该项目由赠款卫生NIAID（用于AJB和LGA，R01 AI076246）全国学院部分支持。 沙门氏菌突变收集部分被卫生补助国立研究院（对于MM们，U01 A152237-05，R01 AI07397-01，R01 AI039557-11和R01 AI075093-01），部分原因是对健康补助国立研究院（HAP的，R21支持AI083964-01，1R0 1AI083646-01，1R56AI077645，R01 AI075093）。我们感谢斯特芬Prowollik的副本电镀和确认集合中的突变体。

1，小鼠巨噬细胞巨噬细胞培养<ol><li>成长低通道数小鼠巨噬细胞，RAW264.7（ATCC得到®数，TIB-71）中的T-75细胞培养瓶中排出过滤器中的Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清的帽（FBS）的，0.5％的NaHCO 3，和1％的100×非必需氨基酸（NEAA）在37℃下，在5％CO 2的培养箱中培养。 </li><li>一旦细胞达到60-80％汇合烧瓶中，用细胞刮刀收获细胞，细胞计数的血球并计算细胞浓度。 </li><li>重悬细胞，并稀释细胞浓度为2.5×10 5个/ ml，在新鲜的DMEM细胞培养基中。使用多道移液器于板加入200μl含有5×10 4个细胞在96孔细胞培养板的每个孔的DMEM细胞培养基中。 </li><li>板四口井每沙门氏菌菌株。设立了三个独立的板块为一组吞噬细胞侵袭实验，并与“联想”，“入侵”和“复制”标记它们分别。 </li><li>放置在5％CO 2培养箱中培养板在37℃下接通，使巨噬细胞的细胞附着到孔的底部。 </li></ol> 2，制备的沙门氏菌野生型和突变体<ol><li>上制备的巨噬细胞的RAW264.7细胞的同一天，挑单菌落的野生型（WT）的沙门氏菌血清型鼠伤寒沙门氏菌14028s，DinvA突变，DphoP突变体，并且所期望的测试突变体菌株，并对其进行培养的Luria-Bertani（ LB）肉汤中添加抗生素为宜。使用每一组的吞噬细胞侵袭测定法来测试多达10个突变体菌株（DinvA突变体和DphoP突变体是有缺陷的侵入和细胞内复制，分别）。 </li><li>生长细菌14小时，在37℃以s以220rpm在圆底管克竞在每个LB肉汤5毫升在松瓶盖14毫升聚丙烯。 LB培养基中含有适当的抗生素，在我们的案例中，大多数突变株耐卡那霉素，50微克/毫升</li><li>第二天，另外4小时振摇在220rpm传代培养每一个的导通沙门氏菌菌株的纯5ml的LB培养液以1:50的比例的培养物。 </li><li>阅读外径每个细菌培养价值600分光光度计上，每读取范围应为0.6-1.2，优化沙门氏菌毒力岛1 III型分泌系统的入侵（SPI-1 TTSS）基因的表达。 </li><li>使用1 OD式600 = 7.5×10 8 CFU / ml的估算细菌浓度，然后稀释细菌为5×10 6 CFU / ml，在新鲜的DMEM细胞培养基中的浓度。 </li></ol> 3，侵袭实验在96孔培养板<ol><li>删除3 96孔CEL从CO 2培养箱升培养板和洗涤各孔一次，用200微升的1×PBS缓冲液中。 </li><li>洗涤并完全除去PBS后，加入200μl的细菌在DMEM培养基（见上文）到每个孔中。这导致在1×10 6个沙门氏菌属细胞在各孔中，感染复数为20的（MOI）：1。 </li><li>离心反应板在1,000 XG在一个密封的载体为10分钟，然后在37℃，5％CO 2培养箱更换板（零时间）为30分钟。对于每种菌株，设置至少四个重复孔中。 </li><li> 30分钟后，从培养箱中取出盘子和洗三次，加入200μl1×PBS中，以除去未关联的细菌。 </li><li>洗涤后，保存标有“协会”接受进一步治疗的板块。加入200微升含有100微克/毫升庆大霉素到标有“入侵”和“复制”的板的每个孔中的DMEM培养基，以吨Ø杀死沙门氏菌外，返回板块至37℃，5％CO2 培养箱额外小时。 </li><li>保存一个孔从每个感染的菌株，用于记录巨噬细胞数，治疗的“关联”板用200μl1×PBS中的含有1％Triton X-100的10分钟，孔的剩余部分。海卫的解决方案将裂解巨噬细胞的细胞并释放相关的沙门氏菌 。 </li><li>嘉实沙门氏菌从每口井，把它们用1.5毫升的Eppendorf管中。从每个孔收获的样品进行3个10倍连续稀释液与900μL的1×PBS中，每个稀释度之间进行涡流。 </li><li>板第三稀释，10-3，在任磅（WT）或LB侃（突变体）板。标记与相应的沙门氏菌菌株名称，稀释号码，时刻和日期的平板上。 </li><li>涡旋第三稀释管后，取10微升样品在琼脂和重复f的表面上我们的时代，共5滴。请务必空间五10微升滴均匀地涂在培养皿板7。 </li><li>滴后浸泡到琼脂中，在转动板孵育过夜，在37℃，5％CO 2的培养箱中培养。 </li><li>治疗保存为巨噬细胞用含有0.25％胰蛋白酶-EDTA的10分钟加入150μl1XPBS的数量的井。 </li><li>胰蛋白酶消化后，将巨噬细胞用1.5毫升的Eppendorf管中，并把它们与50微升的FBS中和胰蛋白酶/ EDTA溶液，将细胞染色用0.4％台盼蓝，并通过使用血球得分它们。 </li><li>当1小时孵化过后，从CO 2培养箱中删除“侵略”和“复制”板块，并洗好每一个为“3.4步”。 </li><li>拯救“入侵”板块作进一步处理“工序3.6-3.12”。 </li><li>加入200μl的DMEM培养基中含有10微克/毫升庆大霉素到在“复制”板块，以维持细胞外介质中沙门氏菌的间隙。返回板以37℃，5％CO 2培养箱中培养额外22.5小时。 </li><li>第二天，37℃，5％CO2培养箱中取出“复制”字牌，而每次洗以及“3.4步”，并把他们当作“步骤3.6-3.12”。 </li><li>最后，过夜培养后取出琼脂平板在37℃培养箱中培养和得分的细菌菌落的数量。 </li><li>良好的菌落分布应为10至100个菌落，每个点约含有2〜20个菌落，这将是很难的得分，如果有超过20个菌落在一个点上。如果计数太低或太高，replate与样品需要10倍的更高或更低的稀释液。 </li></ol> 4，数据分析<ol><li>计算基于所镀的菌落形成单位（每毫升菌落形成单位），每块板的体积和稀释因子。 </li><li>通过从每种菌株的记录，巨噬细胞的细胞计数确定每巨噬细胞的沙门氏菌的数量。 </li><li>从为小区关联，浸润或复制，至少三次独立实验计算每巨噬细胞菌落形成单位的几何平均数分别，进一步正常化到WT的相对值。 </li><li>分别分析用Student t检验进行关联，侵袭和复制的数据，通过比较各菌株与WT，并确定由p值的统计学意义。 </li></ol>

<ol>
	<li>Haraga, A., Ohlson, M. B., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonellae+interplay+with+host+cells.">Salmonellae interplay with host cells.</a> Nat Rev Microbiol. 6, 53-66 (2008).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria+and+antibiotics.">Interaction of intraleukocytic bacteria and antibiotics.</a> The Journal of clinical investigation. 52, 1673-1679 (1973).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-420 (1994).</li><li>Elsinghorst, E. A., Weitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+invasion+and+adherence+directed+by+the+enterotoxigenic+Escherichia+coli+tib+locus+is+associated+with+a+104-kilodalton+outer+membrane+protein.">Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein.</a> Infect Immun. 62, 3463-3471 (1994).</li><li>Behlau, I., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PhoP-repressed+gene+promotes+Salmonella+typhimurium+invasion+of+epithelial+cells.">A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.</a> J Bacteriol. 175, 4475-4484 (1993).</li><li>Hueck, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+secreted+invasion+determinants+are+homologous+to+Shigella+Ipa+proteins.">Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.</a> Mol Microbiol. 18, 479-490 (1995).</li><li>Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+epithelial+cells+with+Salmonella+enterica.">Infection of epithelial cells with Salmonella enterica.</a> Methods Mol Biol. 431, 201-211 (2008).</li><li>Foster, J. W., Spector, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Salmonella+survive+against+the+odds.">How Salmonella survive against the odds.</a> Annu Rev Microbiol. 49, 145-174 (1995).</li><li>Edwards, A. M., Massey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+cells+by+a+bacterial+pathogen.">Invasion of human cells by a bacterial pathogen.</a> Journal of visualized experiments JoVE. 10, (2011).</li><li>Molinari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+played+by+the+group+A+streptococcal+negative+regulator+Nra+on+bacterial+interactions+with+epithelial+cells.">The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.</a> Mol Microbiol. 40, 99-114 (2001).</li><li>Van der Velden, A. W., Lindgren, S. W., Worley, M. J., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+pathogenicity+island+1-independent+induction+of+apoptosis+in+infected+macrophages+by+Salmonella+enterica+serotype+typhimurium.">Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium.</a> Infect Immun. 68, 5702-5709 (2000).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet Pathol. 39, 200-215 (2002).</li><li>Lawhon, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+SPI-1+secreted+effectors+in+acute+bovine+response+to+Salmonella+enterica+Serovar+Typhimurium+a+systems+biology+analysis+approach.">Role of SPI-1 secreted effectors in acute bovine response to Salmonella enterica Serovar Typhimurium a systems biology analysis approach.</a> PLoS One. 6, e26869 (2011).</li><li>Drecktrah, D., Knodler, L. A., Galbraith, K., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Salmonella+SPI1+effector+SopB+stimulates+nitric+oxide+production+long+after+invasion.">The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion.</a> Cell Microbiol. 7, 105-113 (2005).</li></ol>

We have nothing to disclose.

在庆大霉素保护法被广泛地用于研究的侵袭宿主细胞内的细胞内细菌病原体和复制，并且它是特别重要的生物学工具用于研究的病原体，如沙门氏菌 ，其侵袭是用于建立感染1的前提步骤。 沙门氏菌研究界的标准庆大霉素保护法在24孔培养皿5来实现。虽然使用48或者甚至96孔板中之前讨论的用于高通量能力9，没有实际的协议已被证明在细节和成功地用于测试?...

非伤寒沙门氏菌是所有脊椎动物肠道疾病的重要原因。沙门氏菌在人类是顶级的细菌经由食物传染的疾病，1间。该支撑沙门氏菌的互动与他们的动物宿主的分子机制的表征是通过沙门氏菌血清型鼠伤寒沙门氏菌（STM）在感染的组织培养和动物模型的研究，主要实现的。争取在STM-宿主相互作用的见解将帮助我们了解沙门氏菌如何生存和发展的宿主细胞内。在研究这些相互作用的第一个挑战是要找出尽可能多的参与因素可能来自宿主与病原体，但这些努力在很大程度上阻碍了两个独立的复杂生物系统同时处理， 即主机和沙门氏菌的显著困难， 在生理条件下。此外，大repert沙门氏菌 oire和宿主基因可能编码参与宿主相互作用的因素需要高通量生物平台来解决这个难题。 一种改进的，标准化的检测表型沙门氏菌的关联，入侵和复制的巨噬细胞具有高吞吐能力的开发，研究大组基因可能参与沙门氏菌 -host互动。庆大霉素保护法于1973年2开发的，但第一次彻底Elsinghorst在1994年3,4描述。如今，它已成为研究细胞内的许多细菌病原体在体外 ，包括沙门氏菌 5,6的标准工具。内在的细菌避免被打死一些抗生素，如庆大霉素，无法穿透真核细胞3。通过利用这种现象的优点，该庆大霉素保护法测量细胞内巴的存活和生长cterial病原体。在感染过程中三个事件， 即与真核细胞浸润和复制相关联，可以评价为基于感染，庆大霉素处理，并进一步温育（ 图1）之间的时间间隔内的细菌病原体。真核细胞系提供了生理环境比相关的动物模型宿主 - 病原体相互作用的研究那么复杂。 在庆大霉素保护测定是适当的平台来研究STM-宿主的相互作用，但在24孔培养皿中的标准测定具有低吞吐量。 体内数据集的计算分析确定的被预测到大约300宿主基因产物（未发表数据）进行交互149 沙门氏菌基因产物。标准庆大霉素保护测定不具有有效地表现型的突变体的这一数量的能力。 在乳房清洁度离子，庆大霉素保护法理论上可以检测到入侵，甚至一个单一的细菌。因为这个固有的敏感性，当在不同时间重复的原始数据是容易的技术差异。内部控制及标准化后的相对数据呈现对于结果的有意义的解释是必不可少的。鉴于这些考虑，改良，标准化庆大霉素保护法的开发，以提高检测能力，提高精确度。 下面的协议是详尽和图示来执行使用96孔培养皿中，并在小鼠巨噬细胞的RAW264.7细胞系的改性庆大霉素保护测定。相比于在24孔培养皿中的标准协议，修改后的协议具有以下优点：1）使用96孔培养皿允许多达10个不同的突变体菌株进行表型，包括以足够的统计功率内部阳性对照和阴性对照;2）结果的方差显著降低，因为该突变株在相同的培养皿中，并在同一时间进行测试; 3）采用多道移液器的增加吞吐量，同时减少操作员的疲劳。最后，比较于24孔培养皿中，每孔少的宿主细胞在96孔培养皿中的关注通过协议优化和标准化得到解决。 在摘要中，改性的，标准化的检测，以在体外表现型沙门氏菌或其他细菌的关联，侵袭和复制在吞噬细胞中增加精度，并实现高通量的能力，同时降低了操作者的疲劳。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:173px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11965</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Fetal bovine serum</td>
			<td style="width:173px;">HyClone</td>
			<td style="width:173px;">SH30910.03</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">T-75 cell culture flask vented filter cap</td>
			<td style="width:173px;">Nest Biotechnology</td>
			<td style="width:173px;">708003</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">100X Non-Essential Amino Acids</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11140</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Cell scraper</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">353086</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">96-well cell culture plate</td>
			<td style="width:173px;">Corning Incorporated</td>
			<td style="width:173px;">3595</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">Luria-Bertani (LB) broth</td>
			<td style="width:173px;">MP Biomedicals</td>
			<td style="width:173px;">3002-075</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">352059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">PBS pH7.4 (1x)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">10010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Triton X-100</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Kanamycin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">K0254</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Gentamicin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">G1272</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">0.25% Trypsin-EDTA</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">25200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Trypan blue</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput assay to phenotype salmonella enterica typhimurium association, invasion, and replication in macrophages

沙门氏菌是人畜共患的病原体，并在人类和牲畜1食源性疾病的主要原因。了解潜在的沙门氏菌 -host相互作用的机制是很重要的澄清沙门氏菌感染的分子发病机制。在庆大霉素保护测定表型沙门氏菌协会，侵袭和复制在吞噬细胞被适配为允许高通量筛选中使用的RAW 264.7鼠巨噬细胞宿主的相互作用限定的沙门氏菌血清型鼠伤寒沙门氏菌的缺失突变体的作用，在这种协议中，方差在测量相对于标准协议显著降低，因为野生型和多种突变株所用的相同的培养皿中，并在同一时间进行测试。使用多道移液器的增加的吞吐量并提高精度。此外，关注与使用较少豪每孔ST细胞在96孔培养皿中分别处理。在此，修改后的体外沙门氏菌侵袭使用吞噬细胞检测的协议被成功地应用到表型38个人沙门氏菌缺失突变体的关联，入侵和细胞内复制。 在体外表现型呈现，其中一些随后被证实具有体内表型的动物模型。因此，修饰的，标准化的检测，以表型沙门氏菌协会，侵袭和复制中的巨噬细胞与高通量的能力，可以利用更广义地来研究细菌-宿主相互作用。

见代表性结果（ 图2）中的数据是基于改性的吞噬细胞侵袭测定作图后。该数据包括五个不同的菌株，WT，ΔinvA，ΔphoP，突变体A和突变体BΔinvA的 ，已知为有缺陷的入侵，并ΔphoP已知为有缺陷的复制8，被用作阳性对照，以评估试验有效性。的确，在本变形侵袭测定法，ΔinvA突变甚少内在由RAW264.7细胞和ΔphoP突变已24小时后减少复制。突变体A和B?...

Watch this Scientific Journal Video about High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages at JoVE.com

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

高通量测定法在体外表现型沙门氏菌或其他细菌的关联，侵袭和吞噬细胞与高通量能力的复制被开发。该方法来评估沙门氏菌基因敲除突变株的宿主-病原体相互作用的参与。

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

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

high-throughput-assay-to-phenotype-salmonella-enterica-typhimurium

Research

JoVE Journal

Immunology and Infection

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

12.5K 次观看.  Texas A&M University. 高通量测定法在体外表现型沙门氏菌或其他细菌的关联，侵袭和吞噬细胞与高通量能力的复制被开发。该方法来评估沙门氏菌基因敲除突变株的宿主-病原体相互作用的参与。 

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

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

一个用于标识Allelotyping PCR扩增沙门氏菌肠炎血清型，哈达尔，海德堡和鼠伤寒沙门氏菌

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

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 ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

一个协议感染<em&gt;秀丽隐杆线虫</em&gt;与<em&gt;鼠伤寒沙门氏菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

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 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 ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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 Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...