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

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	<li>Mould, A. P. et al. Conformational changes in the integrin a domain provide a mechanism for signal transduction via hybrid domain movement. Journal of Biological Chemistry. 278 (19), 17028-17035 (2003).</li>
	<li>Chigaev, A. et al. Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation. Journal of Biological Chemistry. 284 (21), 14337-14346 (2009).</li>
	<li>Njus, B. H. et al. Conformational mAb as a tool for integrin ligand discovery. Assay and Drug Development Technologies. 7 (5), 507-515 (2009).</li>
	<li>Chigaev, A., Wu, Y., Williams, D. B., Smagley, Y., Sklar, L. A. Discovery of very late antigen-4 (VLA-4, &#945;4&#946;1 integrin) allosteric antagonists. Journal of Biological Chemistry. 286 (7), 5455-5463 (2011).</li>
	<li>Ghigo, A., De Santi, C., Hart, M., Mitash, N., Swiatecka-Urban, A. Cell signaling and regulation of CFTR expression in cystic fibrosis cells in the era of high efficiency modulator therapy. Journal of Cystic Fibrosis. 22, S12-S16 (2023).</li>
	<li>Van Goor, F., Yu, H., Burton, B., Hoffman, B.J. Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function. Journal of Cystic Fibrosis. 13 (1), 29-36 (2014).</li>
</ol>

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。

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			<td>APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24</td>
			<td>BioLegend</td>
			<td>363410</td>
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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

1.5K 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 

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

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

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 ...

科研

免疫与感染

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

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

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 Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

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

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

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

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

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. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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 Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

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

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

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 ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...