Name: high throughput, real-time, dual-readout testing of intracellular antimicrobial activity and eukaryotic cell cytotoxicity
Uploaded: 2016-11-16T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity at JoVE.com

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?...

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><tbody><tr><td>J774A.1 cells</td><td>American Type Culture Collection</td><td>TIB-67</td><td>Host cell</td></tr><tr><td>ACES</td><td>Sigma-Aldrich</td><td>A9758&amp;nbsp;</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Yeast extract, ultrafiltered</td><td>Becton-Dickinson/Difco</td><td>210929</td><td>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><td>Alpha-ketoglutaric acid, monopotassium salt</td><td>Sigma-Aldrich</td><td>K2000</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Potassium phosphate, dibasic</td><td>Thermo Fisher Scientific</td><td>P288-500</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>L-cysteine</td><td>Sigma-Aldrich</td><td>C-7755</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Ammonium iron(III) citrate</td><td>Sigma-Aldrich</td><td>F5879</td><td>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><td>Potassium hydroxide solution, concentrated</td><td>Thermo Fisher Scientific</td><td>SP236-500</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Deonized water</td><td>N/A</td><td>N/A</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Thymidine (tissue culture grade)</td><td>Sigma-Aldrich</td><td>T1895</td><td>For supplementing both RPMI 1640 and buffered yeast extract agar/medium &amp;mdash; 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><td>RPMI 1640, standard formulation</td><td>Corning via Thermo Fisher Scientific</td><td>10-040-CV</td><td>For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine</td></tr><tr><td>RPMI 1640 lacking phenol red</td><td>Corning via Thermo Fisher Scientific</td><td>17-105-CV</td><td>For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine &amp;mdash; supplement to 2 mM before use</td></tr><tr><td>L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade)</td><td>HyClone via Thermo Fisher Scientific</td><td>SH30034.02</td><td>For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration</td></tr><tr><td>Iron-supplemented calf serum</td><td>Gemini Bioproducts</td><td>100-510</td><td>For supplementing RPMI 1640, to 9.1% final concentration</td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td>For staining for J774A.1 cell death determination while counting cell density</td></tr><tr><td>SYTOX Green, 5 mM solution in DMSO</td><td>Thermo Fisher Scientific</td><td>S7020</td><td>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><td>Cell culture incubator</td><td>Thermo Fisher Scientific</td><td>13-255-26</td><td>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><td>Orbital shaker</td><td>BellCo Glass</td><td>7744-01010</td><td>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><td>Shaker flasks (250 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-2038-04</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker clamps for flasks (250 ml)</td><td>BellCo Glass</td><td>7744-16250</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker flasks (1,000 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-2038-07</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker clamps for flasks (1,000 ml)</td><td>BellCo Glass</td><td>7744-16100</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Sponge foam caps for flasks (250 ml-1,000 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-1490-038</td><td>For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps</td></tr><tr><td>MultiDrop Combi programmable multichannel peristaltic pump</td><td>Thermo Fisher Scientific</td><td>5840300</td><td>For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes</td></tr><tr><td>Combi standard bore manifold</td><td>Thermo Fisher Scientific</td><td>24072670</td><td>Default predispense volume of 20 &amp;#956;l is insufficient to compensate for settling &amp;mdash; increase to 80 &amp;#956;l</td></tr><tr><td>White 384 well dishes treated for tissue culture</td><td>Corning</td><td>3570</td><td>For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully</td></tr><tr><td>DMSO (tissue culture grade, in sealed ampoules)</td><td>Sigma-Aldrich</td><td>D2650</td><td>For dissolving positive control and test compounds</td></tr><tr><td>Azithromycin</td><td>Sigma-Aldrich</td><td>PHR1088</td><td>Antibiotic positive control</td></tr><tr><td>Saponin (from Quillaja bark)</td><td>Sigma-Aldrich</td><td>S-4521</td><td>Cytoxicity positive control</td></tr><tr><td>Multichannel pipettor</td><td>Thermo Fisher Scientific</td><td>Finnpipette</td><td>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><td>Epson pin transfer robot</td><td>Epson/ICCB-L</td><td>(Custom equipment)</td><td>For transfer of fixed amounts of test compounds from library arrays</td></tr><tr><td>D300 digital dispensing system</td><td>Hewlett-Packard via Tecan</td><td>D300</td><td>For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 &amp;micro;l</td></tr><tr><td>T8+ cartridges for D300 digital dispensing system</td><td>Hewlett-Packard via Tecan</td><td>T8+</td><td>For dispensing test compounds</td></tr><tr><td>Epifluorescence microscope with computer-connected digital camera</td><td>Nikon</td><td>Ti</td><td>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><td>Glass-bottom tissue culture dishes</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td>For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1,000X magnification through use of an inverted epifluorescent or confocal microscope.&amp;nbsp; 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><td>Photoshop CS6</td><td>Adobe</td><td></td><td>Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images.</td></tr><tr><td>Mathematica 10</td><td>Wolfam</td><td></td><td>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<strong...

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

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

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

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

识别影响培养哺乳动物细胞在低到中度通量下细胞生长和生存的化合物的策略

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

生物化学

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

自动差分核染色测定，准确测定三元细胞毒性

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

癌症研究

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

医学

评估表达 CAR 的 Jurkat 细胞对肿瘤细胞的定量杀伤

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

作者聚焦：CAR T 细胞构建体的高通量筛选以增强细胞毒性和免疫记忆 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

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 ...

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

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此视频中的章节

定量高通量单细胞的细胞毒性T细胞的测定

单细胞测量液泡破裂引起细胞内病原体

识别影响培养哺乳动物细胞在低到中度通量下细胞生长和生存的化合物的策略

自动差分核染色测定，准确测定三元细胞毒性

HIV逆转录酶和整合酶抑制剂的快速筛选

实时的细胞毒性分析在人全血

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat

系统抑制剂靶向细胞内的疗效和毒性筛选