这项研究得到了德州癌症预防研究所 (CPRIT) 奖 RR150044、韦尔奇基金会研究补助金 C-1930 以及由国立卫生研究院 K22 AI110552 授予 NVK 的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。

注意:铜绿假单胞菌和粪肠是生物安全2级病原体, 必须采取适当的安全措施预防意外感染, 并尽量减少表面污染。与病原体接触的所有介质和材料都必须消毒和/或丢弃。在《微生物和生物医学实验室》 (BMBL) 第五版中, 可提供进一步的指导方针。
1. 铜绿假单胞菌的制备与维护
<ol><li>从冷冻的股票上的绿脓杆菌到一磅 (溶源性肉汤) 琼脂盘子。孵育16到24小时在37°c 注: 在 BSL-2 生物安全柜内进行铜绿假单胞菌实验, 以确保不孕。使用适当的安全预防措施, 尽量减少致病感染或污染的几率。</li>
	<li>把这个盘子转移到4摄氏度。该板块可保留在摄氏4摄氏度, 并用于接种文化长达一周。一个星期后, 净化和丢弃的盘子, 并作出一个新的 LB 条纹板。 注:铜绿假单胞杆菌毒力在长时间贮存后减少。</li>
	<li>两天前建立化验板, 接种 3-5 毫升不育 LB 汤与单一殖民地的铜绿假单胞菌从板材制成的1.1。孵育37°c 12 至16小时。 注: 不要孵化长于16小时。在富含液体的培养基中, 铜绿假单胞菌PA14 开始溶解。</li>
	<li>用慢速杀灭培养基制备无菌10厘米板 (3 克氯化钠, 3.5 克蛋白胨, 1 毫米 CaCl2, 1 毫米 MgSO4, 25 毫升磷酸盐缓冲器 (每公升: 132 毫升的 K2HPO4 (1M) 和 868 ml 2 毫升4 (1M)) 和18克琼脂/升)。 注: 提前准备板材。它们可以在4摄氏度的密闭容器中储存长达3周。</li>
	<li>种子每10厘米慢杀板 (SK 板) 与 350 ul 的铜绿假单胞菌从新鲜的隔夜磅文化。使用无菌的细菌摊铺机, 在介质表面均匀传播细菌, 并允许在 BSL-2 生物安全柜中干燥。</li>
	<li>孵育板在37°c 为24小时。</li>
</ol>2. rna 干扰细菌的制备
<ol><li>在含有适当抗生素的 LB 琼脂上 (羧苄青霉素100µg/毫升和四环素, 在 Ahringer 和维达图书馆的15µg/毫升) 中, 将所需的含有干扰的细菌的菌株从冷冻的库存中取出或引脚转移。 注意: 在与致病细菌合作时, 使用 BSL-2 a/B 生物安全柜或 BSL-1 层流罩, 以确保文化的不育, 并尽量减少图书馆交叉污染的几率。</li>
	<li>孵化板为24小时在37°c。</li>
	<li>将盘子存放在4摄氏度, 最多两周。<ol>
<li>两周后, 扔掉盘子, 用刚制成的盘子代替它们。</li>
	</ol>
</li>
	<li>通过在一个24井深井板的单个井中单独接种单个菌落, 并将其分为4毫升的羧苄青霉素补充磅, 并在一个无菌试管中, 对多个干扰菌株进行平行测试。 注意: 据报道, 四环素可降低 rna 干扰的效率。不要在这个媒体中使用它。</li>
	<li>将24井深井板块放入一个为多井板优化的震动孵化器中, 并在37摄氏度处孵育16小时, 而在950转时晃动。 注意: 使用常规的振动孵化器很难在多井板上获得均匀的细菌生长。震动孵化器需要能够至少 750 rpm 的震动, 以使文化能够正常生长。在没有专门的振动筛的情况下, 可以在单个管中培养每种文化。文化可以被转移入24井板材为离心以后, 如果需要。</li>
	<li>用离心收集细菌5分钟, 在 2000 x g。</li>
	<li>醒酒上清, 用力摇动板。并用重悬在100µL 的基部 (5.85 克氯化钠, 1 克 K2HPO4, 6 g, 2 PO4溶解成1升的超纯水和灭菌的高压釜) 的 rna 干扰细菌。</li>
	<li>吸管悬浮细菌进入适当数量的多井 NGM 板 (线虫生长培养基, 3 克氯化钠, 2.5 克蛋白胨, 1 毫米 CaCl2, 1 毫米 MgSO4, 25 毫升磷酸盐缓冲 (每公升的数量: 132 毫升 K2HPO4 (1M)并且868毫升2PO4 (1M)) 和 18 g 琼脂每公升水) 补充与 IPTG (1 毫米最后的集中) 和羧苄青霉素 (100 微克或 mL 最后集中)。<ol>
<li>使用3.5 毫升的 NGM 介质, 每井6井板, 1 毫升每井24井板, 或150µL 每井96井板。</li>
		<li>使用 100 ul/良好的细菌为6井板;50 ul/井为 24-好;或 20 ul/井为96井板。允许干燥。 注: 干燥通常在无菌流动罩中进行, 以促进快速, 均匀干燥。均匀干燥是非常重要的。避免过度干燥, 因为琼脂中的裂缝促进了蠕虫的挖洞。这导致了蠕虫的损失和液体处理系统的潜在堵塞。</li>
	</ol>
</li>
	<li>立即使用准备好的盘子, 或者将它们储存在4摄氏度, 供以后使用。 注: 为防止板材干燥, 可以用塑料石蜡膜密封, 或置于密封的容器中, 用湿纸巾。</li>
</ol>3.线虫的维护和制备
注: 在开始实验前, 生成妊娠、成年雌雄同体蠕虫的同步种群, 如下所示。
<ol><li>从 4-6 10 厘米 NGM 板到15毫升圆锥管中, 将妊娠成人 (即,有多个胚胎可见), 通过加入4毫升的 S 基板, 轻轻旋转, 然后倒入15毫升圆锥管。</li>
	<li>颗粒蠕虫通过离心在 1000 x g 30 秒。</li>
	<li>吸入上清液, 注意不要扰乱蠕虫颗粒。</li>
	<li>添加3.5 毫升的蠕虫漂白溶液 (最终浓度0.5M 氢氧化钠, 1% 次氯酸钠, 在无菌水中) 到蠕虫颗粒, 并混合通过反转1分钟。</li>
	<li>简要地漩涡和离心机在 1000 x g 30 秒。</li>
	<li>吸入或醒酒上清, 注意不要扰乱蠕虫颗粒。</li>
	<li>加入3.5 毫升新鲜的蠕虫漂白溶液的蠕虫颗粒。</li>
	<li>在漂白剂溶液中大力混合蠕虫。定期 (大约每十秒) 目视检查蠕虫在解剖显微镜下。观察蠕虫角质层打破, 释放鸡蛋。一旦大多数成年蠕虫被溶解 (~ 95%), 稀释漂白剂到15毫升以 S 基部停止消化。 注: 蛋壳比成人组织更耐漂白, 但在长时间暴露时可以溶解。为了避免卵子溶解, 不要将鸡蛋暴露在蠕虫漂白溶液中超过7分钟。如果蛋壳被过度破坏, 胚胎就会死亡。</li>
	<li>颗粒胚在 1000 x g 30 秒, 吸或醒酒上清, 而不去除胚胎颗粒。</li>
	<li>并用重悬蠕虫在 S 基部到最后容量15毫升稀释剩余的漂白剂解答。</li>
	<li>重复洗涤步骤 (3.9-3.10) 三多次, 总共4连续洗涤。</li>
	<li>第四洗涤后, 吸入上清, 并增加5或6毫升不育的基部到15毫升圆锥管。目标是并用重悬胚胎的浓度为每µL 50 蠕虫。 注: S 基的数量取决于蛋粒的大小;球团越大, 基部就越有必要。</li>
	<li>在室温下或48小时在15摄氏度上, 在工作台顶部的转子上过夜。幼虫将孵化, 但幼虫发育将在 L1 阶段 (L1 滞育) 中停止, 由于养分匮乏。</li>
	<li>检查解剖显微镜下的胚胎, 以验证它们中的大多数在发育的 L1 阶段已经孵化和被逮捕。</li>
	<li>用20µL 的蠕虫作准备, 用80µL 的 S 基来稀释它, 以确定蠕虫的计数。吸管10µL 稀释的蠕虫溶液直接到一个空白的 NGM 板。重复2次。<ol>
<li>计算每个点的幼虫数并确定平均值。将这个平均值除以 2, 以获得蠕虫准备中每µL 的蠕虫的近似计数。蠕虫眼影可以用于传播板或实验。 注: 4-5 天后, 饥饿幼虫将开始死亡;在这一点上放弃准备。</li>
	</ol>
</li>
	<li>为传播菌株, 吸管适当数量的 L1 蠕虫 (5000-6000) 到 3-4 10 厘米 NGM 板上发现与集中 OP50大肠杆菌- "超级食品" (大肠杆菌OP50 发现25X 浓度 (即, 1 升的隔夜 OP50在 LB 中生长的培养40毫升的 25X OP50 超级食品);2毫升的这种浓缩细菌悬浮被发现到每10厘米 NGM 板)。</li>
	<li>要准备用于实验的板材, 请执行下列操作之一 (对于 "基本协议" 使用3.17.1 设置, 对于 "rna 筛选设置" 使用步骤 3.17.2 3.17.4):<ol>
<li>吸管 ~ 5000 worms/10 厘米板种子有 rna 干扰或 OP50 超级食品。</li>
		<li>吸管 ~ 500 蠕虫/井在6井板种子与 rna 干扰。</li>
		<li>吸管 ~ 300 蠕虫/井在24井板种子与 rna 干扰。</li>
		<li>吸管 ~ 100 蠕虫/井在96井板种子与 rna 干扰。 注: 有关如何进行 rna 干扰的说明, 请参阅步骤 2.7-2.9 在制备 rna 干扰细菌。</li>
	</ol>
</li>
	<li>如果使用了一种肥沃的蠕虫菌株, 在25摄氏度处孵化出 44-48 小时的蠕虫, 在人口达到适当的年龄后尽快使用这些蠕虫。这就最大限度地减少了卵孵化在蠕虫的影响, 在试验期间。</li>
	<li>如果使用的应变是无温度的 (例如, 15 (b26);有限元-1 (hc17)或glp-4 (bn2)), 孵化蠕虫在15°c 为 ~ 16 小时, 然后转移到25摄氏度为 44 h 完成开发和防止胚胎发生。</li>
</ol>4. 液体杀灭试验装置 (基本协议)
注意: 这是一个细菌菌株和一个蠕虫来源的协议。
<ol><li>使用细胞刮板, 从 SK 板中取出并用重悬, 并在 S 基部的5毫升内去除. 如果需要, 请放大。用分光光度计测量细菌悬浮液的光学密度 (OD600)</li>
	<li>在600 ≈ 0.09 (3X 最后浓度) 下, 在 S 基部制备24毫升的铜绿假单胞菌稀释库存。查看4.6.2 的最终内容, 并相应地缩放细菌稀释量。</li>
	<li>添加21毫升的液体慢杀培养基 (3 克氯化钠, 3.5 克的蛋白胨/L 补充与 CaCl2和 MgSO4到最后浓度1毫米)。使用多通道吸管, 将45µL 的细菌和介质转移到384井板的每一个井中。</li>
	<li>将蠕虫从源头 (即步 3.17.1) 中洗涤成50毫升锥形管, 并允许蠕虫在引力作用下安定下来。将上清液吸入5毫升。并用重悬共50毫升的基部。</li>
	<li>重复步骤4.4 两次。</li>
	<li>使用蠕虫分类器, 将大约22条蠕虫放入384井板的每个井中。 注: 安装程序会将每条蠕虫滴在1.1 µL 的液体中。22蠕虫将共计25µL, 使总化验量达到70µL/井。<ol>
<li>每个井的最后组成包括70µL. 45 µL 的这个体积添加为细菌培养基 (0.03 OD600细菌在24µL 的基础, 和21µL SK 补充 CaCl2和 MgSO4)。另外25µL 添加了22蠕虫。</li>
		<li>如果这里使用的分拣机 (见材料表) 是不可用的, 蠕虫可以稀释到最终浓度的1蠕虫/µL 在 S 基部和 pipetted 25 µL/井使用多通道吸管。然而, 结果可能表现出增加的变异性。另外, 如梁和同事19所述, 可以使用蠕动液体分配器。</li>
	</ol>
</li>
	<li>在蠕虫被分类到384井板之后, 用透气膜封住盘子, 在25摄氏度处孵化出 24-48 小时的板材。</li>
	<li>在所需时间, 使用微板块垫圈清洗384井板与 S 基础共5次。<ol>
<li>第二次冲洗后, 吸入大部分介质, 留下20µL. 用微板块 vortexer 用力摇动板材, 至少30秒钟, 以松开井底的任何碎片)。</li>
	</ol>
</li>
	<li>最后洗完后, 将上清液吸入20µL. 添加50µL 0.98 µM 核酸染色 (见材料表)/井的384井板, 为最终浓度0.7 µM。</li>
	<li>室温孵育 12-16 小时。这种染料只会染死蠕虫。在理想的潜伏期后, 用微板块垫圈清洗盘子, 去除多余的污渍 (至少3洗涤)。</li>
	<li>对于数据采集, 使用分光光度计或自动显微镜来图像透射光和荧光 (531 nm 激发和593发射)。<ol>
<li>使用低放大倍数目标, 以使整个井的图像被捕获。</li>
		<li>或者, 使用流 vermimetry。该技术使用大型对象流式细胞仪作为蠕虫 fluorimeter, 测量整个生物体 (即线虫) 的大小和荧光, 这一方法与流动的超流化术有着强烈的相似性。</li>
	</ol>
</li>
	<li>使用自动图像分析软件 (如 CellProfiler, 一个基于 MatLab <a href="http://cellprofiler.org/">http://cellprofiler.org/</a>的自由图像分析工作室), 以无偏见的方式计算每一个良好的荧光和总蠕虫面积。 注: 这些数字的商数代表每个井的分数死亡。如果一个井有7只被染色的蠕虫20总, 则分数死亡包括0.35 或35%。<ol>
<li>在第一次使用之前, 自动图像处理管线必须通过手动评分来验证。这是通过比较自动管道的结果和通过目视检查图像获得的分数, 并记录死蠕虫的分数。验证过程确保自动成像处理的参数准确, 并正确地表示数据。</li>
	</ol>
</li>
</ol>5. 适应筛查多种C 线虫菌株或 Knockdowns (rna 屏蔽设置)
注意: 这是一个为24井板安装而描述的 rna 干扰屏幕。
<ol><li>添加1毫升的 S 基部的每井24井板 (见步骤 3.17.3)。通过摇动盘子轻轻地搅动蠕虫, 然后将蠕虫转移到一个空的、无菌的24深井板上。允许蠕虫解决引力 (5 分钟)。</li>
	<li>吸入上清, 留下大约1毫升。添加7毫升的 S 基向每个井。</li>
	<li>重复步骤 5.1-5.2 至少2次。最后洗净后, 吸入上清, 留下大约400µL。</li>
	<li>利用蠕虫分拣机的 Resampler 功能, 将24井板蜗杆悬浮液中的22种蠕虫从384井板中的每一个井中分类 (24 井板的每一个井产量 8-12 井板)。<ol>
<li>为了避免饥饿, 将细菌注入 384-井板 (在排序前, 可以只作为食物的一种菌株, 如 OP50 或致病性菌株)。 注: 病原体可通过以下步骤 2.3-2.5 或 6.4-6.5 添加, 而食物源细菌可以稀释, 并按照与铜绿假单胞菌相同的方案添加。</li>
	</ol>
</li>
	<li>在蠕虫被分类入384井板之后, 加入小分子或其他实验特定材料。</li>
</ol>6. 对粪肠的适应
注: 仅描述与铜绿假单胞菌的差异。
<ol><li>在37摄氏度的 BHI (脑心输液) 琼脂板和孵化16至24小时后, 通过将冰冻的储存细菌从冷冻的库存中裸奔, 制备出一种新的粪肠源. 注: 在 BSL-2 生物安全柜内进行粪肠试验, 以确保不孕。使用适当的安全预防措施, 尽量减少致病感染或污染的几率。</li>
	<li>盘子可以储存在4摄氏度, 长达一周。这段时间后, 更换一个新鲜的盘子。</li>
	<li>在分类蠕虫之前的晚上, 接种 3-5 毫升无菌 BHI 与一个单一的大肠杆菌群.孵育 12-16 小时在37°c 与鼓动。</li>
	<li>将细菌添加到水井中作为铜绿假单胞菌 (3x 细菌在 S 基部, OD600≈0.09)。 注: 对于粪肠, 最终的井组成是:大肠杆菌(OD600≈0.03), BHI = 10% 伏/五, 其余的体积由 S 基部组成。请记住, 25 µL 的基础将被交付与蠕虫。 注:粪肠产生厚的生物膜, 吸收荧光染色, 造成模糊图像。广泛的洗涤可能不足以去除这种生物膜。在这种情况下, 蠕虫可以被转移到一个空的盘子使用多通道吸管。为便于转移, 补间20可以添加到 s 基部到最终浓度为 0.1% (v/v) 补间20在 S 基部。这有助于清除生物膜中的蠕虫。</li>
</ol>

<ol>
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提交人宣布, 他们没有任何利益冲突可供披露。

这种化验 (或类似的化验, 其他病原体被替代的铜绿假单胞菌或大肠杆菌) 是有用的各种目的, 包括药物发现。它还有助于解决基本的生物学问题, 如确定毒力因素, 阐明宿主防御通路, 并确定参与宿主-病原体相互作用的调控机制。
虽然铜绿假单胞菌液体杀灭试验具有较强的鲁棒性, 但有几点需要认真注意, 以尽量减少失败的几率。首先, 如前所述, 对铜绿假单胞杆菌的细菌源板必须保持新鲜, 以免细菌丧失毒性, 尽管隔夜生长在磅。第二, 确保钙和镁加入到铜绿假单胞杆菌的实验中是关键。没有它, 毒力将会受到严重损害。最后, 值得注意的是, 在 HT115 上饲养的蠕虫 (用于基于 rna 检测的化验) 会比 OP50 上饲养的蠕虫 (n Kirienko, 个人通讯) 的死亡显著晚。由于这个原因, 如果切换到基于 rna 干扰的研究, 这是很重要的经验, 以确定最佳的时间点的化验。
对于用铜绿假单胞菌或粪肠杀灭化验, 关键是要确保有足够的食物, 以发展蠕虫从 L1 阶段, 直到它们准备用于化验。饥饿, 甚至在短期内, 是已知的激活一些宿主防御途径, 如 DAF-2/DAF-16 胰岛素/胰岛素样生长因子信号网络27。我们的数据表明, DAF-16/FOXO 已经被培养在液体20轻微激活, 进一步活化是强烈不可取的, 因为 DAF-16/FOXO 促进广谱病原体耐药性23。
最后, 在任何依赖死亡的化验中使用完全不育蠕虫是至关重要的。如果蠕虫是肥沃的, 在液体中会导致产卵的困难。因此, 一些胚胎在母体内孵化, 最终导致其死亡。因此, 我们通常使用遗传损伤, 如glp-4 (bn2)28,这表明了完全渗透不育表型的这些化验。应避免使用防止胚胎发育但仍允许产卵的化学物质, 如 5-fluorodeoxyuridine (FUdR), 因为它们也可能干扰细菌的生长和发育。
总的来说, 我们发现计划几个时间点进行分析是很有帮助的。虽然该检测是稳健的, 时间通常是一致的, 随机和不知不觉的变化可以改变死亡的时间在 2-4 小时内检测。当需要进行故障排除时, 首先考虑最简单的答案往往是有帮助的;例如,准备新鲜的培养基, 丢弃最近的细菌培养, 再从冰冻的储存中重新条纹细菌菌株等. 这些措施很少能将化验恢复到功能上。
由于该方法相对简单, 因此可以很容易地进行多种修改。从统一的4 (bn2)背景中更改蠕虫, 用于高通量的 rna 干扰屏幕 (如本文中描述的), 对于识别各种外来化合物和有毒化学物质的宿主响应路径非常有用。例如, 通过将相同的化学物质或毒素添加到每一个干扰板 (例如, Cry5B 毒素、邻菲啉等) 中, 可以很容易地确定改变这些毒素敏感性的途径。这些类型的屏幕也可以用来识别防御网络对任何感染C. 线虫的病原体。

在近一个世纪以前, 确定和发展有效的广谱抗生素导致了公共卫生的一个分水岭时刻, 人们普遍认为, 传染病将是过去的祸害。在短短的几十年里, 这种乐观情绪开始消退, 因为病原体形成的抗病机制限制了这些曾经神奇的治疗。一段时间内, 药物发现努力与病原体之间的军备竞赛似乎是平衡的。然而, 滥用抗菌药物最近终于出现了耐药菌株的肺炎克雷伯杆菌, 不动杆菌鲍曼,沙雷质, 和铜绿假单胞菌1, 2,3,4。
铜绿假单胞菌是一种机会主义, 革兰阴性, 多宿主病原体, 严重威胁严重烧伤患者, 那些谁是免疫力低下, 或有囊性纤维化。它也日益被认定为严重医院感染的致病剂, 特别是由于其持续的耐药性的获得。为了应对这一威胁, 我们使用了记录良好的C.铜绿假单胞菌感染系统5。我们的实验室利用这个系统开发了一种基于液体的高通量高含量的筛选平台, 以确定新的化合物, 限制病原体杀死宿主6的能力。有趣的是, 这些化合物似乎属于至少三个一般类别, 包括抗菌素7和毒力抑制剂8。 tuberculosum 分枝杆菌、沙眼衣原体、鼠疫耶尔森菌、李斯特氏细胞增生、土拉弗 tularensis、金黄色葡萄球菌、白念珠病及粪肠球菌, 除其他9,10,11,12,13,14,15,16。这些类型的化验有几个公认的优势, 如限制假阳性命中可能对宿主和病原体, 增加生物有效性的可能性相比, 化学屏幕, 并有能力识别命中超出简单地限制微生物的生长, 例如抗 virulents, 免疫刺激分子, 或其他使宿主-病原体相互作用的平衡倾斜的化合物, 有利于前者。此外, 在这些屏幕上发现的化合物通常在哺乳动物宿主中有效。
值得注意的是, 至少有另外两种化验17,18可以在液体中的C. 线虫中进行高通量屏幕。然而, 每一个这些化验是一个修改, 允许原型肠道殖民化试验, 称为慢杀, 以液体进行, 增加的吞吐量, 并允许化合物更容易筛选。仔细的描述表明, 细菌毒力的机制是不同的, 这些化验和我们的液基筛7。由于两种类型的毒力都观察到哺乳动物系统, 重要的是要考虑哪些毒力决定因素是最相关的实验者的利益之前的化验选择。
在这里, 我们演示了一个优化版本的液态C 线虫-铜绿假单胞菌。我们还报告了我们的液基检测方法的适应性, 以适应革兰氏阳性菌粪肠球菌.与铜绿假单胞菌一样,粪肠球菌越来越多地被认定为严重的医院威胁, 并不断增加的武器耐药性途径1。尽管以前的高通量筛选方法存在14, 但它需要 preinfection 病原体, 这就使程序复杂化, 增加了诸如 COPAS FlowSort 等污染设备的可能性。我们的协议消除了感染前的需要, 改善了安全配置。最后, 我们报告一种方法, 其中任一检测可以结合喂养 rna 干扰, 允许用户搜索的宿主因素, 发挥作用, 建立或抵抗, 感染。

<table>
	<tbody>
		<tr>
			<td>COPAS FP BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>Large object flow cytometer/worm sorter</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron Pro</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Deep-Well RB Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>CS15124</td>
			<td></td>
		</tr>
		<tr>
			<td>384-Well plate</td>
			<td>Greiner Bio-One</td>
			<td>MPG-781091</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) plates</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) media</td>
			<td></td>
			<td></td>
			<td>Amount per liter:&#160; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>USBiological Life Sciences</td>
			<td>L1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Brian Heart Infusion broth (BHI)</td>
			<td>Research Products International Corp</td>
			<td>50-488-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Bleach Solution</td>
			<td></td>
			<td></td>
			<td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>USBiological Life Sciences</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>USBiological Life Sciences</td>
			<td>S5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>USBiological Life Sciences</td>
			<td>P3300</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>USBiological Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffer</td>
			<td></td>
			<td></td>
			<td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>USBiological Life Sciences</td>
			<td>P5100</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium Hypochlorite Solution</td>
			<td>BICCA</td>
			<td>7495.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Fisher Scientific</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P. aeruginosa (PA14)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. faecalis(OG1RF)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli superfood (OP50)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAi expressing bacteria (HT115)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

传统的、体外筛查到的新药数量已经减退, 这就减少了这种方法在寻找新武器以对抗多种耐药性方面的成功。这就得出结论, 研究人员不仅需要找到新药, 还需要开发新的方法来找到它们。其中最有希望的候选方法是全有机体,在体内的检测, 使用高通量, 表型读数和寄主, 范围从秀丽线虫到斑马斑马。这些主机有几个强大的优势, 包括显着减少假阳性命中, 因为有毒的化合物, 对主机和/或 biounavailable 通常下降在最初的屏幕上, 在昂贵的后续行动之前。
在这里, 我们展示了我们的检测如何被用来审问寄主变异的良好记录的C. 线虫-铜绿假单胞菌液体杀死 pathosystem。我们还演示了这种精心编制的技术的几个扩展。例如, 我们能够使用24或96井板格式的 rna 干扰来进行高通量的基因筛, 以查询宿主-病原体相互作用中的宿主因素。使用这项化验, 整个基因组屏幕只能在几个月内完成, 这可以大大简化确定药物目标的任务, 可能不需要费力的生化纯化方法。
我们还报告了我们的方法的变化, 取代革兰氏阳性菌肠球菌的革兰氏阴性病原菌P.与铜绿假单胞菌的情况一样, 由大肠杆菌杀死是有时间依赖性的。与以前的 C.粪肠球菌化验不同, 我们对粪肠球菌的化验不需要 preinfection, 改善其安全状况, 减少污染液体处理设备的几率。该检测具有高度健壮性, 显示95% 死亡率 96 h 后感染。

测定性能的重要参数
正确理解这项化验的生物学基础是解决问题和优化化验的必要。为此, 我们首先提到几项关键文件, 阐明了在液体7,20中, 铜绿假单胞杆菌介导的杀死的发病机制。前提是遵循上述步骤 (见图1所示的检测协议示意图) 只有在病原体存在的情况下, 才会观察到与时间相关的C. 线虫的杀戮 (图 2A)。相比之下, 在缺乏关键的营养补充剂 (例如,如果蛋白胨是留出的媒体, 只有 S 基础上添加), 很少对没有杀害将被观察 (图 2B)。有趣的是, 两步孵化的铜绿假单胞菌(24 小时在37摄氏度, 然后24小时25°c), 这是关键的常规慢杀化验, 并最初实施的液体杀死21, 是可有可无的这项试验 (图 2C)。虽然有可能从隔夜磅培养直接添加铜绿假单胞菌, 但这样做会显著改变致死动力学, 不推荐使用。
了解化验生物学也允许简化化验, 如果适当和可取的。例如, 在这项试验中, 宿主杀死的最重要的驱动因素是铁载体 pyoverdine, pyoverdine 所造成的宿主毒性取决于其对铁7的束缚能力。因此, 可以通过替代富含 pyoverdine 的滤液、纯化的 pyoverdine, 甚至一些合成的铁螯合化学物质 (例如110-邻菲啉) 对活菌进行简化, 作为线虫的转录反应。对这些治疗是非常相似的 (图 3)22。
几种不同的证据表明了实验装置的健壮性。首先, 该设置容忍了广泛的初始细菌浓度。浓度低如 OD600 = 0.0025 (大约10倍低比推荐) 仍然陈列时间和集中依赖的杀害, 虽然计时做变动 (图 4A)。第二, 该试验的重现性是这样的, 只有4口井往往足以获得统计上的重要数据 (图 4B)。
我们不建议进行一些更改。例如, 使用最初的细菌菌浓度远远高于议定书所列;这样做的结果是厚, 生物膜样的材料, 是很难或不可能有效地冲洗, 这使得分致死率复杂化。此外, 高浓度细菌菌也可以与氧气竞争, 并触发非特定宿主杀死7。
另一个重要的注意事项是限制停止化验和成像死蠕虫之间的时间段。请注意, 这个时间框架必须包括染色, 所以它是至关重要的是有效的。死后, 蠕虫体内的生物物质开始挤压和/或被细菌消耗。在 24-32 小时的情况下, 只有角质层残留。角质层的污渍非常糟糕, 是非常轻, 使容易失去在洗涤过程中。重要的是要注意的是, 不同的杀伤动力学的菌株或应变/rna 干扰条件可能会复杂的这种现象 (即,一些蠕虫可能仍然活着, 而其他人已经失去了他们的内容, 是不可能的图像)。图 4A显示这一现象, 因为在板块左侧的蠕虫, 接种了非常低的初始细菌浓度, 仍然主要是活的, 而蠕虫在右侧的板块, 这是暴露在更高的浓度细菌, 已经被冲走了。
检测成功的一个非常重要的决定因素是收集和分析数据的方法。在我们的实验室中, 我们使用了至少三种方法从这些化验中收集数据: 分光光度法、自动显微术和流 vermimetry (即流式细胞技术对线虫的适应; 从字面上看, 测量在流动溶液中的蠕虫)。第一种方法使用 spectophotometer 来读取有关蠕虫的染料或记者的荧光。这种方法具有使用相当普遍的设备 (标准分光光度计与微板块阅读器) 的优点, 是获取数据的最快方法。然而, 它缺乏从自动化显微镜和流量 vermimetry 的统计能力提供的信息内容。
自动显微镜是另一种可行的选择。一些微板块成像系统目前在市场上, 范围内的价格, 可负担的一个单一的调查员 (例如,生物 Cytation5) 到更大, 更昂贵和更高品质的机器, 更常见的核心设施 (如分子设备 ImageXpress 显微镜)。在实践中, 大多数这些解决方案都适合在大多数屏幕和检测中得分。大多数自动成像平台也可以耦合到下游软件进行图像处理 (例如, MetaMorph、ImageJ、Gene5 或单元格探查器), 以进一步实现信息的收益率。图 5和图 6显示了处理实用程序的示例。当信噪比较高时 (如图 5所示), 分析简单, 判别正负条件是微不足道的。在这些情况下, 即使是微弱的命中也可以容易地被辨认。然而, 许多化验都有微弱的信噪比。例如, 治疗 PINK-1::GFP22蠕虫 (其 pharynges 的本构 mCherry 表达) 与 110 phenathroline 增加 PINK-1::GFP 水平。对于数据分析, 诱导的 GFP 报告被规范化为本构 mCherry 信号 (图 6)。在这种情况下, 记者有较弱的表达和/或没有激活在大多数蠕虫。因此, 实现附加的图像处理工具 (如单元格探查器) 可以减少背景和放大信号, 这可能是有利的。
最后, 如果 COPAS FlowPilot 可用, 则可用于通过流 vermimetry 获取数据。就像流式细胞术的更熟悉的概念, 该仪器可以用来测量至少三个通道的C. 线虫的荧光。该方法对数据的采集具有较高的统计意义, 但与自动显微镜相比, 其吞吐量大大降低。流 vermimetry 最显著的优点是能够将整个蠕虫种群作为一个单一的复制群进行分析, 而不是将它们分解成多个井, 并分析井的平均值。以这种方式分析大型群体可以显著提高统计能力, 并允许检测甚至小的效果。
液体杀灭试验的改进
最近的研究进展扩大了它的用处, 包括化验荧光记者的活化 (图 6), 使用中-高通量的干扰技术 (图 3), 甚至替代原病 原 体。
使用 rna 干扰设置的最明显的原因是测试宿主防御通路对铜绿假单胞菌(或其他病原体)。图 3显示了与生存相关的基因的 rna 干扰击倒的例子。与先前的观察结果一致,20、23、24、25、气-2 (rna)增强的线虫在暴露于铜绿假单胞菌时存活, 而daf-16 (rna 干扰),邮编-2 (rna 干扰)和cebp-1 (rna 干扰)缩短了它。如上所述, rna 干扰最简单地包括在多井板上的生长蠕虫中, 每个井都含有不同的 rna 克隆。由于琼脂的粘度高、体积小, 难以实现无专用设备的96井板均匀无气泡充填。此外, 干燥的细菌菌株携带的干扰也可能是一个问题, 因为外水井干燥速度比内水井, 导致不均匀性和巨大的潜力, 琼脂裂解。如上所述, 这鼓励蠕虫挖洞, 减少可供筛查的动物数量和堵塞液体搬运机械的危险。由于这两种原因, 24 井板比96井板更容易使用。虽然这降低了检测吞吐量, 但它提高了可靠性, 并建议, 特别是对于初始屏幕。
最后, 对该法进行了改良, 以取代铜绿假单胞菌. (原则上, 如果确定适当的培养和暴露条件, 则与其他细菌种类的替代不应提供不当的困难。要考虑的最重要的因素是媒介要求的病原体, 无论是对生长和发展和诱导的毒力。例如, 革兰阳性, 机会主义致病菌大肠杆菌需要富含营养的培养基 (如 BHI) 和在较高温度下的孵化, 与14,26的铜绿假单胞菌相比。通过考虑这些因素, 对使用大肠杆菌的化验的适应是直接的。正如对铜绿假单胞菌的注意, 我们看到了依赖时间的杀戮 (图 7)。有趣的是, 死亡的时间和以前发表的化验结果相似, 尽管没有这一步骤, 但在琼脂板上使用了前感染26, 这可能表明发病机制不同。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58068/58068fig1.jpg"> 图 1: 液体基线虫的方案-铜绿假单胞菌感染试验.有关线虫繁殖和制剂的步骤在左侧, 细菌制剂在右侧。涉及两者的步骤都居中。<a href="https://www.jove.com/files/ftp_upload/58068/58068fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58068/58068fig2.jpg"> 图 2: 铜绿假单胞菌杀死C 线虫是具体的, 需要适当的细菌营养。(A)大肠杆菌和铜绿假单胞菌存在时的线虫死亡。(B) 在含有铜绿假单胞菌(右) 和无 (左) 慢杀介质的情况下,线虫死亡 (即仅限于基底)。S 基部是一个极小的媒介, 并且不包含细菌生长所需的营养素并且排除了 pyoverdine 的生产。(c) 在存在不同制备的铜绿假单胞菌的情况下,线虫死亡。*p < 0.01, 根据学生的t测试。误差线表示 S.E.M. 10 井每生物复制使用每个条件。<a href="https://www.jove.com/files/ftp_upload/58068/58068fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58068/58068fig3.jpg"> 图 3:线虫对铜绿假单胞菌的抗性取决于宿主免疫通路。用 knockdowns (a) 或 110-邻苯二氮 (一种化学离子螯合剂, 模拟接触液体中的铜绿假单胞菌), 对选定的 rna 干扰剂进行了处理 (B)。*p < 0.01, #p < 0.05, 基于学生的t检验。误差线表示 S.E.M. 10 井每生物复制使用每个条件。<a href="https://www.jove.com/files/ftp_upload/58068/58068fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58068/58068fig4.jpg"> 图 4: 铜绿假单胞菌对线虫的杀灭是健壮的, 取决于细菌浓度。(a) 有代表性的图像 (a) 和量化 (B) 的线虫死亡后, 暴露在不同浓度的铜绿假单胞菌的 72 h. p值计算的基础上, 学生的t测试。(A) 中的刻度条为1毫米, 每个条件下每生物复制使用16口井。BF: 明亮的领域, 佛罗里达州: 荧光。<a href="https://www.jove.com/files/ftp_upload/58068/58068fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58068/58068fig5.jpg"> 图 5: 具有强信号的检测数据的采集和图像处理.(A) 有代表性的活体 (阴性对照) 和死 (阳性对照)线虫的图像。(B) 对基于数据获取方法、处理方式和井数分析的负和正控制进行统计分析。(C) 对96井板 (两个正和两个负控井) 的四个单独井中的蠕虫进行了荧光分析。每个井包含了100蠕虫。图上的每个点都代表一个蠕虫, 显示了飞行时间 (与此相关, 但是, 由于卷取活蠕虫, 不完全代表大小) 和被测量的荧光 Sytox 橙染色的蠕虫。积极控制蠕虫是预先杀死的热暴露。由于死蠕虫的张力和形状的变化 (如在 (A) 中可见, 死蠕虫看起来更多的棒状, 并有很少的身体弯曲) 相比, 他们的生活相对, 死蠕虫有一个更长的飞行时间。对p值进行了基于学生t检验的计算。刻度条在 (A) 是1毫米.<a href="https://www.jove.com/files/ftp_upload/58068/58068fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58068/58068fig6.jpg"> 图 6: 具有中等信号的检测的数据采集和图像处理.(A) 带有诱导性 PINK-1::GFP 记者和 mCherry 本构标记的C. 线虫的代表性图像 (用于正常化染色体外阵列中基因的表达)。暴露于亚砜 (负控制) 或 110-邻菲咯的线虫(阳性对照)。(B) 对基于数据获取方法、处理方式和井数分析的负和正控制进行统计分析。(C) 采用流动 vermimetry 分析了96井板 (两个正、两个负控井) 的四个井。对p值进行了基于学生t检验的计算。刻度条在 (A) 是1毫米.<a href="https://www.jove.com/files/ftp_upload/58068/58068fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58068/58068fig7.jpg"> 图 7:大肠杆菌的线虫杀死是健壮的, 时间依赖。(a B) 有代表性的图像 (a) 和量化 (B) 的线虫死亡后, 暴露于大肠杆菌。*p < 0.01, 根据学生的t测试。每个条件下每生物复制使用10口水井。误差线表示 S.E.M. 刻度条 (A)为1毫米.<a href="https://www.jove.com/files/ftp_upload/58068/58068fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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A High-throughput, High-content, Liquid-based C. elegans Pathosystem

在这里, 我们描述了一个协议, 它是一个适应性强, 全宿主, 高内容筛选工具, 可用于研究宿主病原体相互作用, 并用于药物发现。

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

a-high-throughput-high-content-liquid-based-c-elegans-pathosystem

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

14.3K 次观看.  Rice University. 在这里, 我们描述了一个协议, 它是一个适应性强, 全宿主, 高内容筛选工具, 可用于研究宿主病原体相互作用, 并用于药物发现。

视频: A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

科研

免疫与感染

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 ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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 ...

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. ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...