这项工作的财政支助由欧洲共同体（ERC-STG INTRACELLTB赠款n°260901，MM4TB赠款n°260872）、国家复兴银行、联邦（12001407（D-AL）Equipex想象生物医学）和加莱地区提供。我们感谢加斯帕德·德洛伊森、伊丽莎白·韦克迈斯特、安东尼诺·邦焦瓦尼和弗兰克·拉方特从BICEL平台获得的技术援助。

1. 高通量全基因组硅纳筛查
在感染Mtb H37Rv表达绿色荧光蛋白（GFP）时，在人类II型肺炎细胞模型A549细胞系中进行筛查。此过程在图 1A中概述。
<ol><li>用 1x siRNA 缓冲器重新存放在母板 （96 井板） 中的干燥硅纳库，以达到 4 μM 的浓度，然后将 10 μl 的混合物转移到 384 井女儿板（女儿板 1）中。</li>
	<li>在女儿板 1 中加入 10μl 的 1x siRNA 缓冲区，将 siRNA 稀释 2 倍。siRNA 悬念后，板材用可剥落的铝密封密封，可存储在 -20 °C 至少 6 个月和长达 2-3 年，但存储时间可能会因 siRNA 库制造商的建议而异。</li>
	<li>将女儿板 1 中的 siRNA 稀释成女儿板 2，以达到 500 nM 的浓度。siRNA 悬念后，板材用可剥落的铝密封密封，可存储在 -20 °C 至少 6 个月和长达 2-3 年，但存储时间可能会因 siRNA 库制造商的建议而异。</li>
	<li>使用前，在室温下解冻女儿板2。</li>
	<li>从女儿板 2 中取 2.5μl 的 siRNA，放入 384 井检测板中。</li>
	<li>在第 1.5 步的同一 384 井检测板中，在各自的井中添加 2.5μl 负和正控制 siRNA。</li>
	<li>将转染试剂稀释为 1x D-PBS，以产生足够的溶液，在每口井中提供 0.1μl 变异试剂，并在室温下预孵化稀释的输血液 5 分钟。</li>
	<li>在检测板中将 7.5 μl 的转染试剂/PBS 溶液混合物添加到每个井中，并在室温下孵育 30 分钟。</li>
	<li>加入40μl的A549细胞（1，500个细胞/井）悬浮在RPMI 1640中等补充10%胎儿牛血清（FBS）。在含有5%CO2的大气中，将细胞保持在37°C的3天潜伏期。这些细胞每24小时分裂一次，因此大约12，000个细胞在输血后三天在井中。</li>
	<li>通过离心处理4，000 x g 5分钟，用D-PBS（从MgCl2和CaCl2免费）清洗两周前的GFP表达Mtb H37Rv培养物，并丢弃洗涤。重复此步骤 3 倍（对于GFP-Mtb H37Rv 文化条件，请参阅协议 3）。</li>
	<li>将细菌颗粒悬浮在 10 毫升 RPMI 1640 中等，并辅以 10% FBS，在室温下减速 1 小时，使细菌聚集物沉积物。</li>
	<li>收集细菌超自然物，并使用微板读卡器测量 OD600（OD 600应在 0.6-0.8 之间）和 GFP 荧光 （RFU 值），以确定细菌浓度。使用显示 RFU 值 = f （CFU 值） 的参考回归线计算悬架的滴定器，该线是在对在相同条件下准备的另一种文化进行实验之前生成的。准备含有2.4 x 106细菌/毫升的细菌悬浮，这相当于5的多种感染（MOI）。</li>
	<li>取出 384 井检测板中的介质，并加入 25 μl 的新鲜制备细菌悬浮剂。</li>
	<li>在含有5%CO2的大气中，在37°C的384井检测板中孵化5小时。</li>
	<li>取出介质，轻轻清洗细胞与RPMI介质补充10%FBS 3倍。</li>
	<li>要杀死剩余的细胞外细菌，请在含有 5% CO 2 的大气中，在含有 5% CO2的大气中，用含有 50μg/ml 的阿米卡辛的新鲜 RPMI-FBS 介质治疗细胞，时间为 1 小时。</li>
	<li>去除中等含阿米卡辛，并加入50μl的新鲜RPMI介质补充10%FBS。在含有5%CO2的大气中，在37°C下孵化384井检测板5天。</li>
	<li>在图像采集之前，在 PBS 中加入 10 μl 的新鲜制备的 30 μg/ml DAPI（最终浓度 5 μg/ml），并在 37 °C 下孵育 10 分钟。</li>
	<li>将板加载到自动对焦显微镜中。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>设置曝光参数。使用激发激光 405 nm 记录 DAPI 荧光，使用发射滤光片 450 nm，使用激发激光 488 nm 记录 GFP 荧光，发射滤波器 520 nm。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>选择每口油井中的油井和要收购的油田，然后称为布局和子布局参数。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>使用步骤 1.20 和 1.21 中的参数生成实验文件，并运行自动采集。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>将图像传输到远程服务器。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>使用图像分析软件评估图像。使用核检测算法从DAPI通道检测细胞核，使用像素强度属性算法（图4A）从GFP通道检测细菌区域。</li>
</ol>注意：此协议进行了优化，以研究基因沉默对细胞内 Mtb 生长的影响。 Mtb 是一种生长缓慢的细菌，每 20 小时在最佳条件下分裂一次。感染后5天，在没有细胞裂解的情况下，细胞外 Mtb 的量仍然很低，不影响分析的质量。该协议必须优化抗生素治疗的长度和潜伏时间，以便使用快速生长的细菌，如 小分枝杆菌和大肠杆菌 ，广泛释放，可以感染新的细胞，以适应siRNA屏幕。
2. 高通量化合物筛选
在受感染的宿主细胞上进行筛查。此过程在图 1B 中概述。
<ol><li>解冻含有复合库的 384 井母板， 溶解在 DMSO 100% 中。在含有 10 μl 的 RPMI 1640 介质中，在 384 井女儿板中转移 0.5 μl 化合物，并辅以 10% FBS。</li>
	<li>通过离心处理4，000 x g 5分钟，用D-PBS（从MgCl2和CaCl2免费）清洗两周前的GFP表达Mtb H37Rv培养物，并丢弃洗涤。重复此步骤 3 倍（有关GFP-Mtb H37Rv 文化条件，请参阅协议 3）。</li>
	<li>将细菌颗粒悬浮在 10 毫升 RPMI 1640 中等，并辅以 10% FBS，在室温下减速 1 小时，使细菌聚集物沉积物。</li>
	<li>收集细菌超自然物，并使用微板读卡器测量 OD600（OD 600应在 0.6-0.8 之间）和 GFP 荧光 （RFU 值）。使用显示 RFU 值 = f （CFU 值） 的参考回归线计算悬架的滴度，该线在实验前已生成。典型浓度为1×108细菌/毫升。</li>
	<li>收获6天大的原发性人类巨噬细胞在4×10 5 细胞/毫升RPMI 1640中等补充10%胎儿牛血清（FBS）和50 ng/ml重组人类M-CSF（对于人类周围血单细胞细胞净化和巨噬细胞分化见协议4）。</li>
	<li>在不同的 MOI 中用巴西利孵育稀释的原细胞，范围从 1-5，在 90 rpm 温和摇晃，在 37 °C 时 2 小时。</li>
	<li>通过离心清洗受感染的细胞350 x g，去除细胞外细菌。每次离心步骤后，在RPMI 1640中等中重新吸收颗粒，并辅以10%FBS。重复此步骤2倍。</li>
	<li>暂停在RPMI 1640中等感染细胞补充10%FBS和阿米卡辛在50μg/ml和孵育悬浮与轻度摇晃1小时在37°C。</li>
	<li>通过离心350 x g去除含有阿米卡辛的细胞培养介质，用完整的RPMI 1640介质清洗受感染的细胞，并辅以10%FBS和50 ng/ml重组人类M-CSF。重复一次。</li>
	<li>在第 2.1 步中，在同一检测板中加入 40 μl 受感染的巨噬细胞悬浮液，该板已包含 10 μl 的复合稀释。目前，每口油井的DMSO最终浓度已达1%。</li>
	<li>在含有 5% CO2的大气中，在 37 °C 下孵化检测板 5 天。</li>
	<li>用细胞渗透的远红荧光染料染色活细胞。</li>
	<li>将板加载到自动对焦显微镜中。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>设置曝光参数。使用激发激光 640 nm 记录远红荧光，使用发射滤光片 690 nm，使用激发激光 488 nm 记录 GFP 荧光，发射滤波器 520 nm。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>选择每口油井中的油井和要收购的油田，然后称为布局和子布局参数。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>使用步骤 2.14 和 2.15 中的参数生成实验文件，并运行自动采集。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">单击此处查看更大的图像。</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>将图像传输到远程服务器。 <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">单击此处查看更大的图像。</a></li>
	<li>使用图像分析软件评估图像。使用像素强度属性算法检测远红色通道的细胞区域，使用像素强度属性算法（图 4B）从 GFP 通道检测细菌区域。</li>
</ol>注意：此协议可以通过用表达荧光蛋白（一口井/一个突变体）的突变体取代化合物来适应 Mtb 突变库筛选（图 1C，另见布罗丁等人） 。4）。 荧光突变体首先在井中播种（每口井20微克细菌悬浮）。然后，细菌通过30微克的细胞悬浮恢复。在离心350 x g下1分钟后，板在含有5%CO2的大气中以37°C孵育。孵化时间和 MOI 取决于检测。例如，对于早期细胞事件（如噬菌体酸化）的可视化，这些细胞可以感染 2 小时，MOI 范围为 1-20。溶酶体在含有 5%CO 2 的大气中使用 Lysotracker 染料在 2 μM 上染色 1.5 小时，在 37 °C 处，然后用 10% 甲醛或 4% 甲醛 （PFA） 固定。使用具有适当算法的图像分析脚本获取并最终分析共焦图像，用于溶酶体检测和亚细胞本地化4。
3. 绿色荧光蛋白表达结 核杆菌 H37Rv（GFP-H37Rv）培养条件
对于长期存储，GFP-H37Rv 被冻结在 D-PBS 中（每小瓶约 1 x 108 分枝杆菌）。
<ol><li>在含有 50 毫升 7h9 肉汤介质的 Erlenmeyer 烧瓶中补充一个冷冻库存的 GFP-H37Rv 小瓶，辅以米德尔布鲁克 OADC 浓缩 10%、甘油 0.5%、Tween-80 0.05% 和 Hygromycin B（50μg/ml）。</li>
	<li>在37°C孵化8天。</li>
	<li>测量 GFP-H37Rv 文化的 OD600。</li>
	<li>稀释 GFP-H37Rv 文化，以获得 OD600 = 0.1 在新鲜的 7H9 汤介质补充米德尔布鲁克 OADC 浓缩 10%， 甘油 0.5%， 特温 - 80 0.05% 和海格罗霉素 B （50μg/ml）.</li>
	<li>在用于检测之前，在37°C下孵化GFP-H37Rv 8天以上。</li>
</ol>4. 人类外周血单细胞细胞从全血或布菲涂层制剂中净化
<ol><li>将血袋稀释2倍于1x D-PBS（不含MgCl2 和CaCl2），包含1%FBS。</li>
	<li>通过菲科尔密度梯度离心分离单核细胞400 x g 20分钟。</li>
	<li>收集孤立的单核细胞。</li>
	<li>用 1 倍 D-PBS（从 MgCl 2 和 CaCl2 中免费）清洗单核细胞3倍，在室温下在 400 x g 处离心 10 分钟，含有 1% FBS。</li>
	<li>将细胞浓缩至 1 x 107 细胞/毫升。</li>
	<li>根据制造商的协议，使用 CD14 磁珠净化单核细胞（参见材料）。</li>
	<li>CD14单核细胞纯化后，在RPMI 1640中以1.5×10 6 细胞/毫升的种子将细胞播种，辅以10%FBS和40 ng/ml的人类巨噬细胞群细胞刺激因子（hM-CSF），在含有5%CO2的大气中以37°C孵育4天。</li>
	<li>4天后，用新鲜的RPMI 1640取代介质，辅以10%的FBS和40 ng/毫升的hM-CSF，并在含有5%CO2的大气中以37°C孵育2天。</li>
	<li>6天后，细胞可用于检测。</li>
</ol>

<ol>
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未声明任何利益冲突。

我们在这里描述使用 GFP 表达 Mtb H37Rv 菌株感染荧光标记主机单元所需的表型检测方法，使其适合高含量/高通量屏幕。该协议可应用于广泛的化合物、荧光探头和 Mtb 突变体。对于上述每个协议，可以在图像采集之前执行固定和免疫标记步骤。我们使用配备 20X （NA 0.70） 或 60X （NA 1.2） 水透镜的自动荧光同焦显微镜来获取图像。共焦显微镜配备了405、488、561和640纳米激发激光器。发?...

在过去几年报告的新兴和重新出现的传染性病原体中，结核病分枝杆菌（Mtb）在2011年造成140万人死亡和870万新感染（2012年全球结核病报告，www.who.int/topics/tuberculosis/en/）中占有突出地位。尽管有多种药物疗法，感染人数仍在上升，耐多药（MDR）以及广泛耐药（XDR）Mtb正在迅速蔓延到世界各地1。 此外，在考虑Mtb抗原的存在时，很明显，全球三分之一的人口被认为是潜在的感染Mtb。 据统计，在十分之一的病例中，有1个病例正在向该病的活跃形式发展，随后出现临床症状2。因此，迫切需要新的手段来打击Mtb。在此背景下，我们开发了一种体外视觉表型检测，依靠自动共聚焦荧光显微镜3监测Mtb入侵并繁殖成宿主细胞。将检测结果与自动图像采集和分析相结合，在 384 井微晶板中进行适应，允许对化合物、硅RNA 和细菌突变体的中尺度库进行高含量/高通量筛选 （HC/HTS）。因此，在这种表型检测上筛选一个基因组宽的RNAi库，能够识别与Mtb贩运和细胞内复制有关的关键宿主因素，并能够阐明块茎杆菌利用的宿主通路。这种特殊的表型检测的另一个适应是识别细菌因素，这些细菌因素对Mtb内法戈索马尔的持久性至关重要。例如，逮捕法戈索姆成熟被认为是促进Mtb在巨噬细胞中生存和复制的主要机制之一。监测荧光标记酸性隔间中Mtb淘汰突变体的亚细胞定位，使参与生存过程的细菌基因得以识别。最后，Mtb的高含量成像也为量化药物效率提供了一个很好的方法，用于抑制细胞内细菌生长3等各种现象。总之，这种高通量表型检测可以加速对结核病的药物发现，这些不同方法收集的数据有助于更好地了解Mtb施加的宿主操纵。

<table border="0" cellpadding="0" cellspacing="0" width="1010">
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		<col /> 
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#181;clear-plate black, 384 well</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781091</td>
			<td style="width:388px;">127.8/86/15 MM with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CellCarrier 384 well plate</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;">6007550</td>
			<td>Black, Clear Bottom, with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">V-bottom white , 384 well-plate</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
			<td style="width:160px;">Corning</td>
			<td style="width:175px;">3345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMax</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">13778150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">34943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI 1640 + GlutaMAX-I</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">61870-010</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [-]MgCl2/[-]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-094</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [+]MgCl2/[+]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-091</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Fetal bovine serum</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">2610040-79</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">FICOLL PAQUE PLUS</td>
			<td style="width:160px;">DUTSCHER</td>
			<td style="width:175px;">17-1440-03</td>
			<td>Ficoll for Peripherical Blood Monocyte Cells purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD14 MicroBeads, human</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-050-201</td>
			<td>Purification of CD14+ Monocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Human M-CSF, premium gr. (1000 &#956;g)</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-096-493</td>
			<td>Macrophage Colony Stimulating Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS Columns</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-042-401</td>
			<td>Columns for CD14+ Monocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tween 80</td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">2002-A</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glycerol high purity </td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">50405-EX</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;"> Middlebrook OADC enrichment</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">211886</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7H9</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">W1701P</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Versene 1X</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">15040033</td>
			<td>Non enzymatic cell dissociation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DAPI</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hoechst 33342</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">H3570</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syto60</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">S11342</td>
			<td>Nuclei/cytoplasm dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formalin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">HT5014</td>
			<td>Cell fixation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siRNA targeting Dectin-1</td>
			<td style="width:160px;">Santa-Cruz</td>
			<td style="width:175px;">sc-63276</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">*siGenome* Non targeted siRNA pool </td>
			<td style="width:160px;">Dharmacon</td>
			<td style="width:175px;">D-001206-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Rifampicin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">R3501</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoniazid (INH)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">I3377-50G</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hygromycin B</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">10687-010</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Amikacin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">A1774</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Automated Confocal Microscope OPERA</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Columbus 2.3.1 Server Database</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Data transfert, storage and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acapella 2.6 software</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image-based analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GraphPad Prism5 software</td>
			<td style="width:160px;">GraphPad </td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:287px;">Excel 2010</td>
			<td style="width:160px;">Microsoft</td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening

尽管有治疗和疫苗，结核病（TB）仍然是世界上最致命和最普遍的细菌感染之一。几十年来，多重耐药菌株的突然爆发对结核病的控制构成了严重威胁。因此，必须确定新的靶点和途径，对结核病的致病剂，结核分枝杆菌（Mtb）至关重要，并寻找可能成为结核病药物的新化学物质。一种方法是建立适合大型图书馆的基因和化学屏幕的方法，以便在大海捞针。为此，我们开发了一种表型检测，依靠使用自动共焦显微镜检测荧光标记的Mtb。这种体外测定允许对Mtb的殖民化过程进行基于图像的定量，并针对 384 井微板格式进行了优化，该格式适合锡RNA、化合物或Mtb突变库的屏幕。然后对图像进行多参数分析处理，从而提供主机单元内Mtb发病机理的读取推断。

高通量全基因组硅纳筛查
Mtb 能够 在体外 和其他几个肺上皮细胞中殖民免疫细胞。例如 ，Mtb能够感染和破坏通常用作人类 II 型肺细胞5-7模型的 A549 上皮细胞。Dectin-1被报告为主机细胞受体，参与 Mtb 吸收，增生反应和抗菌作用细胞内分枝杆菌生长在A549细胞8。第 1 号协议中描述的 siRNA 条件导致 85% 的沉默效率（未显示数据）。用siRNA沉...

Watch this Scientific Journal Video about A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening at JoVE.com

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

在这里，我们描述了一种表型检测，适用于小干扰合成RNA（siRNA）、化合物和 结核 分枝杆菌突变库的高通量/高含量屏幕。该方法依靠使用自动共聚焦显微镜检测荧光标记的荧光结 核分枝杆菌 。

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 ...

a-microscopic-phenotypic-assay-for-quantification-intracellular

Research

JoVE Journal

Immunology and Infection

7.6K 次观看.  Université de Lille. 在这里，我们描述了一种表型检测，适用于小干扰合成RNA（siRNA）、化合物和 结核 分枝杆菌突变库的高通量/高含量屏幕。该方法依靠使用自动共聚焦显微镜检测荧光标记的荧光结 核分枝杆菌 。

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

耐高温溶菌酶的定向进化的一个新的筛选方法

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

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 ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

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

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 combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

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

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

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 ...