研究这个手稿报告是由过敏和美国国立卫生研究院传染病研究所根据奖号R01AI099122到JEK的内容完全是作者的责任的支持，并不一定代表全国学院的官方意见健康。我们想从体外培育牛黄，朗伍德筛选基金和/或国家筛选实验室感谢詹尼弗·史密斯，大卫·罗贝尔，苏蒋道格洪水，肖恩·约翰斯顿，珍妮弗·纳莱，斯图尔特鲁德尼茨基，保仁，理查德兆，和Rachel监狱长卓越的生物防御和新兴他们在开发和高通量筛选试验的表现帮助传染病（由U54AI057159支持）新英格兰地区中心。我们还要感谢肯尼斯·P·史密斯对稿件有益的意见。

1.实时细胞内生长和真核细胞毒性测定<ol><li>准备宿主细胞（J774A.1细胞） <ol><li>培养J774A.1 小家鼠巨噬细胞样细胞悬浮在RPMI 1640用9％铁补充的牛血清。最初通道在组织培养瓶中。细胞在15ml培养基中已经成为一个75cm 2的组织培养瓶中铺满后，通过刮擦和稀释分裂到65毫升的相同类型的介质中，其中15毫升返回到组织培养瓶和50毫升转移到250毫升细菌摇瓶中。 </li><li>对于规模化，文化悬浮于250毫升和/或填充到五分之一体积组织培养基千毫升细菌的烧瓶。通过以大约每分钟120转设定转速通气。对于持续增长，孵化正好5％的CO 2和37℃。 </li><li>当他们在范围达到密度收获细胞2.5×10 6个细胞/ ml至5×10 6个细胞/ ml。确保死细胞百分比（通过台盼蓝染色最容易测定）不超过25％，如死细胞会在细胞毒性测定增加的背景噪声。 </li><li>板在白色，组织培养处理过的，384孔微孔板在30微升组织培养基中5×10 4 J774A.1细胞每孔。孵育微孔板过夜，以实现对实验的每天90％汇合。 </li></ol></li><li>准备发光或荧光嗜肺军团菌 <ol><li>通过嗜肺军团菌 （LP02 :: flaA基因::勒克斯8）适宜的培养基上的细菌， 也就是缓冲木炭酵母提取物（BCYE）中。如果使用胸苷营养缺陷型菌株，补充介质用100微克/毫升胸苷。通过在一个新的BCYE板厚厚扩频生物体和（从冷冻的储存如果）（从以前的盘通路如果）孵育一天或两到三天，以获得汇合准备细菌实验的补丁增长。 <ol><li>通过将10克酵母提取物，10个克N-制备BCYE板- （2-乙酰氨基）-2-氨基乙烟酸，0.35g K 2 HPO 4，和950毫升去离子水1克二氢钾α酮戊二酸。调整与氢氧化钾（≥2.5毫升11.9摩尔溶液）pH值6.9。 </li><li>加入琼脂15克和2克活性炭;并带来至1L总体积。加入磁力搅拌棒和高压灭菌。至55℃，冷介质和含有0.4克L-半胱氨酸和0.42克铵铁（III）柠檬酸溶解在去离子水添加过滤灭菌的溶液。使用磁搅拌盘前浇培养皿混合。 </li><li>对于无菌生长培养基嗜肺军团菌生长的测试，并用于所有BCYE化学试剂相结合，除了木炭准备ACES，酵母提取物肉汤（AYE）和琼脂。过滤消毒，并立即使用，或者冻结以后的实验。 </li></ol></li><li>在SA悬浮生物用于J774A.1细胞用100μg/ ml的胸苷补充适当我组织培养基。 </li></ol></li><li>巨噬细胞感染<ol><li>添加感兴趣的测试化合物（筛选化合物，以及阳性和阴性对照细菌生长抑制和真核细胞裂解）。优选地，在≥500x溶解储备溶液在DMSO（二甲基亚砜）或水溶液，以允许车辆的足够稀释。 <ol><li>虽然库存溶液可以冷冻贮存，避免不稳定化合物和抗微生物剂冻融循环。 </li></ol></li><li>稀释发光细菌的目标的在组织培养基中2.5×10 6 CFU（菌落形成单位）/米和荧光报道标记的细菌到1.0×10 7 CFU / ml和添加适当的非毒性，膜不通透性，核酸-在2.5倍最终测定浓度结合染料。 </li><li>加入20微升混合物到每个J774A.1文化很好，最终测定体积50微升，最终菌浓度为1×10 6 CFU /毫升（勒克斯操纵子记者）或4×10 10 6 CFU /毫升（荧光蛋白报告）。 </li><li> CO 2的100％在37℃下1-3天，在5％的相对湿度，以防止蒸发的边缘效应。 </li></ol></li><li>检测读数<ol><li>阅读微孔板发光仪和荧光为适合记者细菌的生长和真核细胞的毒性被使用。 <ol><li>为了避免温度相关的边缘效应，热微孔板发光读数之前通过放置在一个单一的层平衡一个实验台上用盖子微开大约20分钟（或在有盖的生物安全柜关闭大约10分钟）。 </li></ol></li><li>返回如果在稍后的时间点实时读出希望微孔板孵化器。 </li></ol></li></ol> 2.数据分析<ol><li>正常化数据正负CONtrols计算百分比或折叠抑制细胞内细菌的生长和细胞毒性百分比为真核细胞。 </li><li>为了评估测定鲁棒性，计算为细菌生长抑制和细胞毒性阳性和阴性对照之间的统计分离使用Z因子（Z&#39;）的分析，其中Z&#39;= 1 - 3（σP +σN）/ |μ 的p +μÑ |。在此公式中，σp和σn分别为正和负对照孔，分别标准偏差; μp和μn分别为正和负对照孔的平均值，分别为。 <ol><li>用于高通量筛选测定法，计算标准z得分（或其它替代方案中，定量的统计度量），以评估感兴趣的测试化合物的作用。另外，计算简单折叠减少或登陆倍减少作为一个潜在的生理上更相关<html>影响幅度为衡量几何复制细菌。 </li></ol></li></ol> 3.单和多维（ 即协同）剂量反应测试及数据解读<ol><li>如在协议第1节准备的巨噬细胞和细菌。 </li><li>感染或无菌孵化只是之前，在高端加实验兴趣的抗菌剂以串行，二倍稀释系列，跨越的目标，完全消除生长的浓度（即最小抑菌浓度或MIC）和低结束浓度没有显示出明显的活性。 </li><li>使用自动液体处理系统，以促进通过直接，自动除了根据制造商的协议抗菌稀释的单剂量 - 反应和组合协同作用的测试设置。 </li><li>考虑使用子二倍稀释的，例如，841 / 54841eq1.jpg"/&gt; - 倍稀释系列，和测定复制以增加数据的准确性和可重复性。 </li><li>对于巨噬细胞的感染，稀发光细菌以在组织培养基中的目标2.5×10 6 CFU（菌落形成单位）/毫升。加入20μl混合物的每个J774A.1培养孔，最终测定体积50微升，最终菌浓度为1×10 6 CFU / ml的;并在37℃下在5％CO 2，在100％相对湿度下孵育。 <ol><li>为无菌增长试验，稀发光细菌在AYE介质1×10 6 CFU / ml的终浓度，加入50微升到384孔板的每个孔中并在5％CO 2在37℃孵育 在100％的相对湿度。 </li></ol></li><li>计算抑制细菌生长的抗菌组合分数抑制剂浓度指标。在组合导致细菌○完全或&gt; 99％抑制每种药物（A，B，。。n）的"一个"本身FIC 一个 =化合物的浓度"a"的通过的最小抑制剂浓度分为:rowth，计算各个部分抑菌浓度（FIC 的a，b，正。）如下。 。使用相同的公式，计算出FIC B， 等，然后计算组合FIC指数或<img alt="公式（2）" src="/files/ftp_upload/54841/54841eq2.jpg" /> FIC = FIC A + FIC B +。 。 .FIC N每个抑制组合。 <ol><li>使用偏离最远的从1.0到评估协同作用的组合索引。考虑≤0.5的截止作为协同效应保守指示和≥4，拮抗剂效果。 12 </li><li>情节等效线图，等高线等效线图，和/或等值面等效线图的组合效应为6替代图形表示。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们描述了细胞内的细菌的生长和宿主细胞的细胞毒作用的同时检测的实时检测。有在协议中的几个关键步骤。首先，对于健壮测定性能，必须有细菌和细胞毒性读数之间有足够的光谱分离。这样的分离是本征为萤光素酶操纵子记者和荧光DNA结合染料的组合。然而，根据我们的经验（ 表1-3， 图2），使用双荧光读出要求的不重叠的荧光信号（ 例如 ，利用一个远红色荧光蛋白报告和绿色核酸染色的），以获得健壮，基于溶液的数据。此外，以往的经验，这可能涉及到读者提供光学或解决方案荧光性质，只能用绿色或橙色发射DNA污渍可用了数据的细胞毒性， 也就是说 ，Z&#39;大于&gt; 0.5，表示斯特龙措施摹检测性能。 4因此，基于解决方案的，双读出试验是有点在什么可以用来记者条款的约束。 另一个关键步骤是感染细菌的巨噬细胞的比率。细菌感染诱导宿主细胞死亡。因此，过高的接种物可能导致早期宿主细胞死亡，相对低水平细菌复制的，并且在研究抗微生物剂直接细胞毒性效应的潜力模糊处理。适当的感染比率可以凭经验确定，并可能取决于所研究的特定的细菌菌株的毒力。约1的低比例:1是一个很好的起点。实验如在图2中所示的那些与表1 - 3与若干感染比率进行将允许在将来测定使用条件的评估和优化。值得注意的是， 军团菌 ，特别是使用的菌株背景具有NAT的ively降低宿主细胞毒性（鞭毛蛋白突变体）8显著贡献获得了高含量的鲁棒性。此外，双读出技术对其他细胞内的翻译，病原体主机系统可能也需要对感染协议额外的调整。 军团不会在组织培养基中生长，使我们能够增加记者信号单独归因于细胞内的生长。然而，许多细胞内病原体（ 如 ， 布鲁氏 ）既可以在细胞内和在组织培养基中生长到不同程度。因此，附加的步骤，如初始感染后庆大霉素治疗和宿主细胞的洗涤可能是必要的，以减少外复制和允许的胞内复制的能力抽查。 22 在高通量筛选和协同测试试验规模扩大使用都与它自己的挑战有关。最特别LY，我们发现，所需的J774A.1细胞的大量可能最好在细菌而不是传统的组织培养瓶（参照图1）进行培养。然而，为了维持培养无菌，海绵泡沫或其他过滤器帽这些烧瓶发现优选标准细菌烧瓶帽。一个标准的组织培养箱里面的小轨道摇床平台的放置允许我们手头有设备进行规模化的文化。最后，使用自动点胶系统（ 图1A，B），极大地促进了实验设置和组合系列稀释的测试。 值得注意的是，细菌复制的适当检测依赖于充分地表达任一勒克斯操纵子或荧光蛋白在感兴趣的细菌菌株的能力。为此，我们已经创建了一组由强组成启动子应该允许发驱动荧光灯和勒克斯操纵子记者转座子CILE多样种类的细菌的标记（康和卡比，数据未显示）。可替代地，对于病原体的细胞内复制证明对于宿主细胞的细胞毒性，单独的实时的细胞毒性的测量可以用作细胞内复制的替代品。然而，在这种情况下，细胞毒性和非活性化合物不能在单一测定可靠地区分。 实时读出的可用性提供了用于高通量筛选测定法特别有利。具体来说，避免细菌生长（菌落形成单位决定）7和细胞毒作用（除了细胞活力试剂在试验终止）23,24消除了几个实验步骤和相关的劳动端点评估。此外，课程的时间可以很容易地遵循相同的试验井实验努力增加最小。重要的是，两种不同类型的细菌复制了记者的可用性（细菌升勒克斯操纵子和荧光蛋白）具有高通量筛选试验发展特别重要的意义。具体来说，从勒克斯操纵子或荧光报告输出杂散干扰造成的误报抗菌读数可以通过同时使用记者依次在初筛和复筛，互补正交试验分别为25处理。值得注意的是，我们发现，细菌勒克斯操纵子是细菌生长的更敏感的报告一个大约20倍到100倍比我们的荧光蛋白的记者的检测下限。然而，勒克斯输出很可能受到基于所述多个基因产物和用于光输出所需的步骤多误报。 26更具体地说，在相对于真核单基因萤光素酶的记者，细菌勒克斯操纵子是，对萤光素酶底物的内源性合成编码两双组分萤光素酶和基因五基因簇。它会有意义因此使用勒克斯操作者记者在主屏幕，以确保足够的灵敏度，并且所述荧光蛋白记者在次级测定法，以确保特异性。 两者合计，所描述的方法提供了实时的，非破坏性的替代终点测定法用于测定细菌复制和真核细胞毒性，因此，代表用于评估对胞内病原体的抗菌效果的方法简单和通用的方法。

在车厢内临时增加或居住病原体很难根除治疗。预留或相对专性细胞内的病原体，如嗜肺军团菌 ， 立克次体 ， 布鲁氏菌。 ，土拉弗朗西斯菌 ， 结核分枝杆菌。往往需要长时间抗菌治疗疗程治愈，可能范围从个月甚至数年。此外，细胞外的病原体可能暂时占据利基内并以这种方式通过抗生素治疗一般疗程逃脱通关，后来冒出来启动毒感染的新一轮。 金黄色葡萄球菌和1肠杆菌科细菌尿路感染2,3两日益认识到的例子。因此，从根本上药物发现的目标是，以确定渗透到细胞内的区室新颖抗微生物剂。最佳治疗迅速消灭胞内生物体和防止通过亚抑制抗微生物暴露性的发展是特别理想的。 为此，我们开发了一种高通量筛选技术来识别细胞内渗透剂抗菌药物靶向模型病原体， 嗜肺军团菌的细胞内生长。 4既往临床观察表明，标准的药敏试验并没有准确地对这种微生物体内的治疗功效预测。 5具体地，这是因为主要类别抗微生物剂，如β内酰胺类和氨基糖苷类的，虽然针对无菌生长军团菌非常有效，没有充分渗透到其中， 军团驻留的胞内区室。 5,6后来的证据表明，在技术上更复杂的细胞生长测定有效预测临床疗效。 7 </sup&gt;不幸的是，这些测定是极其费力的端点测定法，要求感染巨噬细胞，用抗微生物剂处理过的，在不同的时间点被裂解为菌落形成单位计数。此类测定法是不实际的大规模做，不适合于高通量药物筛选。 因此，我们开发的技术为细胞内细菌生长的实时确定。 6这是通过使用或者通过一个细菌萤光素酶操纵子8的集成改性的细菌菌株的9记者（第二代，正交检测，这里描述的）来实现（第一代测定中，如前所述）4或荧光蛋白到细菌染色体中。以这种方式，发光或荧光信号提供细菌数的替代，实时读数。 然而，这些属性没有解决在细胞内infectio一个主要混杂因素Ñ测定法，对宿主细胞的脱靶效应。具体地，宿主细胞的死亡固有地限制细胞生长和导致的抗微生物效果的假阳性鉴定。在筛选库，许多化合物是真核细胞的毒性，例如假阳性将压倒真抗微生物剂，因此需要大量的后续，端点细胞毒性测定法进行解析。 因此，这是极大的兴趣的，以便能够同时评估真核细胞生存力和细胞内生长。值得注意的是，非存活的真核细胞的特征是细胞膜完整性的损失。该测试细胞膜的渗透性探针可以因此用于评估细胞生存力。我们以前的特点一系列推定细胞膜非透性，荧光，DNA结合染料的访问和染色死细胞的核DNA的能力。 4在结合核DNA，这些染料显示quantu大幅增加米荧光产量造成了后台解决方案增加了荧光信号。因此，这些染料提供真核细胞死亡的定量读数。 4值得注意的是，我们发现几个人正在无毒自己长期合作孵育J774巨噬细胞中。当最初的感染过程中加入的，它们提供了一种实时的，可以由一个微板荧光计来测量或观察到的微观真核细胞死亡的荧光读数。 因此，通过组合使用细菌记者和无毒，膜不通透性的，DNA结合染料，我们能够发展一种简单的，非破坏性的，实时的检测，以测量细菌负荷，同时真核细胞的细胞毒性。此法使我们能够在384孔板筛选〜万知的生物活性物质，包括〜250抗菌剂和&gt; 24万的小分子与功能上未鉴定的活性，抑制细胞内生长的能力嗜肺军团菌 ，而在同一时间产生用于每个化合物的真核细胞的细胞毒性数据。 6我们对军团菌生长的细胞内已知的抗菌药物分析是这种类型迄今为止最全面的探索。 6 根据我们的测定形式的效率，在组合使用时，我们也随之探讨已知抗微生物剂的潜在的协同作用。其中最常见的协同作用试验中，所谓的棋盘测定中，被标准地通过评估两个或更多抗微生物剂的两倍连续稀释的组合效果进行。 10在这些试验中，当两个或多个抗微生物剂都大于每个单独施加的效果的总和施加一起协同作用是由更大的效果的观察中定义。值得注意的是，迄今为止，只有专注和反对细胞内嗜肺军团菌进行选择性的协同测试</eM&gt;因为涉及传统的端点检测乘以所需的组合排列的巨大努力。 为了方便协同测试，我们在自动化数字胶技术相结合6利用我们的实时细胞生长/真核细胞毒性实验的。这种自动化允许我们以分配溶于DMSO或单独或在384孔格式组合水溶液的化合物的系列稀释。 11此外，这种强大的液体处理技术，使我们能够轻松地执行更高的分辨率，平方根的二（而不是标准的，较低的分辨率，加倍）稀释组合在我们的二维实现更高水平的特异性，棋盘的协同作用分析。用两倍稀释系列12时，该分辨率是在解决有关再现性的协同作用字段关注特别有价值。最后，我们的分析是定量的，也疗法安伏测量抑制等级。其结果，在测定捕获的抑制信息的全部，在等高线等效线图表达的，其中isocontours与生长抑制的类似水平连接的组合的浓度。 6该绘图策略允许组合剂量-反应曲线的可视化。为了说明我们的方法，我们描述我们的协议进行这些检测并显示代表性的结果。

<table border="0" cellpadding="0" cellspacing="0" width="1458">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">J774A.1 cells</td>
			<td style="width:248px;">American Type Culture Collection</td>
			<td style="width:161px;">TIB-67</td>
			<td style="width:627px;">Host cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">ACES</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">A9758&#160;</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Yeast extract, ultrafiltered</td>
			<td style="width:248px;">Becton-Dickinson/Difco</td>
			<td style="width:161px;">210929</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium; lower grades may cause impaired growth and/or alter sensitivity of Legionella to growth inhibitors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Alpha-ketoglutaric acid, monopotassium salt</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">K2000</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium pyruvate</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">P5280</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Potassium phosphate, dibasic</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">P288-500</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">L-cysteine</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">C-7755</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Ammonium iron(III) citrate</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">F5879</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium; ferric pyrophosphate may be used instead but is more difficult to weigh accurately</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Potassium hydroxide solution, concentrated</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">SP236-500</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Deonized water</td>
			<td style="width:248px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Thymidine (tissue culture grade)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">T1895</td>
			<td style="width:627px;">For supplementing both RPMI 1640 and buffered yeast extract agar/medium &#8212; lower grade thymidine may be used for the latter, but may cause impaired cell growth and/or cell death in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">RPMI 1640, standard formulation</td>
			<td style="width:248px;">Corning via Thermo Fisher Scientific</td>
			<td style="width:161px;">10-040-CV</td>
			<td style="width:627px;">For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">RPMI 1640 lacking phenol red</td>
			<td style="width:248px;">Corning via Thermo Fisher Scientific</td>
			<td style="width:161px;">17-105-CV</td>
			<td style="width:627px;">For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine &#8212; supplement to 2 mM before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade)</td>
			<td style="width:248px;">HyClone via Thermo Fisher Scientific</td>
			<td style="width:161px;">SH30034.02</td>
			<td style="width:627px;">For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Iron-supplemented calf serum</td>
			<td style="width:248px;">Gemini Bioproducts</td>
			<td style="width:161px;">100-510</td>
			<td style="width:627px;">For supplementing RPMI 1640, to 9.1% final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Trypan Blue solution</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">T8154</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination while counting cell density</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">SYTOX Green, 5 mM solution in DMSO</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">S7020</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination by fluorescence reading or epifluorescence microscopy (in conjunction with orange-red or far red fluorescent bacteria). Use at 125 nM final concentration.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">GelRed, 10000X solution in water</td>
			<td style="width:248px;">Biotium</td>
			<td style="width:161px;">41003</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination by epifluorescence microscopy (in conjunction with green fluorescent bacteria). Use Gel Red&#160; at 1X final concentration.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Cell culture incubator</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">13-255-26</td>
			<td style="width:627px;">For incubation of J774A.1 cells (both before and after infection); can also be used for incubation of bacteria, or a standard atmosphere incubator can be used instead)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Orbital shaker</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-01010</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection; fits inside cell culture incubator; includes shaker base 7744-01000 and tray 7740-01010 (these are also available separately)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker flasks (250 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-2038-04</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker clamps for flasks (250 ml)</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-16250</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker flasks (1000 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-2038-07</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker clamps for flasks (1000 ml)</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-16100</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Sponge foam caps for flasks (250 ml - 1000 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-1490-038</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">MultiDrop Combi programmable multichannel peristaltic pump</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">5840300</td>
			<td style="width:627px;">For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Combi standard bore manifold</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">24072670</td>
			<td style="width:627px;">Default predispense volume of 20 ml is insufficient to compensate for settling &#8212; increase to 80 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">White 384 well dishes treated for tissue culture</td>
			<td style="width:248px;">Corning</td>
			<td style="width:161px;">3570</td>
			<td style="width:627px;">For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">DMSO (tissue culture grade, in sealed ampoules)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">D2650</td>
			<td style="width:627px;">For dissolving positive control and test compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azithromycin</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">PHR1088</td>
			<td style="width:627px;">Antibiotic positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Saponin (from Quillaja bark)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">S-4521</td>
			<td style="width:627px;">Cytoxicity positive control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Multichannel pipettor</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">Finnpipette</td>
			<td style="width:627px;">For transfer of fixed amounts of positive control compounds; pipettor must have digital dispensing with detents to enable repetitive fixed volume dispensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Epson pin transfer robot</td>
			<td style="width:248px;">Epson/ICCB-L</td>
			<td style="width:161px;">(Custom equipment)</td>
			<td style="width:627px;">For transfer of fixed amounts of test compounds from library arrays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">&#160;D300 digital dispensing system</td>
			<td style="width:248px;">Hewlett-Packard via Tecan</td>
			<td style="width:161px;">D300</td>
			<td style="width:627px;">For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 microliters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">T8+ cartridges for D300 digital dispensing system</td>
			<td style="width:248px;">Hewlett-Packard via Tecan</td>
			<td style="width:161px;">T8+</td>
			<td style="width:627px;">For dispensing test compounds</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:423px;">EnVision multi-mode plate reader</td>
			<td style="width:248px;">Perkin-Elmer</td>
			<td style="width:161px;">(Contact manufacturer)</td>
			<td style="width:627px;">For optimal detection of SYTOX Green fluorescence, use excitation filter 485/14, emission filter 535/25, and dichroic mirror 505 nm, with selection of minimum gain and transmittance, and &#8220;high concentration mode. For luminescence detection, use the &#34;USLUM&#34; protocol for high-sensitivity detection. For mNeptune2 detection, use excitation filter 600/8, emission filter 665/7.5, and dichroic mirror 658 nm, with selection of gain and transmittance to achieve the highest maximum signal possible without saturating the photomultiplier.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Epifluorescence microscope with computer-connected digital camera</td>
			<td style="width:248px;">Nikon</td>
			<td style="width:161px;">Ti</td>
			<td style="width:627px;">For live cell imaging; any standard fluorescent microscope can substitute, with phase contrast or DIC optics, capable of imaging green (fluorescein), orange-red to red (Texas Red), and far-red (Cy5) fluorescence, with 100X oil objective for highest resolution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:423px;">Glass-bottom tissue culture dishes</td>
			<td style="width:248px;">MatTek Corporation</td>
			<td style="width:161px;">P35G-1.5-20-C</td>
			<td style="width:627px;">For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1000X magnification through use of an inverted epifluorescent or confocal microscope.&#160; These specific dishes are 3.5 cm nominal diameter, 3.3 cm inside diameter, with 20 mm diameter #1.5 thickness cover slips inserted into the bottoms.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Photoshop CS6</td>
			<td style="width:248px;">Adobe</td>
			<td style="width:161px;"></td>
			<td style="width:627px;">Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Mathematica 10</td>
			<td style="width:248px;">Wolfam</td>
			<td style="width:161px;"></td>
			<td style="width:627px;">For generation of two-dimensioonal isocontour isobolograms and three-dimensional surface isobolograms.</td>
		</tr>
	</tbody>
</table>

high throughput, real-time, dual-readout testing of intracellular antimicrobial activity and eukaryotic cell cytotoxicity

细胞内的抗菌活性和真核细胞的细胞毒作用的传统措施，依靠端点检测。这样的端点检测要求，如细胞裂解，菌落形成单位的确定，或试剂除了读出之前一些额外的实验步骤。时，例如，在高通量筛选执行数以千计测定的，对于这些类型的测定所需要的下游努力是相当大的。因此，为了便于高通量抗菌发现，我们开发了实时测定以同时识别的细胞内细菌生长抑制剂和评估的真核细胞的细胞毒性。具体地讲，实时的细胞内细菌生长的检测用标记与任一细菌勒克斯操纵子（ 第一代测定法）或荧光蛋白记者（ 第二代，正交测定）细菌筛选菌株已启用。无毒的，细胞膜非透性，核酸结合染料巨噬细胞的最初感染过程中也被加入。这些染料被排除在活细胞。然而，非存活的宿主细胞失去膜完整性允许进入和核DNA的荧光标记（脱氧核糖核酸）。值得注意的是，DNA结合与荧光量子产率，提供了宿主细胞死亡的基于溶液的读出一个大的增加有关。我们已经使用该组合测定法来执行在微孔板格式的高通量筛选，并通过显微镜来评估细胞内的生长和细胞毒性。值得注意的是，抗微生物剂可显示协同作用，其中当一起施用两种或更多种抗微生物剂的组合效果是，当分别施加大于。测试体外抗细胞内病原体的协同作用，通常是一个很大的任务，因为在不同浓度的抗生素的组合排列必须评估。然而，我们发现，我们的实时检测与自动化，数字化配送技术相结合permitted轻便协同测试。使用这些方法，我们能够系统地调查了大量抗菌药物单独的行动，并结合对细胞内病原体， 嗜肺军团菌。

微孔板细胞生长试验 图1图中的检测步骤。示出的自动化的步骤可以手动完成。然而，可以通过使用液体处理系统大大地促进。 图2示出了从384孔微孔板，双读出，实时的细胞内生长，并用军团菌菌株（LP02）标有任一个勒克斯操纵子（ 图2A，2B）或mNeptune2荧光蛋白（真核细胞的细胞毒性测定法的代表性结果图2C，2D）记者。阳性和阴性对照化合物（DMSO，抗生素，皂素）通过使用销搬运机器人的加入到板中的高通量筛选的设置，以模拟性能。显著军团 </青霉&gt;生长（ 图2A，2C）可以在比较皂素裂解和抗生素处理的对照在24至72小时为发光细菌和48到72小时为mNeptune2标记细菌容易区分，分别。 军团通常裂解复制为48到72小时后的宿主细胞。这可以在图1B和1D，其示出随着时间的推移增加SYTOX绿（有代表性的，不通透性，核酸结合染料和宿主细胞死亡的标志物）的荧光可以理解。细胞毒性最终到达在洗涤剂（皂苷）中观察到的最大量，宿主细胞裂解，阳性对照。在比勒克斯操纵子标记的细菌四倍的高接种添加mNeptune2标记的生物，可能占更快速的宿主细胞的杀伤和更早版本相关的上升荧光信号mNeptune2实验。正如预期的那样，有效的抗菌药物治疗（左氧氟沙星或阿奇霉素）上一页ented细菌生长和宿主细胞裂解。相反地，通过在宿主细胞的破坏局限于胞内生长的细胞毒性化合物（ 例如 ，皂苷）可以很容易地通过低发光或mNeptune2荧光，和高的细胞毒性相关的信号的组合来标识。此外，在这两个发光和mNeptune2荧光（细菌生长）合并的减少，和细胞毒性的观察提供合理的信心的化合物是一个真正的细胞内生长抑制剂（ 例如 ，阿奇霉素，左氧氟沙星阳性对照），而不是假阳性从寄生干扰产生细菌记者信号。 表1 - 3显示了三种不同的细菌记者代表Z&#39;（勒克斯操纵子，mNeptune2，tdTomato）和相应的荧光读数相对于对照。一个Z&#39;&gt; 0.5表示一个国家统计LLY鲁棒测定适合于高通量的设置;然而，低级Z&#39;可以根据实验的目标， 即 ，为了可靠地辨别试验化合物或扰动的或多或少微妙的影响的能力，是合适的。基于从图2的结果，mNeptune2是不敏感了记者的细菌荧光素酶操纵子。因此，正如所料，mNeptune2信号之间在负（DMSO）中健壮差和正（左氧氟沙星，阿奇霉素，皂苷）生长抑制剂的对照中直到孵化的第2天或第3天观察到，与此相反的早期，勒克司的合理可靠的信号感染后第1天操纵报告细菌。相比之下，tdTomato，另一种细菌荧光报告，显示整个过程的传染性次优Z&#39;。次优的特点荧光灯的部分原因来自用于tdTomato的S读出最佳的激发和发射到尾的范围侵占ignal。这种侵蚀可以从tdTomato皂苷与左氧氟沙星对比发现正Z&#39;可以理解（在两种情况下这意味着在细菌数的显著差异，细菌不应该复制）。这个结果可以通过皂苷裂解引起强烈的荧光信号，其尾部被检测为tdTomato发射，并因此导致皂素和抗生素对照之间的乱真差值进行说明。因此，当细菌勒克斯和mNeptune2记者在荧光组合出现可用于双读出，基于溶液的，实时测定，所述tdTomato和荧光结合，至少在没有进一步的数学信号去卷积，则不会。 图3示出以前发表的使用勒克斯操纵子，记者标记生物体已知抗微生物剂的剂量-反应曲线的例子。 6值得注意的是， 军团菌不在组织培养基中不能生长。因此，在巨噬细胞感染检测细菌的复制仅仅代表了细胞内生长。然而， 军团将在专门，低钠axenically增长，液体ACES（N - （2-乙酰氨基）-2-氨基乙磺酸）的酵母提取物培养基。对细胞内和无菌生长20以下的效果进行比较。使用这两种生长系统中，我们观察到，极性抗菌素，如β内酰胺（美罗培南，头孢三嗪）和氨基糖苷类（数据未显示）是胞内生长的差抑制剂，而相比之下，在无菌生长的强效作用。想必在巨噬细胞感染试验疗效不佳是基于无法进入细胞内的复制军团利基。相反，真核细胞的渗透抗微生物剂如喹诺酮类（左氧氟沙星）和阿奇霉素，表现出对细胞内和细胞无菌细菌有效的作用。 50值（浓度为50％的细菌生长抑制），并可以被确定CC 50（浓度诱导50％的真核细胞死亡），和选择性，CC 50 / IC 50，进行计算。所有这三个措施是在药物发现工作进展化合物的重要标准。 图4示出了响应于抗生素强力霉素这种双重，剂量-反应曲线的一个例子，随着时间的推移从同一筛选井获得的数据。在图4A中 ，上感染了1天，约一百倍细菌复制与抗生素观察抑制的最高水平相比处于零抗生素测试井明显。什么都有，除了在非常高的doxycy最小的相关真核细胞的毒性克莱因浓度（≥10微克/毫升）。第2天（ 图4B），细菌勒克斯信号是在低的抗微生物浓度观察显著细菌复制相关的细胞毒性大约10倍超过1天以上。正如所料，细菌复制诱导宿主细胞死亡在整个抗微生物剂量 - 响应曲线的细菌数（勒克斯信号）密切跟踪。在较高的抗微生物浓度的细胞毒性降低至基线，直到非常高的浓度，在第1天观察到，其中强力霉素再次出现毒性朝着与基于荧光细胞毒性测定稍高的抗微生物浓度的发光剂量 - 反应的表观移位是有些夸大对数和线性刻度，密谋发光和细胞毒性分别时，如图所示。集成电路50细菌生长是约100纳克/毫升，而CC 50约为10微克/毫升， </suB&gt;预先与CC 50一致描述为强力霉素治疗白细胞谱系，HL-60细胞系。 21因此，在该双读出实验确定总选择性约为100。 图5显示了先前公布的与该抗生素对组合进行了测试反对细胞内嗜肺军团菌增强效果二维协同测试相关的等高线等效线图。 6虽然标准等效线连接的抗生素完全抑制增长，我们选择的基础上，可量化的数据抑制，来分用isocontours抑制相似的水平连最低的组合的浓度。在这些图中最右边的等高线对应于标准等效线图线。凹等效线图通常表明协同作用，而凸等效线图generallŸ表示冷漠或拮抗作用。协同作用（板AC）和漠不关心的例子示（DF），在对应于分别&lt;0.5和≥1，FIC指数值这种情况。 6 值得注意的是，阿奇霉素，米诺环素和利福平对组合表现出协同作用。因此，这些试剂都在三元组合一起测试， 如图6 6。在这里，表面被吸引到三个抗菌药物，导致&gt; 99％的细菌细胞生长抑制的最低浓度组合连接。所观察到的凹形表面，由本身提示三维协同作用，在相当低的FIC指数（0.325）表示的实质的协同效应。 <img src="/files/ftp_upload/54841/54841tbl1.jpg" table="" /> 表1:Z&#39;为勒克斯操纵子报告呃的阳性和阴性对照 军团 infection-比较。 Z&#39;对应于图2A，2B中所示的数据点。该表已经从Chiaraviglio和柯比2014年4正面发光Z&#39;&gt; 0.25的修改突出了在黑色背景上的黄色字符。 <img src="/files/ftp_upload/54841/54841tbl2.jpg" table="" /> 表2:Z&#39;为mNeptune2-记者， 军团菌 感染-的阳性和阴性对照的比较。 Z&#39;对应于图2C，2D示出的数据点。正mNeptune2荧光Z&#39;&gt; 0.25高亮显示红色的字符。 <img src="/files/ftp_upload/54841/54841tbl3.jpg" table="" /> 表3:Z&#39;为tdTomat邻记者， 军团菌 感染-阳性和阴性对照比较。正tdTomato荧光Z&#39;&gt; 0.25高亮显示红色的字符。假阳性Z&#39;&gt; 0.25重叠SYTOX绿色tdTomato排放相关的突出显示绿色字符。 <img alt="图1" src="/files/ftp_upload/54841/54841fig1.jpg" /> 图1: 双报告实验设置的图示。 （A）生长在悬浮液于384孔的菜肴J774A.1细胞Replating。 （B）除了微孔板的目标化合物。 （C）的同时感染和除核酸结合染料的;孵化;和读出。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig1large.jpg" target="_blank">请点击此处查看该图的放大版本</a><html> URE。 <img alt="图2" src="/files/ftp_upload/54841/54841fig2.jpg" /> 图2:双，在高通量形式的细胞内的细菌生长和真核细胞毒性的实时读出。 J774A.1细胞内感染勒克斯操纵子表达嗜肺军团菌在对应发光（A）和荧光（B）的信号。 J774A.1细胞内感染mNeptune2表达嗜肺军团杆菌中相应的荧光蛋白（C）和荧光（D）的信号。四个处理显示:阴性DMSO对照（ <img alt="公式3" src="/files/ftp_upload/54841/54841eq3.jpg" /> ）;阳性细胞毒性控制，皂甙（ <img alt="公式5" src="/files/ftp_upload/54841/54841eq6.jpg" /> ）;和阳性细菌生长抑制控制，阿奇霉素（化4"SRC ="/文件/ ftp_upload / 54841 / 54841eq4.jpg"/&gt;）和左氧氟沙星（ <img alt="公式5" src="/files/ftp_upload/54841/54841eq5.jpg" /> ）。数据点表示的一式两份，384孔板中进行96次重复的平均值和标准偏差。注意，对于一些数据点的标准偏差误差棒是最小的，因此，隐蔽数据点符号内。面板A和B已经从Chiaraviglio和柯比2014年4修改<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54841/54841fig3.jpg" /> 图 3: 一维，剂量响应测定法的代表性例子。 J774A.1细胞感染勒克斯操纵子表达， 嗜肺军团菌和治疗用两倍稀释系列表示抗微生物剂的（拉贝莱德为"细胞内的"）。并联，勒克斯操纵子表达， 军团稀释到在ace球酵母提取物培养基（赞成）相同的终浓度，并用相同的，两倍稀释系列（标为"无菌"）处理。发光读了后两天的细菌接种和作图的抗菌浓度。 A组包括抗菌药物常用于治疗军团菌感染。 B组比较了不同的大环内酯类抗生素活性。 C组包括与不同的，有时对比的细胞内和细胞无菌效力几个不同的类抗菌剂。数据点表示平均值和每个条件三个独立试验的井的标准偏差。这个数字已经从Chiaraviglio和柯比2015年6修改<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig3large.jpg" target="_blank">请点击这里查看大图</a>版本这个数字。 <img alt="图4" src="/files/ftp_upload/54841/54841fig4.jpg" /> 图4: 用于确定IC 50，CC 50 和选择性 的双读出，剂量反应实验的实施例 。分别在1和2天感染后测定对细胞内生长和J774A.1细胞毒性多西环素的影响。数据点表示平均值和每个条件三个独立试验的井的标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54841/54841fig5.jpg" /> 图5: 二维协同测定的实施例 。组合系列稀对抗菌药物的以及英反对在巨噬细胞J774A.1 军团内生长的协同作用进行了测试。 Isocontours线连接类似的抑制百分比的点。最右边的等效线图解连接具有完整的细胞内生长抑制（&gt;发光减少99％相比，未处理的对照组）的组合和对应于标准等效线图。阴影表示抑制百分比通过在每个图中的颜色编码的密钥作为划定。 Isocontours从左向右对应于100，90，80，70，60，50，40，30，20，生长相对于未处理的对照10，图5，和1％。这个数字已经从Chiaraviglio和柯比2015年6修改<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54841/54841fig6.jpg" /> <strong&gt;图6: 一个三维协同测定的实施例。阿奇霉素，米诺环素和利福平，导致&gt; 99％生长抑制的最低组合浓度相连，形成一个等效线图表面图。这个数字已经从Chiaraviglio和柯比2015年6修改<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54841/54841fig6large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity at JoVE.com

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

high-throughput-real-time-dual-readout-testing-intracellular

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  Beth Israel Deaconess Medical Center. A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

视频: High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

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 ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

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

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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, ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

使用实时细胞分析监测宿主外的流感病毒存活率

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...