我们感谢康涅狄格大学健康中心流式细胞术核心的 Evan Jellison 博士和 Li Zhu 女士在流式细胞术方面的帮助，感谢康涅狄格大学健康中心免疫学系的 Lynn Puddington 博士对仪器的支持，感谢康涅狄格大学健康中心临床研究核心的 Slawa Gajewska 女士和 Paul Appleton 博士在获取血液样本方面的帮助。我们感谢康涅狄格大学医学院的 Christopher "Kit" Bonin 博士和 Geneva Hargis 博士在科学写作和编辑本手稿方面提供的帮助。这项研究得到了美国国立卫生研究院、美国国家心脏、肺和血液研究所 （R01HL145454）、美国国家普通医学科学研究所 （P20GM121176）、美国心脏协会职业发展奖 （18CDA34110426） 和康涅狄格大学健康中心的启动基金的支持。 图 1 是使用 BioRender.com 创建的。

根据《赫尔辛基宣言》的原则，在获得知情同意后，从去识别化的健康人类供体处获得肝素化全血样本，经康涅狄格大学健康机构审查委员会批准。已获得所有捐赠者的知情同意。本研究的纳入/排除标准经过精心制定，以确保受试者的适用性并将潜在风险降至最低。符合条件的参与者年龄在 18 至 65 岁之间，不分种族，英语流利，能够提供知情同意。被排除在外的受试者包括那些无法为自己提供知情同意的人，例如那些需要合法授权代表的人、18岁以下或65岁以上的个人、被监禁的人和孕妇。此外，参与者必须没有使用抗炎药物和炎症。当前感染或持续的慢性或急性炎症性疾病也是排除标准。最后，当前或近期有 COVID-19 感染史的个体不符合该研究的资格。这些标准旨在确保参与者的安全性和适用性，同时最大限度地减少可能影响研究结果的潜在混杂因素。
1.试剂的制备
<ol><li>中性粒细胞培养基:通过向不含酚红的RPMI-1640中加入2%人血清白蛋白来制备中性粒细胞培养基（参见 材料表）。</li>
	<li>fMLP溶液:用不含酚红的RPMI 1640从10mM fMLP二甲基亚砜（DMSO）储存溶液（参见 材料表）中稀释100倍来制备fMLP溶液，得到100μM fMLP溶液。</li>
	<li>抗体溶液:在 10 mL 中性粒细胞培养基中稀释 120 μL 别藻蓝蛋白 （APC） 偶联单克隆抗体 mAb24 （mAb24-APC）（参见 材料表），该抗体报告了 β2 整合素的高亲和力构象，形成 1.2 μg/mL mAb24-APC 溶液。</li>
	<li>化合物库:通过使用液体处理器将 5 μL 的 10 mM 文库（参见 材料表）加入 384 孔板中的 45 μL DMSO 中，将浓度为 10 mM 至 1 mM 的初始化合物库稀释至 1 mM。随后，使用液体处理器将每孔 5 μL 的 1 mM 化合物库转移到空的 384 孔板中。</li>
	<li>如图1所示，四列（ 1、2、23、24）仅含有DMSO，不含化合物，在筛选中作为阳性和阴性对照。通过加入 45 μL 不含酚红的 RPMI-1640 进一步稀释每个孔，使所有化合物的最终浓度为 100 μM。</li>
</ol>2. 从人体血液中分离中性粒细胞
<ol><li>使用血清移液管，在15mL离心管中小心地将4mL血液分层在8mL市售密度梯度培养基（参见 材料表）（图2A）中。</li>
	<li>将血液在20°C下以550× g 离心30-50分钟。 逐渐使转子减速（减速系数为1）。 注意:中性粒细胞（ 图2B中的条带2）从红细胞中成功分离可能因供体而异。对于大多数供体，30 分钟的离心就足够了。然而，对于一些供体，如果分离不成功，可能需要额外的10-30分钟离心（图2C）。</li>
	<li>小心地去除血浆（顶部的黄色液体）和单核细胞（上部混浊带; 图2B中的PBMC条带）使用1 mL移液管。</li>
	<li>将中性粒细胞从下部混浊带（ 图2B中的中性粒细胞带）收集，并在透明液体下方约3-4mL收集到含有10mL磷酸盐缓冲盐水（PBS）的15mL离心管中。将中性粒细胞悬浮液倒置2-3次，轻轻混合。</li>
	<li>将悬浮液在400× g 下在20°C下离心10分钟，然后通过倾析小心地除去上清液。</li>
	<li>用5mL PBS轻轻重悬沉淀，并在20°C下以300× g 离心5分钟。</li>
	<li>通过倾析除去上清液，并使用真空抽吸小心地清除管口周围和管壁上的任何残留上清液。将沉淀重悬于 1 mL 中性粒细胞培养基中。</li>
	<li>使用血细胞计数器计数细胞数。通常，从 8 mL 人血中获得 1 至 4 ×10 7 个中性粒细胞。 注意:中性粒细胞悬浮液中可能存在红细胞污染。红细胞不会显著影响大多数检测，并且可以防止中性粒细胞活化/启动27.红细胞在计数前需要裂解，以获得准确的中性粒细胞浓度。将 10 μL 细胞悬液加入 891 μL 去离子水中 10-30 秒以裂解红细胞，然后加入 99 μL 10× PBS 以平衡渗透压，防止中性粒细胞裂解。</li>
	<li>通过加入中性粒细胞培养基，将细胞密度调节至 6.25 ×10 5 个细胞/mL。</li>
</ol>3. 384孔板的制备
<ol><li>在 1.5 mL 离心管中，将 1 mL 抗体溶液 （1.2 μg/mL mAb24-APC） 与 0.2 mL 中性粒细胞培养基混合，制备 1 μg/mL mAb24-APC 溶液。然后，将 25 μL 该混合物加入 384 孔板（ 图 1 中的蓝色孔）中的阴性对照孔中。</li>
	<li>在 15 mL 离心管中，将 9 mL 抗体溶液与 21.6 μL fMLP 溶液 （100 μM） 和 1.7784 mL 中性粒细胞培养基混合，以制备含有 1 μg/mL mAb24-APC 和 200 nM fMLP 的溶液。接下来，将 25 μL 该混合物加入 384 孔板（ 图 1 中的红色和蓝色孔）中的阳性对照和测试孔中。</li>
	<li>使用多通道移液器将 5 μL 的 100 μM 化合物溶液从化合物库板转移到 384 孔板中。</li>
	<li>在室温下以500× g离心液体1分钟。 注意: 要离心板，需要一个水平桶转子和板桶。</li>
</ol>4.细胞处理
<ol><li>使用 16 通道移液器（5-50 μL 范围，384 孔板的每列）向每个孔中加入 20 μL 中性粒细胞悬浮液。使用移液管轻轻混合 5-10 次，每次移液之间保持一致的时间间隔。 注:孔中的最终浓度如下:中性粒细胞 2.5 × 105 个细胞/mL（所有孔）;mAb24-APC 0.5 μg/mL（所有孔）;fMLP 100 nM（阳性对照和测试孔）;化合物 10 μM（测试孔）。</li>
	<li>在室温（RT）下，在300rpm的振荡器上孵育板10分钟。</li>
	<li>使用16通道移液管（最终PFA浓度~0.91%）向每个孔中加入3μL的16%多聚甲醛（PFA）来固定细胞。然后，在冰上孵育10分钟。 注意:建议在添加中性粒细胞和PFA时，不同色谱柱之间保持一致的时间间隔。这确保了每个孔中的中性粒细胞与 fMLP 和 mAb24-APC 具有相同的孵育时间。</li>
</ol>5. 流式细胞术
<ol><li>打开流式细胞仪（见 材料表），并确保连接到仪器的计算机也已打开。</li>
	<li>确保鞘液容器已装满，废液容器为空并正确连接到仪器。</li>
	<li>将 384 孔板加载到流式细胞仪的样品加载器上。确保板的 A1 孔与装载机上的 A1 标记对齐。</li>
	<li>打开软件并创建一个新实验。选择 384 孔 并选择所需的荧光基团。接下来，单击" 板设置"，拖动以选择所有孔，然后单击 "样品"。打开"高吞吐量"开关，然后在速率面板下选择 高 。<ol><li>在音量框中，输入 50。在奇数列中选择样品，然后激活"搅拌"开关。最后，在右侧面板中命名示例。</li>
	</ol></li>
	<li>单击 "绘图和门"。然后，单击 "应用 "按钮（绿色填充圆圈上的两个箭头），随后单击 播放 按钮（三角形）。等待几秒钟以观察第一个孔中的一些细胞，然后单击 暂停 按钮。<ol><li>调整 FSC 和 SSC 电压，以确保图上细胞的正确可视化。单击 "采集"，然后单击 播放 按钮（三角形）以开始检测。</li>
	</ol></li>
	<li>流式细胞仪将依次从每个孔中取样 ~50 μL 的 1000-3000 个中性粒细胞。完成整个 384 孔板大约需要 3 小时。</li>
	<li>完成所有示例后，导出 .fcs 文件以供下一步使用。</li>
</ol>6. 数据分析
<ol><li>打开流式细胞术数据分析软件（参见 材料表）。选择包含所有 .fcs 文件的文件夹并将其拖动到软件窗口中，然后松开以将数据导入软件。</li>
	<li>双击一个样品，在弹出的窗口中基于FSC和SSC28,29,30的单个中性粒细胞进行门控，如图3所示。选择门控行并将它们拖动到文件夹中，然后松开以将门控应用于该文件夹中的所有样本。</li>
	<li>使用软件的表格编辑器功能计算并导出每个孔中嗜中性粒细胞的 APC 中值荧光强度 （MFI）。单击"编辑"选项卡下的"添加列"。在统计选项卡中选择中位数，选择门控群体中性粒细胞，然后选择 APC 作为参数。单击"确定"按钮。</li>
	<li>返回"表格编辑器"选项卡，在"组"选项卡中选择 "所有样品 "，然后按 "创建表格 "按钮。将出现一个新窗口，显示创建的表。将其另存为文本、CSV 或 Excel 文件。</li>
	<li>打开导出的表格，计算阳性和阴性对照样品的APC MFI的平均值和标准偏差（图4）。</li>
	<li>使用以下公式计算板的 Z' 因子 （Z'）: Z' = 1 - 3（σ p + σ n）/（μp - μn）， 其中 σ p 和 σ n 分别是阳性和阴性对照的标准差，μp 和 μn 分别是阳性和阴性对照的平均值31. 注: Z' 因子表示正负对照分布的分离。Z' 为 0 表示没有分离，而 Z' 为 0.5 表示相等的分离。如先前的研究31 所述，Z' > 0.5 表示化合物筛选的极好测定方法。0.5 到 0 范围内的 Z' 是可以接受的，但它可能只能识别检测中的强命中，这需要在二次检测中进一步验证。</li>
	<li>当化合物处理的中性粒细胞的APC MFI低于从阳性对照MFI中减去阳性对照MFI标准差的三倍时，将化合物视为筛选的"命中"（P = 0.0013，由 图4中的虚线表示）。</li>
</ol>

人中性粒细胞中 β2 整合素激活抑制剂的筛选

作者声明没有相互竞争的经济利益。

中性粒细胞刺激和染色的开始和终止由添加中性粒细胞和固定剂PFA来确定。因此，确保将中性粒细胞或PFA移液到每根色谱柱中的时间间隔相同至关重要。这确保了每个孔中嗜中性粒细胞的刺激和染色时间保持一致。由于中性粒细胞的寿命较短，从从供体采集血液到完成流式细胞术的整个实验必须在同一天进行。中性粒细胞对温度变化高度敏感，当暴露于温度快速升高时，例如从4°C过渡到室温或从...

许多炎症性疾病的特征在于中性粒细胞浸润在肿胀或损伤部位1。为了浸润这些组织，中性粒细胞必须完成中性粒细胞募集级联反应，包括阻滞到内皮细胞，外渗穿过血管壁，然后募集到组织中2。循环中性粒细胞需要β2整合素激活才能完成这一级联反应，特别是在停滞阶段。因此，减少中性粒细胞粘附、外渗和募集的整合素抑制药物可有效治疗炎症性疾病 3,4。
β2整合素以前曾被靶向用于炎症性疾病。依法珠单抗是一种直接靶向整合素αLβ2的单克隆抗体，用于治疗银屑病5。然而，依法珠单抗因其致命的副作用而被撤回 - JC病毒再激活导致的进行性多灶性白质脑病6,7。基于整合素的新型抗炎疗法应考虑维持白细胞的抗感染功能，以尽量减少副作用。依法珠单抗的副作用可能是由于单克隆抗体在血液中的循环时间延长，从长远来看可能会抑制免疫功能8.最近的一项研究表明，依法利珠单抗介导 αLβ2 交联和 α4 整合素的不需要的内化，为副作用提供了另一种解释9.因此，短寿命的小分子拮抗剂可能会避免这个问题。
本文介绍了一种使用人中性粒细胞筛选小分子β2整合素拮抗剂的高通量方法。β2 整合素激活需要整合素胞外结构域的构象变化才能进入并增加其与其配体的结合亲和力。在规范的弹簧刀模型中，弯曲闭合的整合素胞外结构域首先延伸到扩展-闭合构象，然后打开其头件以完全激活的扩展-开放构象10,11,12,13。还有另一种途径，从弯曲闭合到弯曲打开和扩展打开，最终是14、15、16、17、18、19。构象特异性抗体 mAb24 与人 β2-I 样结构域中的表位结合，当胞外结构域的头饰打开时20,21,22,23。
在这里，mAb24-APC 用于确定 β2 整合素是否被激活。为了激活中性粒细胞和整合素，N-甲酰甲硫酰-亮氨酰-苯丙氨酸 （fMLP），一种细菌衍生的短趋化肽，可以激活中性粒细胞 β2 整合素24，在该方案中用作刺激物。当 fMLP 与中性粒细胞上的 Fpr1 结合时，涉及 G 蛋白、磷脂酶 Cβ 和磷酸肌醇 3-激酶γ的下游信号级联被激活。这些信号转导事件最终通过由内而外的信号通路18,25 导致整合素激活。除了直接与β2整合素结合并阻止整合素活化构象变化的小分子拮抗剂26外，该方法还可以检测出可以抑制β2整合素由内而外的激活信号通路中成分的化合物。自动流式细胞仪可实现高通量筛选。识别新的拮抗剂不仅可以加深我们对整合素生理学的理解，还可以为基于整合素的抗炎治疗提供转化见解。

<table><tbody><tr><td>16-channel pipettes</td><td>Thermo</td><td>4661090N</td><td>Instrument</td></tr><tr><td>384-well plate</td><td>Greiner</td><td>784201</td><td>Materials</td></tr><tr><td>APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24</td><td>BioLegend</td><td>363410</td><td>Reagents</td></tr><tr><td>Bravo&amp;nbsp;Automated Liquid Handling Platform&amp;nbsp;</td><td>Agilent</td><td>16050-102</td><td>384 multi-channel liquid handler</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Model 5810R</td><td>Instrument</td></tr><tr><td>FlowJo</td><td>Becton, Dickinson &amp;amp; Company</td><td>NA</td><td>Software</td></tr><tr><td>Human Serum Albumin Solution (25%)</td><td>GeminiBio</td><td>800-120</td><td>Reagents</td></tr><tr><td>Lifitegrast</td><td>Thermofisher</td><td>&amp;nbsp;50-208-2121</td><td>Reagents</td></tr><tr><td>Nexinhib20</td><td>Tocris</td><td>6089</td><td>Reagents</td></tr><tr><td>N-Formyl-Met-Leu-Phe (fMLP)</td><td>Sigma</td><td>F3506</td><td>Reagents</td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Reagents</td></tr><tr><td>Plate buckets</td><td>Eppendorf</td><td>UL155</td><td>Accessory</td></tr><tr><td>Plate shaker&amp;nbsp;</td><td>Fisher</td><td>88-861-023</td><td>Instrument</td></tr><tr><td>PolymorphPrep</td><td>PROGEN</td><td>1895 (previous 1114683)</td><td>Reagents</td></tr><tr><td>Prestwick Chemical Library Compound Plates (10 mM)</td><td>Prestwick Chemical Libraries</td><td>Ver19_384</td><td>1520 small molecules, 98% marketed approved drugs (FDA, EMA, JAN, and other agencies approved)</td></tr><tr><td>RPMI 1640 Medium, no phenol red</td><td>Gibco</td><td>11-835-030</td><td>Reagents</td></tr><tr><td>Swing-bucket rotor&amp;nbsp;</td><td>Eppendorf</td><td>A-4-62</td><td>Rotor</td></tr><tr><td>ZE5 Cell Analyzer</td><td>Bio-Rad Laboratories</td><td>Model ZE5</td><td>Instrument</td></tr></tbody></table>

a flow cytometry-based high-throughput technique for screening integrin-inhibitory drugs

该方案旨在建立一种利用构象变化报告抗体和高通量流式细胞术鉴定β2整合素活化的小分子拮抗剂的方法。该方法还可以作为其他基于抗体的高通量筛选方法的指南。β2整合素是白细胞特异性粘附分子，在免疫反应中至关重要。中性粒细胞依靠整合素活化排出血液，不仅可以抵抗感染，还可以参与多种炎症性疾病。控制β2整合素活化是治疗中性粒细胞相关炎症性疾病的可行方法。在该方案中，单克隆抗体 mAb24 与 β2 整合素的高亲和力头片特异性结合，用于定量分离的原代人中性粒细胞上的 β2 整合素活化。N-甲酰甲硫酰-亮氨酰-苯丙氨酸 （fMLP） 用作激活中性粒细胞 β2 整合素的刺激物。本研究使用了能够自动运行 384 孔板样品的高通量流式细胞仪。在3小时内评估320种化学物质对β2整合素抑制的影响。通过这种方法可以识别直接靶向β2整合素的分子或G蛋白偶联受体启动的整合素由内而外的激活信号通路中的靶分子。

来自代表性的 384 孔板筛选的数据（图 4）显示，阴性对照的 mAb24-APC MFI 为 3236 ± 110，而阳性对照的 mAb24-APC MFI 为 7588 ± 858。该板的 Z' 因子约为 0.33，在可接受的范围内31。然而，Z'需要在二次测定中进一步验证。
为了对数据进行归一化，对所有值进行了缩放，以将最大值 1 分配给正均值，将最小值 0 分配给负均值。Z'因子将在二次测定?...

Watch this Scientific Journal Video about Development of a Method for Identifying Small Molecular Antagonists of Î²2 Integrin Activation at JoVE.com

作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

该协议描述了一种基于流式细胞术的高通量筛选方法，用于鉴定抑制人中性粒细胞上β2整合素活化的小分子药物。

一种基于流式细胞术的高通量技术筛选整合素抑制药物

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

我们的研究重点是中性粒细胞整合素，在这项研究中，我们旨在建立一种寻找β-2整合素激活的小分子拮抗剂的方法。我们相信，通过这种方法，我们可以找到新的拮抗剂，这些拮抗剂可能会加深我们对整合素生理学的理解，并为基于整合素的抗炎疗法提供转化见解。未来，我们将专注于发现新的抗炎药并剖析其作用机制。

a-flow-cytometry-based-high-throughput-technique-for-screening

Research

JoVE Journal

Immunology and Infection

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

1.8K 次观看.  UConn Health. 我们的研究重点是中性粒细胞整合素，在这项研究中，我们旨在建立一种寻找β-2整合素激活的小分子拮抗剂的方法。我们相信，通过这种方法，我们可以找到新的拮抗剂，这些拮抗剂可能会加深我们对整合素生理学的理解，并为基于整合素的抗炎疗法提供转化见解。未来，我们将专注于发现新的抗炎药并剖析其作用机制。

视频: 作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

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

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

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

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

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

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

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 ...

生物学

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://app.jove.com/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

使用工程淋巴细胞筛选免疫调节化合物的基于荧光的测定

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

Source: Schoofs, G., et al. <a href="https://app.jove.com/v/57271">A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.</a> J. Vis. Exp. (2018).
In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.

竞争性结合测定，用于鉴定破坏受体-配体相互作用的化合物

Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to ...