SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1，96孔龙引物PCR <ol><li>预冷冻96孔PCR冷却器在-80℃冷冻机中。 </li><li>在一个10毫升管，准备预混一个:2.100微升的DDH 2 O，105微升PCR级DMSO，105微升10毫米的dNTP，105微升PPOT模板（25毫微克/微升）9，每盘2415微升总体积。 </li><li>等分试样23微升到96孔PCR板的每个孔中。 </li><li>加入2微升100μM汇集引到每个加载以及使用P20 12通道多道移液器的。用膜，代替板密封在预冷的PCR冷却器并允许冻结在-80℃至少20分钟。 </li><li>在10ml的管中，制备主混合物A:2048微升DDH 2 O的，525微升10×缓冲液，52.5每板2626微升的总体积微升聚合酶。 </li><li>从-80℃冰箱中取出板和PCR凉爽。 </li><li>随着板块仍然在PCR冷却器，在冻结的顶部加入25微升主混合物BN主控一个混合的50微升反应总体积。这是时间关键的，并且应完成之前混合物A可以解冻。封板与薄膜。 </li><li>设定热循环如下:94℃10分钟，然后94℃30个循环持续15秒，65℃，30秒，72℃2分钟，然后72℃7分钟的最后延伸期间。 </li><li>加载PCR板的块已到达94℃后。 </li></ol> 2. 96-龙引物的PCR验证<ol><li>投大1％（重量/体积）在Tris醋酸EDTA（TAE）琼脂糖凝胶用4×28孔的梳子运行缓冲液，得到一种凝胶具有4行孔中。对准一12信道多道移液器的尖端的梳子的交替齿。仔细淹没在TAE凝胶电泳缓冲液的车道加载后。 </li><li>负载1 kb的DNA梯到1 次和28 次良好的每一行。 </li><li>使用P20 12通道多道移液器，转移2微升PCR产物迪尔ectly到凝胶（ 图1A）的各泳道。为此迅速减少污染扩增子的可能性。 <ol><li>装载从PCR板的行A中的PCR产物到凝胶的第1行的奇数泳道（A1 - 泳道3，A 2 - 泳道5 ...... A11 - 车道23，A12 - 泳道25）和加载PCR产物从PCR板的列B到凝胶的1行的偶数道（B1 -泳道4，A 2 -泳道6 ...... B11 -车道24，B12 -泳道26）。 </li><li>如上所述，与C行到奇数通道及D行到偶数泳道加载来自使用相同的图案的PCR板的行C和D的PCR产物。直到PCR板的每个孔中装载到凝胶继续这个装载模式。 DNA的加样缓冲液不需要加载此凝胶。 </li></ol></li><li>将凝胶进入运行罐和TAE加满油箱运行缓冲液直至凝胶被淹没。在100V进行30分钟运行，并可视化使用UV透照。参见图1B的例子凝胶。 注意:PCR产物可被储存在-20℃下几个星期转染前</li></ol> 3. 96孔转<ol><li>使用T.布氏 procyclic形式的细胞系SMOXP9 10此过程和生长在含有10％FCS 10的SDM-79的介质。 </li><li>使用1×10 7个细胞每转上的C末端标记时（总共1.1×10 9个细胞）的蛋白质的N末端 ​​和每转染2×10 7个细胞标记时（总共2.2×10 9个细胞）的蛋白质。保持在对数中期（1.2×10 6 -1×10 7细胞/ ml）的细胞在转染前几天和收获转染的细胞以5-8×10 6细胞/ ml的密度。 </li><li>允许冷冻PCR产物进行转染，在室温下解冻。 </li><li>使用血红素细胞计数ocytometer或自动细胞计数。 </li><li>在800 XG 10分钟多50ml试管颗粒所需数量的细胞。 </li><li>离心后弃上清，重悬在10毫升的细胞改性cytomix（0.8毫EGTA，24毫米氯化钾，0.15毫氯化钙 ，10mM磷酸钾缓冲液pH7.6的，25毫米的HEPES-KOH pH7.6的，2.6毫MgCl 2的， 0.5％（重量/体积）葡萄糖，每管100微克/毫升BSA，1mM的次黄嘌呤，144毫蔗糖）。传输所有细胞溶液到单个管中并在800 xg离心再次旋转10分钟。 </li><li>离心后，弃去上清，重悬23毫升改性cytomix的细胞的5×10 7个细胞/ ml的终浓度。 </li><li>而细胞被纺纱，加入1 ml的SDM-79的介质，以4×24孔组织培养板的各孔中。标签板AD，绘制出周围环以及A1在永久性标记每块板，以帮助板方向。 </li><li>板处理程序连接到电穿孔。在electropora器单元设置的电压为1500伏，脉冲长度为100微秒和脉冲12的数目和脉冲的时间间隔为500毫秒。上盘处理，设定脉冲计数为1。这确保了电穿孔将应用于单个脉冲到各个电穿孔板的12列。 </li><li>使用P200的12信道多道移液器，从PCR板转移PCR反应，以96孔一次性电穿孔板，4毫米间隙。 </li><li>当细胞已被纺丝并重新悬浮于5×10 7个细胞/ ml（步骤3.7）的最终浓度，转移到试剂容器。吸移管200微升的细胞溶液进入使用P200的12信道多道移液器电穿孔板的各孔中，通过移液混合用的PCR产物。 </li><li>采用组织从板块以避免短路的顶部移除任何液滴。 </li><li>在板包装提供的密封膜适用于电板，POS机的顶部itioning它以便留下在每一列的顶部和底部露出的电极上的孔。避免覆盖用于指导电影提出的问题。 </li><li>加载电板进入平板处理器，并盖上盖子。按电穿孔单位"脉冲"。 </li><li>电穿孔后，迅速从96孔电穿孔板的4×24孔组织培养板转移的细胞。为了转移细胞中，使用P200 12通道多道移液器与所有其他尖端缺失，使得剩余的6个技巧排队与24孔培养板的6行。转移在96孔电穿孔板中的电穿孔细胞在预先定义的模式的24孔组织培养板中（ 图2A）。 </li><li>准备P200枪头 - 每个96孔转染准备96提示2盒拆下隔列。 <ol><li>传递孔A1 / A3 / A5 / A7 / A9 / A11的96孔电穿孔板板24孔培养板的排A，B1 / B3 / B5 / B7 / B9 / B11成排B，C1 / C3 / C5 / C7 / C9 / C11到C行和D1 / D3 / D5 / D7 / D9 / D11进行D.每组6个孔从96孔板的24孔平板的转移后，在96孔板的孔中，然后用培养基洗涤从相应的孔中的24-孔板和洗涤，然后转移回至孔在24孔板中。 </li><li>传递孔的E1 / E3 / E5 / E7 / E9 / E11上的96孔电穿孔板24孔盘B行A，F1 / F3 / F5 / F7 / F9 / F11成列B，G1 / G3 / G5 / G7 / G9 / G11成在96 C行和H1 / H3 / H5 / H7 / H9 / H11到行D.从96孔板每组6个孔的转移到24孔板后，将孔-Well板然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li><li>转让井A2 / A4 / A6 / A8 / A10 / A12的96孔电穿孔板24孔板C列A，B2 / B4 / B6 / B8 / B10 / B12成排B，C2 / C4 / C6 / C8 / C10 / C12到C行和D2 / D4 / D6 / D8 / D10 / D12成排D.从96每组6口井的转移后-Well板至24孔板，在96孔板的孔中，然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li><li>转让井E2 / E4 / E6 / E8 / E10 / E12的96孔电穿孔板24孔板d行A，F2 / F4 / F6 / F8 / F10 / F12到B行，G2 / G4 / G6 / G8 / G10 / G12的进行C和H 2 / H4 / H6 / H8 / H10 / H12到行D.从96孔板每组6个孔的转移到24孔板后，在96孔-Well板然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li></ol></li><li>使用倒置显微镜，研究1井从每列检查细胞。会有活的和死的细胞用的混合物大量的细胞碎片。活细胞可以从死细胞区分开来，因为他们将在相差显微镜明亮，会动。 </li><li>放置在一个干净的，塑料盒24孔板与形成紧密密封的盖。把盒子在28℃培养箱。 </li><li> 6-8之间小时后，加入1 ml SDM-79的含2倍选择性抗生素，每孔10％FCS。根据我们的经验，其中该基因的标签上的N末端细胞系是对较高浓度选择药物的抗性。因此，在使用杀稻瘟素作为一个例子，标记上的N末端要求20微克的终浓度/ ml的杀稻瘟素和标记在C末端需要10微克/ ml的杀稻瘟素的最终浓度。 注:转染后的转基因细胞系将成为可见的9-10天。 </li><li>在新鲜的药物和媒体至少两次分析前通道。 图2B示出从一个典型的96孔transfe结果ction上的N末端标记96个不同的蛋白质。评估每孔用相差显微镜，以确定它是否包含健康，药物抗性细胞。健康以及主要包含（&lt;95％）高活性的同相合同显微镜明亮细胞。通过荧光显微镜或免疫印迹随后的分析是必需的，以确定该蛋白已成功标记。 </li></ol>

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The authors have nothing to disclose.

在蛋白质组学和转录方法在过去的5 - 10年的灵敏度显着改善提供了数以千计的基因及其产物的有价值的数据。然而，这些工具来解决这些蛋白质的功能没有跟上。 标记的蛋白质有利于无数次的实验，以确定其功能。例如，蛋白质可以在各种不同的颜色，以促进定位和共定位研究来融合于荧光蛋白。标签电子显微镜，如APEX2或miniSOG 11,12开发，使标签蛋白的超微结构定位。标签为生物化学，例如在TAP标记，并ProtC-TEV-PROTA（PTP）标记7,13允许与蛋白质的结合伙伴的身份或体外生物化学测定相关联配合物的纯化。 那些对protoc的成功至关重要的具体步骤醇有:二甲基亚砜掺入PCR主混合物1之前，在加入主混合物2中，在主混合物2中使用的商业聚合酶和cytomix电穿孔缓冲液的修饰的冻结PCR主要混合液1。在我们的经验，这是必要的，以便达到阳性孔的相似的比例使用细胞的双数为C末端标记的转染，作为N末端标记的转染。因此，如所描述，应执行的所有步骤。 这种技术只可能转染虫procyclic形式锥虫时获得成功。血液形式具有较低的转染效率14，而且他们很可能是由于这是无关的选择药物的密度依赖毒性选择过程中死亡。因此，我们以前的协议代表了血液形式锥虫9标记当前最先进的技术。它也可能是反式染效率会变化取决于特定的锥虫分离物。其他菌株可能需要额外的优化 - 这个协议使用927 SMOX procyclic形式进行了优化。可能增加成功的概率的措施包括:增加PCR扩增子的量，增加包括在转染细胞的数目。 我们提出了数百蛋白质可以并行标记的方法。这将有利于本地化和互动的大规模研究，补充现有大型数据集，并提供宝贵的信息社会。底漆模板可根据要求和质粒模板列表可以从:http://www.sdeanresearch.com/

布氏锥虫是原生动物寄生虫引起牛非洲人类trypanosomasis和那加那T。布氏是蛋白质功能的分析的理想生物体由于高品质的基因组，众多蛋白质组学和转录数据集和发达分子工具1-3的组合。蛋白质组学和测序的进步导致了突出潜在的有趣的基因4-6大型数据集;然而，许多基因都与他们现有的数据库相关联的最少信息。因此，有需要一种高通量方法，以帮助蛋白质功能表征。 一个标签蛋白的表达能给见解的多样性成蛋白质的功能。例如，标记有荧光蛋白或表位的蛋白质可以通过荧光显微镜，其中给出了关于其中PROT信息进行本地化EIN可能会发挥其生物效应。另外，贴上了TAP 7的蛋白质，HaloTag 8或他的标签可以用于生化分析及其合作伙伴的互动鉴定纯化。 我们最近开发出一种强大的标注方法为T.布氏 9。这在过去很长的引物PCR生成转染DNA，并允许数十种蛋白质并行的标记 - 一个重大改进现有的协议。现在，我们已经通过了一个数量级提高该协议在procyclic形式的可扩展性。这里，我们提出我们的方法，其中，我们执行PCR和转染到96-孔板，以恢复和选择在24孔板中发生。数百蛋白质现在可以在并行进行标记该方法提供用于标记整个锥虫基因组中的具有成本效益的，可行的方法。

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotide</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:119px;">na</td>
			<td style="width:167px;">desalt only (do not use additional purification)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMSO</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">Included with the Expand HiFi pack</td>
			<td>Must be PCR grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP</td>
			<td style="width:115px;">any reputable</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Expand HiFi polymerase</td>
			<td style="width:115px;">Roche</td>
			<td style="width:119px;">11759078001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BTX ECM830</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;"></td>
			<td>Electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HT-200 plate handler</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">HT-200</td>
			<td>Handles the 96-well eletroplates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MOS 96 ELECTROPLATE 4mm</td>
			<td style="width:115px;">Harvard Apparatus</td>
			<td style="width:119px;">45-0452</td>
			<td>Electroplate for the 96-well electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Melford</td>
			<td style="width:119px;">3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin b Gold</td>
			<td>Invivogen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phleomycin</td>
			<td style="width:115px;">Melford</td>
			<td style="width:119px;">P0187</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">225cm TC Flask, canted neck, phenolic cap</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well culture plates</td>
			<td style="width:115px;">Appletonwoods</td>
			<td style="width:119px;">BC017</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eppendorf&#174; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well Multiply&#174; PCR plate with lateral skirt</td>
			<td style="width:115px;">Sarstedt</td>
			<td style="width:119px;">72.1979.203</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

在这个代表转染的引物使用TagIt perl脚本9设计和商业合成。在96孔PCR进行和验证所描述（ 图1A）;在这个例子中，95/96的PCR成功（ 图1B）。在我们的经验，重复或者与原始引物或重新合成的引物失败的反应不会导致成功的PCR。 扩增子被转移到96孔电穿孔板电穿孔（ 图2A），然后转移到用于恢复和选择的24孔培养板中。后有足够的时间以允许发生的选择，各孔打进之前进一步分析存活。在这个例子中，九十六分之八十八井（92％）为15天之后选择阳性。 <i毫克ALT ="图1"SRC ="/文件/ ftp_upload / 54342 / 54342fig1.jpg"/&gt; 图1:长引物的PCR（A）的图案的验证 PCR产物转移从96孔PCR平板的琼脂糖凝胶进行PCR验证。 （B）为在N末端 ​​标记95的基因的代表性96孔PCR验证凝胶，和不含有5&#39;靶向同源充当在转染的阴性对照1扩增子。样品装载到1％（W / V）琼脂糖凝胶，并在100伏解决30分钟。 <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54342/54342fig2.jpg" /> 图2:96孔电穿孔和选择 （A）模式的电穿孔细胞从96孔电开发平台转移。E要24孔培养板。 （B）示意图，显示在后24孔板选择15天一个典型的活/死得分。绿色代表着成功转染，灰色代表也只有死细胞。在这个例子中，井九十六分之八十八（92％）含有成功转染寄生虫。 <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

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-gene-tagging-in-trypanosoma-brucei

Research

JoVE Journal

Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei

7.9K 次观看.  University of Oxford. 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.

视频: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

科研

免疫与感染

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

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 ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...