将对应于 autofluorescent 背景信号 (图 1A)。对于正则控件示例, 请添加50µL CXCL12AF647。阳性控制样本将产生最大荧光信号检测, 因为不包括预先孵化与化合物的潜在抑制 (图 1A)。</li>
	<li>离心96井板在 400 x g 为5分钟在 RT. 通过翻转盘子从颗粒细胞中取出上清。在纸巾上擦干盘子。</li>
	<li>用多通道吸管在试剂库中添加200µL 的新鲜化验缓冲器。立即进行。</li>
	<li>再次离心板5分钟, 在 400 x g 在 RT. 通过翻转盘子, 再将其干燥在组织上, 取出上清液。</li>
	<li>轻轻并用重悬细胞颗粒在200µL 的1% 多聚甲醛溶解在 PBS。此步骤将修复单元格。</li>
	<li>使用流式细胞仪对荧光进行量化, 立即继续该协议。</li>
</ol>4. 流式细胞术对样品的分析
CXCL12AF647染色和固定细胞现在可以使用流式细胞仪进行分析。可以使用几种类型的流 cytometers, 但它们需要配备正确的激光 (即一个红色激光, 励磁范围 ~ 630 nm) 为励磁和适当的过滤器为荧光检测 (发射过滤器 ~ 660 毫微米)。他们需要能够处理样品的96井板格式。在材料和试剂表中给出了合适的流式细胞术装置的例子。
<ol><li>启动设备并打开相应的软件 (请参阅材料和试剂表)。</li>
	<li>选择下面的蜂窝参数以斑点印迹格式进行可视化: 正散射 (FSC)、侧散点 (SSC) 和荧光 (CXCL12AF647) 检测通道。 注: 在 FSC 参数下, 由于检测到的光吸收与细胞直径成正比, 因此根据其大小对细胞进行判别。SSC 参数, 测量在90°角的光散射, 提供了有关细胞粒度的信息。</li>
	<li>选择一个示例 (例如,一个负控制样本), 根据 FSC 和 SSC 参数执行已定义的同质细胞填充的浇口。<ol>
<li>选择自动注射100µL 的固定细胞从这个负控制样本进入流式分析仪。在注入前选择 "混合" 选项, 并使用1.5 µL 的采样流率。</li>
		<li>通过选择 "获取数据" 运行此示例。此示例的 FSC 和 SSC 参数现在将显示在屏幕上。</li>
		<li>选择软件的浇口工具。基于 FSC 和 SSC 斑点杂交可视化, 预先定义了一个均匀和可行的细胞群的门。为此, 请创建一个多边形 (使用软件的浇口工具), 其中包括基于这两个维度的 homogenously 分布式单个单元格 ("事件")。 注: 浇注程序的目的是定义一个均匀和可行的细胞群体, 将用于进一步分析。浇口依赖的假设, 大多数可行的细胞将形成一个同质细胞种群的基础上的 FSC 和 SSC 参数。通过执行这一步骤, 细胞碎片、死细胞和细胞聚集物可以很大程度上被排除在进一步分析之外。在图 1B中给出了浇口过程的说明。</li>
	</ol>
</li>
	<li>选择以分析每个示例的2万个 "事件" (即,单个单元格)。 注意: 这意味着对于每个样本, 在预定义的门内的2万个单元将最终被分析。每个示例的数据获取将继续进行, 直到分析此事件的数量。</li>
	<li>启动运行 (选择 "记录数据")。流式细胞仪现在将通过记录每个样本的平均荧光强度 (多边基金会) 对所有样本进行一次分析。这一多边基金会对应于2万个细胞的平均荧光信号在预定的门之内。</li>
</ol>5. 数据分析
使用为每个样本获得的多边基金会执行所有进一步的计算。对流式细胞仪数据的分析可以通过几个商业上可用的软件包进行 (参见材料和试剂表)。
<ol><li>为了确定荧光结合信号的百分比抑制, 由于复合预孵化, 应用以下公式: <img alt="Equation 1" src="/files/ftp_upload/57271/57271eq1.jpg"> 地方: "多边化" Exp = 实验性 (= 复合处理) 示例的多边化 "多边化" PC = 正控件的多边化 多边化NC = 负控制的多边化</li>
	<li>要确定复合 (即) 的 IC50值, 可将荧光绑定信号减少50% 的化合物的浓度, 请应用以下公式: <img alt="Equation 2" src="/files/ftp_upload/57271/57271eq2.jpg"> 地方: Log轻质. 2 = 一个化合物浓度的对数, 导致不到50% 抑制 PC 和 NC 的多边化值之间的差异 Log轻质 1 = 一个化合物浓度的对数, 导致超过50% 抑制了 PC 和 NC 的多边建筑的价值之间的差异。 注: 另外, 在高活性化合物的情况下, 可以根据每个测试的化合物浓度对应的多边建筑, 生成覆盖数个震级对数的剂量响应曲线。通过使用适当的软件 (参见材料和试剂表) 应用非线性回归曲线, 可以从生成的曲线推断出 IC50值。此分析方法的一个示例显示在图 3中。</li>
</ol>

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G., Nimmagadda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+CXCR4+expression+in+human+cancer+xenografts:+evaluation+of+monocyclam+64Cu-AMD3465.">Imaging CXCR4 expression in human cancer xenografts: evaluation of monocyclam 64Cu-AMD3465.</a> J Nucl Med. 52, 986-993 (2011).</li><li>Hatse, S., Princen, K., Liekens, S., Vermeire, K., De Clercq, E., Schols, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+CXCL12AF647+as+a+novel+probe+for+nonradioactive+CXCL12/CXCR4+cellular+interaction+studies.">Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.</a> Cytometry A. 61, 178-188 (2004).</li><li>Wootten, D., Christopoulos, A., Sexton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+paradigms+in+GPCR+allostery:+implications+for+drug+discovery.">Emerging paradigms in GPCR allostery: implications for drug discovery.</a> Nat Rev Drug Discov. 12, 630-644 (2013).</li><li>Louis, K. S., Siegel, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li><li>Perry, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc:+a+review+of+its+use+in+the+management+of+CCR5-tropic+HIV-1+infection.">Maraviroc: a review of its use in the management of CCR5-tropic HIV-1 infection.</a> Drugs. 70, 1189-1213 (2010).</li><li>Moyle, G., DeJesus, E., Boffito, M., Wong, R. S., Gibney, C., Badel, K., MacFarland, R., Calandra, G., Bridger, G., Becker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+of+activity+with+AMD11070,+an+orally+bioavailable+inhibitor+of+CXCR4-tropic+HIV+type+1.">Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1.</a> Clin Infect Dis. 48, 798-805 (2009).</li><li>Balabanian, K., Lagane, B., Infantino, S., Chow, K. Y., Harriague, J., Moepps, B., Arenzana-Seisdedos, F., Thelen, M., Bachelerie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+SDF-1/CXCL12+binds+to+and+signals+through+the+orphan+receptor+RDC1+in+T+lymphocytes.">The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes.</a> J Biol Chem. 280, 35760-35766 (2005).</li><li>Burns, J. M., Summers, B. C., Wang, Y., Melikian, A., Berahovich, R., Miao, Z., Penfold, M. E., Sunshine, M. J., Littman, D. R., Kuo, C. J., Wei, K., McMaster, B. E., Wright, K., Howard, M. C., Schall, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chemokine+receptor+for+SDF-1+and+I-TAC+involved+in+cell+survival,+cell+adhesion,+and+tumor+development.">A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.</a> J Exp Med. 203, 2201-2213 (2006).</li><li>Stoddart, L. A., Kilpatrick, L. E., Briddon, S. J., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+pharmacology+of+G+protein-coupled+receptors+with+fluorescent+ligands.">Probing the pharmacology of G protein-coupled receptors with fluorescent ligands.</a> Neuropharmacology. 98, 48-57 (2015).</li><li>Vernall, A. J., Hill, S. J., Kellam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+small-molecule+fluorescent-conjugate+toolbox+for+Class+A+GPCRs.">The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs.</a> Br J Pharmacol. 171, 1073-1084 (2014).</li><li>Stoddart, L. A., White, C. W., Nguyen, K., Hill, S. J., Pfleger, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-+and+bioluminescence-based+approaches+to+study+GPCR+ligand+binding.">Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.</a> Br J Pharmacol. 173, 3028-3037 (2016).</li></ol>

作者没有什么可透露的。

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。

<table><tbody><tr><td>BD FACSCanto II</td><td>Becton Dickinson</td><td>Not applicable</td><td>Flow cytometry device</td></tr><tr><td>BD FACSDIVA Software</td><td></td><td></td><td></td></tr><tr><td>BD FACSArray</td><td>Becton Dickinson</td><td>Not applicable</td><td>Flow cytometry device</td></tr><tr><td>BD FACSArray System Software</td><td></td><td></td><td></td></tr><tr><td>Graphpad Prism</td><td>Graphpad</td><td></td><td>software package used for nonlinear regression analysis in Figure 2 and Figure 3</td></tr><tr><td>FlowJo</td><td>FlowJo is now a wholly owned subsidiary of BD.</td><td></td><td></td></tr><tr><td>Vi-CELL</td><td>Beckman Coulter</td><td>Not applicable</td><td>cell viability analyzer</td></tr><tr><td>Sigma 3-18 KS</td><td>Sigma</td><td>Not applicable</td><td>centrifuge</td></tr><tr><td>AMD3100</td><td>Sigma</td><td>A5602-5mg</td><td>specific CXCR4 antagonist</td></tr><tr><td>Maraviroc</td><td>Pfizer</td><td></td><td>antiretroviral drug, CCR5 antagonist, available for research at Selleckchem (cat#S2003), Sigma (cat#PZ0002)</td></tr><tr><td>h-SDF1a (AF647)</td><td>ALMAC</td><td>CAF-11-B-01</td><td>fluorescently labeled CXCL12, CXCL12AF647</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco (Life Technologies)</td><td>10270-106</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A1933-25G</td><td></td></tr><tr><td>HBSS (10x), calcium, magnesium, no phenol red</td><td>Gibco (Life Technologies)</td><td>14065-049</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco (Life Technologies)</td><td>15630-056</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Gibco (Life Technologies)</td><td>14190-094</td><td></td></tr><tr><td>Jurkat cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>Reagent reservoir PP</td><td>Sigma</td><td>BR703411</td><td></td></tr><tr><td>Rapid flow filter: 0.2 &amp;micro;m aPES</td><td>Thermo Scientific</td><td>566-0020</td><td></td></tr><tr><td>Sterilin microtiter plate, 96-well, U bottom, clear</td><td>Thermo Scientific</td><td>611U96</td><td></td></tr><tr><td>Falcon tubes, 50ml</td><td>Greiner Bio-One</td><td>227 261</td><td></td></tr><tr><td>Tissue culture flask (T75)</td><td>Corning</td><td>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 ...

a-flow-cytometry-based-assay-to-identify-compounds-that-disrupt

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.6K 次观看.  KU Leuven. 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。

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

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 ...

科研

免疫与感染

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

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 ...

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 ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

一种基于动力学荧光的 Ca2 +动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

医学

人中性粒细胞中 β2 整合素激活抑制剂的筛选

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

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

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基于细胞的流式细胞仪检测，以测量细胞毒活性

细胞与分子极化的快速和强大的分析趋化因子诱导信号

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

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

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

化学抑制剂库筛选 t 细胞受体信号的中介识别

一种基于动力学荧光的 Ca^{2 +}动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物