Kaloshian实验室的研究经费由美国国家食品和农业研究所（2007-35607-17765）的资助。罗伯茨实验室的研究是由来自美国的资助机构国际开发（GDG - G-00-02-00012-00和EDH-A-00-07-00005）和加州的干豆腐咨询委员会。

1。番茄幼苗生长在花盆和托盘<ol><li>用于盆栽试验，在富含有机质的土壤，如阳光混合的单罐厂番茄种子。在温室保持在22-28°C。发芽后，施肥每周一次奇迹格罗。 </li><li>发芽后约两个星期，在这两个真正的叶阶段，移植到无菌的沙质土壤沙90％和10％的有机组合与充满盆（直径10厘米和17厘米深）单独苗。 Osmocote缓释肥料在温室两周保持在22-28°C。继续奇迹格罗每周一次的植物施肥。 </li><li>对于高通量筛选，直接在托盘在沙质土壤的植物种子，用保鲜膜覆盖的托盘，直到发芽，保持以上。发芽后，加Osmocote缓控释肥，施肥和每周一次的奇迹格罗。 </li></ol> 2。豇豆幼苗生长袋<ol><li>小号电火工品是在培养皿​​中发芽，内衬几层Kimwipe纸，单独转移到袋或直接放在袋纸槽和发芽。 </li><li>挂着一个塑料文件夹，每个文件夹中放置袋，并安排在垂直位置的机架中的文件夹。放置在温度25-28°C和16 h光照/ 8 h黑暗周期保持在一个受控环境室的机架。机架也可以保持在温室，但需要陪衬或植物茎的两侧放置在机架上的僵硬的纸盖，以减少真菌污染的潜力。 </li><li>水袋与反渗透水，每天一次或两次。播种后约10至14天，已开发足够的根系统具有大专根尖（ 图1），苗接种准备。 </li></ol> 3。线虫虫卵提取电子商务的提取感染根GGS修改赫西和巴克（1973）开发了一个协议。 <ol><li>线虫三到四天之前，提取感染番茄根部的根结线虫卵。鸡蛋提取开始之前，工作区用热水洗脸，彻底避免污染。也是在热水冲洗搅拌机，两桶水，丝网的支持下，橡胶锤，三筛425，90和25微米的孔径，量筒和剪刀。 </li><li>堆栈按照下列顺序从上到下筛：425，90和25微米的孔径。 25微米筛上的鸡蛋将被收集在底部。放在水槽支持丝网筛。丝网下把水桶收集贯通的解决方案。 </li><li>作为接种源，切断受感染的植物（S）的上衣，并丢弃。小心取出锅​​厂。根洗净，浸在装满水的塑料桶。下冲洗进一步运行的根源宁水，直到土壤颗粒从根部洗净。 </li><li>切剪刀切成小块的根源。处置的主根。 </li><li>把盖在一个大塑料瓶从单一植物切碎的根，刚够10％的漂白粉溶液覆盖的根源，并盖上盖子。 </li><li>摇动罐子含2分钟的根源。 </li><li>打开罐子，倒入到顶端筛根，并用软管连接到一个喷雾喷嘴。洗井，直到所有的漂白剂气味消失了。使用木槌挖掘，以避免堵塞筛孔筛双方。 </li><li>卸下顶部筛和冲洗的第二个筛子上的杂物。 </li><li>删除第二个筛子，并收取罚款的碎片，其中包括鸡蛋，从去年筛。从一瓶水的温柔流，将杂物筛一侧。收集到一个干净的烧杯中水的最低金额碎片和鸡蛋。 </li><li>筛一次水收集在通过25微米筛收集任何可能逃脱的蛋桶。用水冲洗干净，并收集在同一烧杯。 </li><li>丢弃从顶部筛植物碎片和压水彻底清洗所有三个筛子。请勿触摸网的一部分，因为它可能会扭曲孔径的筛子。 </li></ol> 4。孵化线虫虫卵<ol><li>行清洁的金属与几层Kimwipe纸的篮子，适合在玻璃培养皿。允许一个篮子和盘子的底部之间的1厘米的距离。 </li><li>在纸张上提取的线虫虫卵倒在篮筐。加入足够的液体，使篮筐底部接触水面，但不是浸泡在水中。盖上一个塑料盖的顶部。 </li><li>每天检查水分蒸发，在培养皿中添加一些水，使篮子的底部接触的水。这将防止干燥鸡蛋。 </li><li>隔日，收集佤之三，包含到一个烧杯，培养皿J2S。如果不立即使用，实验室供气或水族馆泵产生的空气通气在室温下使用所收集的接种。使用2天之内的曝气接种。的J2S可能收集超过6-8天期间设立的孵化。 </li></ol> 5。番茄根在花盆或托盘和评估感染感染<ol><li>使用三等分收集线虫计数的幻灯片或菜J2S数量来计算，计算平均值，并确定为接种所需的音量。通常3000 J2S使用单株盆栽植物种植在育苗盘和500 J2S。 </li><li>搅拌磁性板在低速搅拌接种。 </li><li>之前接种植物，确保土壤湿润，但不要太湿。锅接种，在每个番茄根系统使用铅笔周围的沙孔，深度约一半锅（ 图2 </强&gt;）。接种到三个孔用吸管J2S提供每个工厂。此后，包括孔。 </li><li>对于托盘的高通量筛选，用孔上粘到5毫升枪头沿移液器（ 图3），提供入土壤J2S双方修改后的密封尖端针。此外，线虫可交付步骤5.3。 </li><li>温室中的植物保持在24-27°C的六到八周。继续奇迹格罗每月两次施肥。 </li><li>感染的评价，小心取出从盆的植物和洗根（步骤3.3中所述）。 </li><li>染色15分钟，淹没在1毫克/升erioglaucine根蓝色的蛋群众。 </li><li>水冲洗根和评估计数彩绘蛋群众对个别根系统。一个发光的桌子放大镜可以用来帮助可视化的蛋群众。 </li><li>如果筛选的植物都需要进一步Ğenetic研究，不削减洗涤前评价根的顶部。染色和计数的卵块后，移植在有机土壤的树苗，修剪顶部，大量减少蒸腾，并保持在一个温室。 </li></ol> 6。豇豆根感染感染的邮袋和评价<ol><li>计数线虫接种和磁力搅拌板搅拌，如前面所述。 </li><li>删除水平的表面上悬挂的文件夹和地方的邮袋。接种5毫升在1500 J2S每个邮袋。提起袋的塑料盖，并均匀地分布在根表面的线虫。 </li><li> 24小时后接种黑纸覆盖排除光，保持在一个水平位置的袋，然后返回到生长室吊夹。 </li><li>需要水的植物，一次或两次，每天，半强度霍格兰的解决方案（霍格兰德嫩，1950年， 表1 </STRONG&gt;）观察直到化肥的响应，通常增强观叶绿化和新梢生长更加旺盛。一般植物的半强度霍格兰连续3天浇水。此后，保持袋与水的滋润。 </li><li>接种后约30天（范围28-35天），注入约10-20毫升75毫克/升erioglaucine每个邮袋。保持一夜之间淹没在水平位置与染料袋。 </li><li>漏袋和评估计数照明办公桌放大镜下的蛋群众的根系统。 </li><li>如果筛选植物需要进一步的遗传研究或育种工作，精心拉出的根源，从纸张，移植在花盆里的土壤有机和保持在一个温室。 </li></ol> 7。代表结果两个系统的线虫接种番茄和豇豆植物的适当阶段，显示在图1和2。此外，番茄和豇豆感染根的例子在图4和图5所示。至于在大多数抗病屏幕的，最好是使用至少6-10％的基因型植物线虫来计算，平均感染率。相同的基因型，植物之间的感染线虫的变化，可以减少使用统一的工厂规模和准确的数量和交货的接种。 托盘和增长袋的使用，使筛选数百数以千计的植物在一个小的增​​长空间。增长袋也让没有必要洗根（ 图4）快速，高效的根结线虫感染的非破坏性评估。 <img alt="图1" src="/files/ftp_upload/3629/3629fig1.jpg" /> 图1。两个周龄的豇豆植物生长在一个小袋，并准备为根结NEMAtode接种。 <img alt="图2" src="/files/ftp_upload/3629/3629fig2.jpg" /> 图2。三个星期岁番茄根结线虫接种的准备。 <img alt="图3" src="/files/ftp_upload/3629/3629fig3.jpg" /> 图3。一种改进的针头和枪头，用于高通量的线虫接种。一针的底部是密封的，和3套孔钻针在使用激光束。然后，修改后的针粘到5毫升的枪头。 <img alt="图4" src="/files/ftp_upload/3629/3629fig4.jpg" /> 图4。感染根结线虫番茄的根系统。 <img alt="图5" src="/files/ftp_upload/3629/3629fig5.jpg" /> 一个豇豆染色erioglaucine接种后30天内增长的鸡蛋群众的根系统图5。28°C。

<ol>
	<li>Bhattarai, K. K., Xie, Q. G., Mantelin, S., Bishnoi, U., Girke, T., Navarre, D. A., Kaloshian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+susceptibility+to+root-knot+nematodes+requires+an+intact+jasmonic+acid+signaling+pathway.">Tomato susceptibility to root-knot nematodes requires an intact jasmonic acid signaling pathway.</a> Mol. Plant Microbe Interact. 21, 1205-1214 (2008).</li><li>Ehlers, J. D., Matthews, W. C., Hall, A. E., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+broad-based+form+of+root-knot+nematode+resistance+in+cowpea.">Inheritance of a broad-based form of root-knot nematode resistance in cowpea.</a> Crop Sci. 40, 611-618 (2000).</li><li>Hoagland, D. R., Arnon, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+water-culture+method+for+growing+plants+without+soil.">The water-culture method for growing plants without soil.</a> Calif. Agric. Exp. Stn. Circ. 347, (1950).</li><li>Hussey, R. S., Barker, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+of+collecting+inocula+of+Meloidogyne+spp.,+including+a+new+technique.">A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique.</a> Plant Dis. Rep. 57, 1025-1028 (1973).</li><li>Martinez de Ilarduya, O., Moore, A. E., Kaloshian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+Rme1+locus+is+required+for+Mi-1-mediated+resistance+to+root-knot+nematodes+and+the+potato+aphid.">The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid.</a> Plant J. 27, 417-425 (2001).</li><li>Matkin, O. A., Chandler, P. A., Baker, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+U.C.-type+soil+mixes.+In:+The+U.C.+system+for+producing+healthy+container-grown+plants.">The U.C.-type soil mixes. In: The U.C. system for producing healthy container-grown plants.</a> Calif. Agric. Exp. Stn. Manual. 23, 68-85 (1957).</li><li>Omwega, C. O., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+resistance+to+Meloidogyne+spp.+in+common+bean+and+the+genetic+basis+of+its+sensitivity+to+temperature.">Inheritance of resistance to Meloidogyne spp. in common bean and the genetic basis of its sensitivity to temperature.</a> Theor. Appl. Genet. 83, 720-726 (1992).</li><li>Omwega, C. O., Thomason, I. J., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nondestructive+technique+for+screening+bean+germplasm+for+resistance+to+Meloidogyne+incognita.">A nondestructive technique for screening bean germplasm for resistance to Meloidogyne incognita.</a> Plant Dis. 72, 970-972 (1988).</li><li>Perry, R. N., Moens, M., Starr, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Root-Knot Nematodes. , (2009).</li><li>Starr, J. L., Cook, R., Bridge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Resistance to Parasitic Nematodes. , (2002).</li></ol>

我们什么都没有透露。

有两个关键步骤为一个成功的线虫屏幕：一个高度感染接种和使用植物在适当的发展阶段的准备。根结线虫卵孵化率是充满变数和范围介于5 - 50％。因此，要获得最佳水平的舱口和高度传染性J2S必须支付给鸡蛋提取和孵化过程中，密切关注。鸡蛋应为最短的时间内暴露漂白和漂白剂应冲洗根混合。孵蛋时，泥浆含有鸡蛋不应该被淹没在水中。此外，避免蔓延到鸡蛋孵化的幼鱼收集底层的培养皿。避免溢出的鸡蛋在孵化过程中的一种方式是不加水直接到Kimwipe的文件有可能打破。而不是直接加水的培养皿中。 为了达到最佳效果，请使用新鲜孵化J2S。如果在hatch的速度低，需要更多的接种，J2S可以储存在15°C一段较长的时间。暖存储接种到室温复苏之前接种线虫。但是，不要长时间存放，J2S即使在15°C为饥饿J2S不会感染有效。 幼苗是最好的根结线虫接种的发展阶段。然而，这需要形成足够的根系统提供足够数量的根尖作为切入点，J2S平衡。保持最佳的植物生长条件也是至关重要的屏幕。避免过度浇水，特别是那些生长在袋。过度浇水袋，站立袋底部的水，能促进霉菌的生长，减少根健康。 与这两种检测方法的进一步评价线虫感染和鸡蛋/根系统和鸡蛋的数量计算/克的频率SH根可以执行。为番茄的检测中，个别基层称重和鸡蛋中提取描述为接种准备（第3节）。当处理大量的植物样本，个别根系统可以浸渍鸡蛋提取使用搅拌机。鸡蛋的数量应计算在至少三个等分和蛋/克鲜根系统计算。根系统的邮袋检测，被拉到纸张插入，称重，鸡蛋中提取。

<table border="1">
	<tbody>
		<tr>
			<td>Compound </td>
			<td>Concentration </td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Ca(NO3)2 &bull; 4 H2O</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgSO4 &bull; 7H2O</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Hamp-ene Fe EDTA 13.0%</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>46 &mu;M</td>
		</tr>
		<tr>
			<td>MnCl2 &bull; 4H2O</td>
			<td>9 &mu;M</td>
		</tr>
		<tr>
			<td>ZnSO4 &bull; 7H2O</td>
			<td>0.00076 &mu;M</td>
		</tr>
		<tr>
			<td>CuSO4 &bull; 5H2O</td>
			<td>0.00032 &mu;M</td>
		</tr>
		<tr>
			<td>(NH4)6Mo7O24 &bull; 4H2O</td>
			<td>0.00016 &mu;M</td>
		</tr>
	</tbody>
</table>
Table 1. Reagents for Hoagland's solution.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent </td>
			<td>Company </td>
			<td>Catalogue number </td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Erioglaucine</td>
			<td>Fisher</td>
			<td>AC22973-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miracle-Gro for tomato</td>
			<td>Scotts Miracle-Gro</td>
			<td>&nbsp;</td>
			<td>18-18-21</td>
		</tr>
		<tr>
			<td>Osmocote</td>
			<td>Scotts Miracle-Gro</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Seedling growth pouches</td>
			<td>Mega International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sieve 25 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3886</td>
			<td>US standard #500</td>
		</tr>
		<tr>
			<td>Sieve 90 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3880</td>
			<td>US standard #170</td>
		</tr>
		<tr>
			<td>Sieve 425 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3871</td>
			<td>US standard #40</td>
		</tr>
		<tr>
			<td>Sunshine mix</td>
			<td>Sun Gro Horticulture Canada</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Table of specific reagents and equipment.

high and low throughput screens with root-knot nematodes meloidogyne spp.

根结线虫（ 根结线虫属）是专性植物寄生虫。他们是非常杂食性，并认为经济最重要的植物寄生线虫。第二阶段的微观少年（J2）蛋一次molted，是感染阶段。在J2S从卵孵化，在土壤内的水膜自由移动，并找到合适的植物品种的根尖。植物根系穿透后，他们对血管缸迁移，在那里建立一个饲养的网站，并开始喂养使用他们的口针。多细胞喂养的网站是由几个扩大多核细胞被称为“巨细胞”，形成从细胞进行有丝分裂（有丝分裂重复）没有胞质。邻近的中柱鞘细胞分裂和扩大规模产生典型的胆或根结，根结线虫感染的特点症状。一旦开始喂养，J2S成为久坐和联合国的三个额外molts dergo成为成年人。成年雌虫在根的表面上或下方的凝胶状基质150-250鸡蛋。从卵孵化新感染J2S并开始一个新的周期。的根结线虫的生命周期在4-6个星期内完成，在26-28°C。 在这里，我们目前的感染植物，生长在花盆里，根结线虫和两个高通量检测方法，传统的协议。用于小如番茄种子植物的第一个高通量的方法，而第二大种子植物如豇豆和菜豆。大粒种子，支持延长幼苗生长，以最小的营养补充品。第一个高通量检测采用幼苗生长在托盘，在沙子，而在第二个实验植物生长在土壤的情况下袋。幼苗生长袋是由一个15.5×12.5厘米纸灯芯，顶部折叠，形成一个2厘米深的槽，放置在种子或幼苗。 “纸灯芯包含一个透明塑料袋内。这些增长袋允许直接观察线虫感染症状，根和鸡蛋的大规模生产的拉毛，下一个透明袋的表面。这两种方法都允许使用的筛选植物，分型后，横过马路或在种子生产。增长袋使用一个额外的好处是因为袋挂在衣架安排在塑料文件夹中存储的小空间要求。

Watch this Scientific Journal Video about High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp. at JoVE.com

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

屏幕植物根结线虫的两种不同的方法进行了阐述。所述方法包括高通量筛选，在一个非破坏性的方式，促进这些植物育种计划使用的线虫。

根结线虫的高和低通量筛选根结线虫。</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

high-low-throughput-screens-with-root-knot-nematodes-meloidogyne

Research

JoVE Journal

Immunology and Infection

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

27.7K 次观看.  University of California, Riverside. 屏幕植物根结线虫的两种不同的方法进行了阐述。所述方法包括高通量筛选，在一个非破坏性的方式，促进这些植物育种计划使用的线虫。

视频: High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

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, ...

科研

免疫与感染

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

ScanLag：高通量的菌落生长定量和滞后时间问题

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

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. ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

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 ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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 ...