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>

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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细菌突变体的集合。这里，修饰和标准化协议被开发为使用96孔培养皿，以表型沙门氏菌突变体。值得注意的是，该协议只检测沙门氏菌和吞噬细胞，从而改进和优化显然是有必要的行吟诗人<html> ř细菌或宿主细胞。 有几个优点改性庆大霉素保护测定表型沙门氏菌协会，侵袭和复制中的吞噬细胞。首先，在96孔培养皿中的格式允许高通量的检测和分析，多达10种不同菌株能同时具有足够的重复统计分析和内部控制进行测试。第二，因为该测定法的灵敏度高，对庆大霉素保护测定法的原始数据时，可能会在不同的时刻反复易受技术差异。根据此修改方案，一些菌株在相同条件下的表型在整个过程中，降低方差。最后，使用多道移液器的提高效率，降低操作者的疲劳。新协议促进了快速分型众多沙门氏菌突变而产生大量的定量的，可再生的数据。 ove_content"&gt;在做侵袭测定法在96孔培养皿中的一个问题是，各孔含有较少的真核细胞，并且因为这个定量可能会受到影响。为了解决这个问题，进行了大的系列实验，以优化可重复性。初在这个协议中所使用的RAW264.7巨噬细胞中有不到20次传代，并且在测定之前，将细胞接种于96孔培养皿过夜其次，MOI降低到20:从经常使用50 1:1或100:1的MOI 20:1，确保99％的巨噬细胞会通过10至30的细菌，根据上述随机点由泊松分布来计算暴露于感染据认为，不限定于生物因子，细胞损伤可能是从压倒细菌引起的物理触点10此外， 沙门氏菌入侵能诱导细胞凋亡的巨噬细胞，这正与高MOI 11关联使用20的MOI:1，蜂窝damagë被发现是最小的，这可能是由于有限的细菌细胞巨噬细胞的物理接触。第三，温育时间间隔是三个时间点的优化:协会30分钟无庆大霉素;入侵另外1小时（总1.5小时后的感染），100微克/毫升庆大霉素;额外22.5小时（共24小时后感染）的复制与10微克/毫升的庆大霉素。在我们的试验中，5微克/毫升庆大霉素足以杀死所有的沙门氏菌细胞的DMEM，10％胎牛血清（未发表数据），从而，100微克/毫升庆大霉素可以预期，以确保快速杀灭和10克/毫升庆大霉素将维持胞菌在细胞培养基中过夜培养的间隙在体内 ，它需要大约15分钟，以检测组织相关联的沙门氏菌用小牛肠上皮细胞12,13; 在体外孵育30分钟被报告给是足够的，野生型<eM&gt;入侵14。对于标准侵袭测定中，前两个时间点的工作负荷是由于冲洗，连续稀释并镀相当激烈。对每个时间点的精确时间间隔是通过利用多通道移液管来实现，因为该装置显著增加了操作者的工作效率和精度。此外，将样品镀法是改变的，而不是镀覆100微升，并手动散布，5个独立的10微升液滴铺板于井平板上的每个重复这大大降低了电镀时间，并增加了精确度连续稀释后，因为最终计数每孔中拿下5镀样品。总的来说，这些优化和标准化程序增加了测定的重现性，不同的运营商，可以重复获得在不同的时间一致的结果。 该协议采用的吞噬细胞，而不是上皮细胞的该测定在一个较小的96孔格式。在研究沙门氏菌病的发病机制，使用最广泛的上皮细胞系是Caco-2细胞，从异质上皮结肠直肠腺癌而得。使用与Caco-2细胞的常规24孔培养皿中的入侵检测，回收细菌在每个时间点的实际数目，特别是用于表型复制了22.5小时的时间点，比当低得多的吞噬细胞，如J774或RAW264 0.7巨噬细胞中，使用（未发表数据）。因此，这使得它更难以用时各孔中具有较少的宿主细胞的96孔培养皿中进行表型分型。已知的是巨噬细胞能积极地吞噬细菌，这可能导致更多的细菌被内在化，并且它也不能排除沙门氏菌显然在Caco-2细胞复制速度越慢。 沙门氏菌生存和复制的巨噬细胞内的细胞的能力是在发病机制中涉及到的关键角色沙门氏菌病， 即引发大规模的主机炎症反应和促进全身感染1。我们主要采用的改良和标准化庆大霉素保护法，以表型的大集合运算流水线选定的基因他们在宿主相互作用的潜在约定。它变成了接近50％的沙门氏菌突变体的表型被作为参与细胞内的复制中的吞噬细胞的基因。

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><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11965</td><td></td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30910.03</td><td></td></tr><tr><td>T-75 Cell culture flask vented filter cap</td><td>Nest Biotechnology</td><td>708003</td><td></td></tr><tr><td>100x Non-Essential Amino Acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>Cell scraper</td><td>BD Falcon</td><td>353086</td><td></td></tr><tr><td>96-well Cell culture plate</td><td>Corning Incorporated</td><td>3595</td><td></td></tr><tr><td>Luria-Bertani (LB) broth</td><td>MP Biomedicals</td><td>3002-075</td><td></td></tr><tr><td>14 ml Polypropylene Round-Bottom Tube</td><td>BD Falcon</td><td>352059</td><td></td></tr><tr><td>PBS pH 7.4 (1x)</td><td>Life Technologies</td><td>10010</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>Kanamycin solution</td><td>Sigma</td><td>K0254</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1272</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>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是使用此协议检测代表株。突变的A显示显著减少复制（ 图2），类似于ΔphoP突变体 ;有趣的是，突变的B显示（ 图2），在复制过程中的显著增加。这些数据清楚地表明这两个墓塔NTS已经改变表型的入侵和生存的巨噬细胞。 总之，许多沙门氏菌缺失突变体的单独的表型，使用修改后的侵袭测定参与细菌的关联，侵袭和复制在RAW264.7巨噬细胞。 38测试突变体二十迄今在巨噬细胞中（ 表1），由此将改性实验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了精确度和高通量的能力，同时细菌感染显示出的变量，但显著缺陷降低了操作者的疲劳。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51759/51759fig1highres.jpg" width="500" /> 高通量检测图1。工作方案。庆大霉素保护法是莫迪田间，标准化至在96孔板中同时表现型众多沙门氏菌突变体。执行的关键步骤与上述研究协会， 沙门氏菌入侵和复制的巨噬细胞巨噬细胞的细胞。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51759/51759fig2highres.jpg" width="500" /> 图2代表性的成果的高通量测定法进行沙门氏菌菌株- WT，ΔinvA，ΔphoP，突变体A和突变体B -在巨噬细胞巨噬细胞的细胞。对于每个菌株，每个细胞巨噬细胞菌落形成单位的几何装置从为小区关联，侵袭和复制，分别三次独立实验，这是进一步标准化为WT和图形表示示出获得的。误差线表示标准误差，以及各菌株的统计显着性引用向WT则分别通过t检验来确定。 * P &lt;0.05。 MOI = 20。 <img alt="表1" fo:content-width="4in" src="/files/ftp_upload/51759/51759table1.jpg" width="400" /> 表1表型的候选基因。候选沙门氏菌突变体的改良，标准化法的关联，入侵和复制的巨噬细胞巨噬细胞的表型。 20突变体有关联，侵及/或复制显著缺陷。

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

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

12.6K 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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

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

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

免疫与感染

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

正向遗传学屏风使用巨噬细胞识别<em&gt;弓形虫</em&gt;基因的重要的抗IFN-γ依赖性细胞免疫自主

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

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 ...

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. ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

在沙门氏菌感染组织培养细胞中，NF-βB依赖性路西酶的激活和基因表达的定量

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

使用 ImageJ 自动分析 沙门氏菌 的细胞内表型

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

An In Vitro Assay to Quantify the Intracellular Growth of Parasite in Macrophages

Source: Sarkar, A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.</a> J. Vis. Exp. (2018).
This video demonstrates an assay to quantify the intracellular growth of parasites in bone marrow-derived macrophages (BMMs). The macrophage cells are infected with the motile and non-motile invasive stages of Leishmania, metacyclic promastigotes and amastigotes, respectively. The parasites invade the macrophages, getting internalized into a parasitophorous vacuole and multiplying inside. The intracellular parasite growth is quantified using a microscope upon fluorescently staining the macrophage and parasite nuclei.

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

1. Plating BMMs on Coverslips for Infection 

	Prepare 6-well tissue culture plates by placing four (4) sterile 12 mm glass coverslips (autoclaved and ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

Source: Meunier, E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52960">Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization.</a> J. Vis. Exp. (2015).
This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin, leaving the SCV intact. Upon labeling the cytosolic bacteria with a fluorescently tagged antibody, the percentage of vacuolar and cytosolic bacteria is quantified using fluorescence-activated cell sorting (FACS).

高通量检测到的表型

摘要

探索更多视频

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

正向遗传学屏风使用巨噬细胞识别

胞浆的定量与液泡

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

在沙门氏菌感染组织培养细胞中，NF-βB依赖性路西酶的激活和基因表达的定量

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

使用 ImageJ 自动分析沙门氏菌的细胞内表型

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

定量感染原代巨噬细胞中胞质和液泡沙门氏菌的测定法

高通量检测到的表型

摘要

探索更多视频

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

正向遗传学屏风使用巨噬细胞识别

胞浆的定量与液泡

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

在沙门氏菌感染组织培养细胞中，NF-βB依赖性路西酶的激活和基因表达的定量

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

使用 ImageJ 自动分析 沙门氏菌 的细胞内表型

一种定量巨噬细胞中寄生虫细胞内生长的体外测定

定量感染原代巨噬细胞中胞质和液泡沙门氏菌的测定法

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

使用 ImageJ 自动分析沙门氏菌的细胞内表型