We would like to thank the entire Lazzaro lab, and especially Susan Rottschaefer, for their help in both reviewing and testing these protocols. This is a product of their cumulative expertise. Work in the Lazzaro lab is supported by grants R01 AI083932 and R01 AI064950 from the US National Institutes of Health.

1.收集和准备苍蝇<ol><li>后排D.所需的实验条件下果蝇 。小心不要饲养过程中过度拥挤的苍蝇和确保幼虫密度在整个治疗一致的，因为在幼虫阶段的环境条件下可以在成年阶段深刻地影响免疫防御的表型。15 </li><li>收集实验苍蝇0 - 羽化后3天从蛹壳，并将其转移到新鲜的培养基。 </li><li>房子所收集的蝇在所需的温度（温度之间22°C和28°C通常是合适的），直到它们被熟化5 - 后7天羽化。 注：这允许足够的时间过得完成蜕变，成为成熟的成年人，但也衰老开始之前。 </li><li>苍蝇的期望数量的分类到感染前的单独小瓶中，再次注意避免过度拥挤。 注：只有马文件被感染在此示范但同样可能感染使用描述的方法的女性。 </li></ol> 2.文化和准备细菌<ol><li>准备选择的​​细菌至少2天前感染的主板块。条纹从15％甘油贮存在-80℃下无限期储存细菌。存储主板在4℃下长达2周。始终连胜主板直接从冷冻甘油股票。避免细菌的串行通道从板到，在文化路重复可造成细菌进化毒力减弱。 注：本示例利用雷氏普罗威登斯菌和大肠杆菌 。 </li><li>使用以下步骤准备菌悬液注射。 <ol><li>通过接种无菌介质单菌落从主隔离板成长2 ml培养的细菌。生长的细菌固定相（ 如 </em&gt; O / N的增长，在37°C）。 注：两个P.雷氏普罗威登斯菌和大肠杆菌的Luria肉汤中生长良好，在温度为20 - 37℃下柔和摇动。 </li><li>一旦培养达到稳定期，轻轻地从约沉淀细胞。 600微升的培养物在台式离心机（3分钟，在5000×g离心），弃上清，重悬的细菌大约1000微升无菌磷酸盐缓冲盐水（PBS; 137mM的氯化钠，2.7毫米氯化钾，10毫的Na 2 PO 4，1.8毫KH 2 PO 4，pH值= 7.4）。 </li><li>稀释的悬浮细胞在无菌PBS以达到光密度（OD）适当的细菌被使用，用分光光度计作为吸光度在600nm测定。使用此悬挂感染的苍蝇。 注：600 = 0.1或1.0分别对应于约10 8和10 9 雷氏普罗威登斯菌或E.每毫升大肠杆菌 。由于两人住一第二死细菌有助于光密度，它在固定相早期收获培养细菌尸体积累之前和歪曲光密度和感染过程中引入的活菌数之间的关系是很重要的。 </li></ol></li></ol> 3.感染蝇注：由于果蝇的免疫受昼夜节律的影响，它在整个实验重复每天的同一时间进行感染是非常重要的16。 3.1）用Nanoinjector <ol><li>准备玻璃针感染。 <ol><li>通过使用微量拉马拉硼硅玻璃毛细管制成玻璃针。 </li><li>使用镊子，断绝针的尖端，以产生大约50微米直径的开口，以允许液体喷射。 </li></ol></li><li>组装的注射器。 <ol><li>将密封O形环，然后将白色菌宏碁（大酒窝朝外）在金属活塞。 </li><li>填充玻璃针用矿物油使用与地下30针的注射器。 </li><li>把填充玻璃针穿过筒夹，然后将较大的O形环围绕其基部，大约从针的钝端1毫米。 </li><li>针滑入金属柱塞，轻轻拧上夹头，直到安全。 </li><li>喷射大多数矿物油从喷射器，但要确保仍有一小体积的油中的针充当喷射器和细菌悬浮液之间的屏障。确保有矿物油或细菌悬浮液内没有气泡，或两种液体之间。 </li><li>喷射器设置成用于注射的所需体积（9 NL和50 NL之间）。 </li></ol></li><li>产生伤人的控制。 <ol><li>通过仔细插入毛细管针的尖端在媒体和pressi的管填充注射器针头用无菌PBS纳克在喷油器的“填充”按钮。 注：无菌细菌生长培养基也可以使用一个伤人控制，如果实验需要注射的细菌悬浮于它们的生长培养基中。但是，由于细菌的生长介质包含部件可以刺激免疫系统或具有在主机上其它影响，惰性载体如PBS中是优选的。 </li><li>麻醉下的 CO 2的光流的苍蝇的期望数量。 </li><li>注入上腹外侧表面与无菌PBS苍蝇于前腹部。 注意：也可以注入苍蝇在其它部位，如胸廓sternopleural板，但为了保持注射部位各实验内一致是很重要的17。 </li><li>将注入苍蝇到新鲜小瓶新媒体，铺设在自己身边的小瓶，直到所有的苍蝇都从麻醉中恢复，以防止苍蝇从被卡的食物。&lt;BR /&gt;注：建议将注入细菌实验苍蝇所以同样的针可以同时用于治疗前注入PBS控制苍蝇。它并不总是有可能使用相同的针对整个实验。在这种情况下，可能希望记录哪些针被用来与飞并包括作为实验因子在统计分析针身份。 </li></ol></li><li>注入细菌病原体。 <ol><li>喷射从喷射剩余的无菌介质和填充同一针与细菌悬浮液。 </li><li>重复上述步骤（3.1.3），现在的注射用蝇菌悬液的过程中准备2.2。 </li></ol></li></ol> 3.2）感染性针刺<ol><li>准备针刺。 <ol><li>融化200微升微量移液器尖端的端部，并插入一个0.15毫米昆虫minutien销入熔融塑料。让塑料凝固苏通道，所述销被保持在适当位置以从塑料延伸大约0.5厘米销。 </li></ol></li><li>产生伤人的控制。 <ol><li>麻醉下的 CO 2的光流的苍蝇的期望数量。 </li><li>刺苍蝇与针胸廓sternopleural板，避免了翼和腿部的连接位点。如果有必要，温和地去除苍蝇用软钳minutien引脚。 注意：也可以刺破苍蝇在其它部位，如在腹外侧表面的前腹部，但为了保持注射部位各实验内一致是很重要的17至腹部的角质层刺更趋于难比穿刺胸廓的，因此是不常见的。 </li><li>将一竖苍蝇到一个新的小瓶新飞媒介，奠定在其一侧的小瓶，直到所有的苍蝇都从麻醉中恢复，以防止苍蝇从以下到来粘在食品。 </li></ol></li><li>介绍了细菌感染。 <ol><li>麻醉下的 CO 2的光流的苍蝇的期望数量。 </li><li>每一个刺在同一位置作为媒体控制飞行，浸渍销的尖端插入菌悬液各刺起飞前准备程序中的2.2。 </li><li>将一竖苍蝇到一个新的小瓶新飞媒介，奠定在其一侧的小瓶直到所有的苍蝇从麻醉中已经恢复，防止苍蝇从被卡的食物。 </li></ol></li></ol> 3.3）评估的感染剂量交付<ol><li>感染作为任一过程3.1或3.2以上述唯一的而不是返回苍蝇食物小瓶从麻醉中恢复，一组苍蝇，将每个飞入冰上一个离心管中。 </li><li>微升的PBS，加入250至每个管并用杵或珠打浆机均质苍蝇任&lt;/ LI&gt; <li>镀上的LB琼脂平板匀浆，或者使用一个螺旋接种仪或系列稀释。 <ol><li>制版用系列稀释，每飞匀浆转移到96孔板的第一行。 </li><li>填补剩余的行每孔90微升PBS。 </li><li>使用多道移液器，取10微升含有飞匀浆，分装到第二行的第一行。 </li><li>移液器上下至少10次调匀，再取10微升，并转移到第三排。利用剩余的行重复此过程。 </li><li>从底行（最稀的细菌悬浮液）开始，可使用多道移液器采取从每个孔中并沉积10微升在LB板上，小心不使样品被分配作为离散斑点不互相接触。重复，直到所有的井都被取样的每一行，配药他们降序排列稀释的LB平板。 </li&gt; <li>离开板在室温，直至斑​​已经完全浸透到LB平板。 注：干燥的LB平板在室温使用前几天，建议确保液体很容易被吸收，减少的样本点之间的意外接触的机会。 </li></ol></li><li>孵育板O / N，注意不要过度生长板使菌落保持小而分散。 注意：根据所用的细菌生长培养基和温育温度，从果蝇的内源性肠道微生物衍生的菌落，最终可能会出现在平板上。然而，用于病原感染大多数细菌生长速度比在37℃下在LB琼脂肠道菌群快得多。 </li><li>从孵化器中取出印版时，实验细菌生长可见菌落（一般为8 - 12小时），但在果蝇肠道微生物菌落出现（约36小时）。对于生长缓慢的细菌实验，使用选择性抗生素在LB琼脂从肠道微生物除去任何菌落。 </li><li>算为每匀浆菌落数。 <ol><li>为螺旋板，计数生长使用自动菌落计数器，可以每ml匀浆基于在板的菌落数及位置估计细菌负荷的菌落。 注：螺旋计数是最准确的，当细菌浓度在每毫升5×10 2〜5×10 4个细菌的范围内。 </li><li>现货板，手动计数每个飞从哪个稀释后含有30菌落 - 300菌落并计算每毫升原匀浆的细菌数。 </li></ol></li></ol>感染4.检定存活<ol><li>测定死亡率后喷射。 <ol><li>将受感染的苍蝇和受伤的控制在新鲜小瓶中并在培养箱中在期望的温度保持（25℃，12:12韧带HT：暗周期推荐）。不把超过15蝇成单一小瓶当它变得难以计数超过15生活苍蝇。 </li><li>每一天算生活和死苍蝇的数量，或更频繁，如果合适。 </li><li>为了保持食品质量，翻转苍蝇新的食品定期调度。 <ol><li>如果管含有只有男性，他们转移到新鲜小瓶每三天。如果样品瓶中含有女性，转移到新鲜小瓶每隔两天，因为幼虫后代将液化的食品，增加了风险成蝇可能卡住并导致死亡因​​感染高估率。 </li></ol></li></ol></li><li>统计分析数据。 <ol><li>情节生存作为采用Kaplan-Meier曲线，这表明使用对数秩t的个体将生存于测量时间点后喷射。18对于存活曲线不交叉，执行成对比较的概率EST（也称为曼特尔考克斯测试）或执行采用Cox比例风险模型，它可以包含几个因素及其相互作用更复杂的分析。对于跨生存曲线，进行分层Cox分析。17 </li></ol></li></ol> 5.测定细菌负荷感染后<ol><li>测量细菌负荷。 <ol><li>在规定的时间后喷射，重复用于评估感染剂量，以确定细菌负荷在感染过程的程序（见协议3.3）。 注意：如果使用的是螺旋接种仪，稀匀浆使菌悬液落在范围内的1000 - 100000细菌每毫升。 </li></ol></li><li>分析数据。 <ol><li>如果细菌荷载是非正常的，或者变换该数据，以便更接近正态分布或分析使用非参数统计分析（ 例如，采用Mann-Whitney U检验）的数据。使用箱Cox分析到fIND的最优变换;自然对数或碱-10日志转换往往是有效的。19 </li><li>如果数据被充分正态分布和仅存在两个被对比处理，执行t检验 ，以确定两种治疗是否导致不同的平均细菌载量。如果有多个实验变量或其他潜在预测因素，使用方差分析（ANOVA）来确定哪些因素是细菌负荷显著预测因子。19 </li></ol></li></ol>免疫系统的基因分析6转录激活<ol><li>粗略地显现免疫激活感染后，感染的转基因果蝇表达从抗微生物肽基因的启动子的控制下的绿色荧光蛋白（GFP），如前所述。18 </li><li>查看被感染的苍蝇荧光功能的显微镜下; GFP感应应该成为visib在文件中的第一个6脂肪体 - 感染后10小时。 </li><li>对于免疫激活的更精确的量化，使用的cDNA模板（定量RT-PCR）荧光定量PCR来衡量的抗菌肽基因转录的丰度。 注：免疫基因的表达可以在后（或之前）感染的任何时间进行测量。一般来说，感应免疫基因表达的，在未来24小时开始注射，并增加后约4小时。 <ol><li>麻醉蝇使用CO 2。 </li><li>将15苍蝇进入离心管的每个RNA提取。 注：建议收集至少有三个重复样品，每个处理条件。 </li><li>从果蝇提取RNA和使用任何数目的标准方法或市售试剂盒产生第一链cDNA。商店的RNA在-80℃。商店的cDNA在-20℃下的几个星期期间和在-80℃下长期贮存。之前RNA提取，存储˚F在于在-80℃。 </li><li>感兴趣使用cDNA为模板的靶基因进行定量PCR（qPCR的）。请参阅表1引物序列扩增抗菌肽基因Diptericin A，Drosomycin，防御 ， 家蝇Attacin A和Metchnikowin以及管家对照基因RP49（又称RPL32）。 注：编码抗菌肽Diptericin A和Drosomycin的基因D.提供很好的读数果蝇诱导主要通过IMD的和Toll途径免疫活性，分别。为了控制对样本-样本变化在RNA得率和反转录的效率，规范记录表达靶基因与参考管家基因的表达预期不会更改与感染如RP49（也称为RPL 32）。 </li><li>分析数据。 &lt;OL&gt; <li>用于表达的差异粗略近似，计算从测试基因获得的起始量（SQ）的值的比率（ 例如，Diptericin A）中相对于从参考基因得到的SQ值（ 例如，RP49）对于每个样品（换算为SQ - DPTA / SQ - RP49）。这些缩放比率为基线控制处理，如感染处理控制。任意设定控制表达水平等于1.0和可视化实验性治疗的相对倍数变化。估计为针对从系列稀释的已知-量的cDNA产生的标准曲线的每个基因的SQ值。 注：比例的统计分析可以是复杂的，所以这种方式是更好地为快速近似比严格的分析。如果PCR效率低，设计新的引物，以改善的PCR动力学如果可能的话。对于qPCR的反应与近乎完美的效率（广告PCR产物每个周期）的oubling，阈值循环（C T），可被取代的SQ值。 </li><li>对于简单的实验与优化有效扩增，数据可以使用ΔΔCT法进行分析。19对于每个样品，从测试基因的（C T）（ 例如，Diptericin减去参照基因的（C T）值（ 例如，RP49） A）以产生ΔC 吨 。然后减去从ΔCT表示每个实验样品以产生ΔΔC 的T值各样品的基线对照的ΔC 笔 ;这ΔΔCT是指示治疗，其中2（-ΔΔCT）近似表达的倍数变化之间的基因表达水平的相对差异。 注意：此分析可以严重misestimate折叠改变靶基因的表达，如果任一测试基因或参考的PCR基因具有低效率。 </li><li>为最严格的分析，使用测试基因（ 例如Diptericin A）中作为响应变量的和所估计的表达对照基因（ 例如，RP49）和其它实验因素作为解释变量的估计表达式进行方差分析（ANOVA）分析。 注：此方法是在PCR扩增相对强劲效率低下，并允许更复杂的实验设计分析。 </li></ol></li></ol></li></ol>

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T., 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=A+model+of+bacterial+intestinal+infections+in+Drosophila+melanogaster.">A model of bacterial intestinal infections in Drosophila melanogaster.</a> PLoS pathogens. 3 (11), e173 (2007).</li><li>Jaenike, J., Polak, M., Fiskin, A., Helou, M., Minhas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+transmission+of+endosymbiotic+Spiroplasma+by+mites.">Interspecific transmission of endosymbiotic Spiroplasma by mites.</a> Biology. 3 (1), 23-25 (2007).</li><li>Kanbar, G., Engels, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+bacterial+infection+of+wounds+in+honey+bee+(+Apis+mellifera)+pupae+punctured+by+Varroa+mites.">Ultrastructure and bacterial infection of wounds in honey bee ( Apis mellifera) pupae punctured by Varroa mites.</a> Parasitology research. 90 (5), 349-354 (2003).</li></ol>

None of the authors have competing interests or conflicting interests.

这里所描述的过程产生果蝇的严谨和高品质的感染。示出的实施例主要集中在感染雷氏普罗威登斯菌和大肠杆菌杆菌 ，但该协议是适应性强，可以适用于感染多种细菌在一定范围的宿主饲养和维护条件。 的最佳实验方法的细节将取决于用于感染的细菌，宿主的基因型，使整体的实验条件。它发起更雄心勃勃的项目之前，任何新的实验条件下，强烈建议先导性试验。一个好的起点是超过100倍的范围内测试三个剂量的感染。高毒力的病原体通常最好在非常低的感染剂量引入，10的数量级上 - 每个飞100细菌细胞。较为温和的病原体可以在较高剂量的每FL 1000左右的细菌注入y和非病原可能在剂量高达每飞万菌注射。它往往是有益的，通过跟踪病原体负载，主机死亡率，和免​​疫系统活性过一个纵向的时间序列来定义的新颖的感染动力学。因为病原体负载和宿主基因表达的测量是破坏性的测定，有必要对感染不同苍蝇在实验为每个时间点是要测量的开始。 当决定是否使用针刺或微毛细管的注塑，必须注意的有优点和局限性每种方法是重要的。毛细注射引入了一个体积的液体成飞，其中两个略有增加膨压，并引入的盐或悬浮或溶解在载体中的其它分子。毛细管注入也需要访问打针设施所需的设备或购买。化粪池针刺不需要特殊的设备和介绍可忽略的媒体成飞，并且通常为感染大量苍蝇更有效。然而，针刺感染不允许精确控制，可与毛细管注射感染实现剂量。本协议集中在机械调节喷射量的喷射装置，但也有根据加压空气的离散脉冲喷射系统。20,21，这些通常比装置这里展示更昂贵，并且需要空气脉冲到每个校准针，以确保一致的注射体积。 有相当多的争论，但是，苍蝇如何成为全身感染的细菌在野外很少的数据。一些研究者断定，大多数自然感染的发生，当果蝇摄入致病细菌和细菌随后难逃肠道建立一个全身性感染。但是，也有非常菲已知瓦特细菌能够越过D的肠果蝇 ，和那些有这种能力是非常致命的苍蝇22,23。另一种理论是，经常飞过从失败的企图掠夺或攻击外寄生螨虫逃逸维持表皮的伤害。这种假设是支持的频繁收集野生D的果蝇黑色素轴承斑点是指示愈合的伤口（未发表意见）的。螨已显示在果蝇 24发送细菌感染和由螨留下的伤口可在蜜蜂25被二次细菌感染。但是，在D的螨驱动或否则机会性感染的性质的频率通过角质层违反果蝇是未知的。这里所描述的协议允许引入细菌直接进入通过定量注射绕过任何障碍上皮或行为IMMU淋巴无穷大。方法用于馈送致病菌D.果蝇已经于Vodovar 等人 22和Nehme 等人 23进行了描述。 许多昆虫细菌造成很少或没有人类健康的风险，使研究人员能够与他们舒适地工作。此外，很少有细菌感染的果蝇实验无接触的干预能力，所以细菌感染的“流行病”蔓延通过实验室通过污染的表面上的风险或逃苍蝇通常是非常低的。不过，最好是确保有足够的遏制措施，以防止感染的苍蝇逃跑和夺回任何逃脱苍蝇。实验室应在生物安全水平与病原体相称的使用进行配备，并在微生物标准的最佳做法应被采用。 实验infecti在这里介绍的方法可以让果蝇与任意细菌任何剂量的感染。一旦感染已经成立，它是直接测量细菌增殖或间隙动力学，跟踪主机的死亡率，并测定诱导宿主免疫系统。受感染的苍蝇可以很容易进行其它的表型分析，包括生理功能可能形成或由感染被成形的试验。描述的方法是便宜的，需要相对小的专门设备，并且很容易了解到，这使得它们适合用于在研究和教学实验室广度在不同项目中使用。

在果蝇是首屈一指的模式生物用于研究功能和免疫防御。 果蝇进化是价格低廉，易于后部之一，具有高度的服从实验操作，并已开发了一系列广泛的科学界的支持阵列的研究工具。先天免疫的许多方面都昆虫和哺乳动物之间是保守，包括信号转导通过Toll样受体和NF-κB家族的转录因子，JAK / STAT信号和JNK途径的反应介导的。1,2-这些基因和途径可以的功能在D查询果蝇使用突变或RNAi击倒该增加或减少通路活动3 - 6此外， 果蝇可用于研究感染和疾病的生理后果，包括在进化生活史理论的前后关系7。- 9所有这些研究，但是，依赖于可靠地感染实验苍蝇定义的处理条件下的能力。此处描述的方法提出了用于递送健壮和可重复的细菌感染到果蝇和随后测量感染的严重程度和量化宿主的免疫反应的方法框架。 果蝇可自然和实验感染通过多种寄生虫和病原体，包括细菌，真菌，病毒，线虫和寄生蜂的。目前的协议是专注于提供全身细菌感染。许多不同的细菌可用于感染苍蝇，和实​​验者的选择应基于精确的科学问题被提出。例如，人的临床分离物可用于研究细菌毒力机制10，或生态相关菌株可以是地优选ð进化研究。11有些细菌是D.主管病原体果蝇，增殖后感染并引起宿主疾病或死亡。其它细菌被宿主的免疫系统有效地管理和在几天内清除。在此示范， 雷氏普罗威登斯菌将用作增殖性病原体，可引起宿主的死亡率和仍然存在于存活宿主。 大肠杆菌将被用来作为一个非病原体是受宿主免疫系统清除。 感染会通过引入细菌直接进入体腔的苍蝇的建立。这种方法绕过上皮屏障和保护行为，使全身感染的调查传输无关的自然方式。有两个主要方法用于实验建立系统性感染。在第一，一个nanoinjector和拉玻璃毛细管针用于注入的精确数细菌带入飞。这种方法具有允许感染剂量的大的动态范围和存在定量高重复性的优点。第二种方法是提供感染化粪池针眼。这种方法具有可快速，不需要特殊仪器的优势。一旦感染建立，变得可以测量系统性病原体负载，主机死亡率，并诱导免疫系统的活性。当然，任何数目的额外的表型可以令人信服地在感染D.测定果蝇 ，包括感染后繁殖力12，学习能力13，代谢状态14，或几乎可以想像的任何其他特征。

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Reagent</td>
			<td style="width:239px;">Company&#160;</td>
			<td style="width:177px;">Part Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Incubator</td>
			<td style="width:239px;">Powers Scientific, Inc</td>
			<td style="width:177px;">DROS52SD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paintbrush</td>
			<td style="width:239px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">CO2 Flypads</td>
			<td style="width:239px;">FlyStuff</td>
			<td style="width:177px;">59-114</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">CO2</td>
			<td style="width:239px;">Airgas</td>
			<td>CD FG50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Drosophila rearing mix&#160;</td>
			<td style="width:239px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">6 oz Square Bottom Bottles, polypropylene</td>
			<td style="width:239px;">Genesee Scientific</td>
			<td style="width:177px;">32-130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nosterile Extra Large Cotton Balls</td>
			<td style="width:239px;">Fisher brand</td>
			<td style="width:177px;">22-456-882</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Microscope</td>
			<td style="width:239px;">Olympus Corporation</td>
			<td style="width:177px;">SZ51</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Drosophila Vials polystyrene</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">89092-720</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nosterile Large Cotton Balls</td>
			<td style="width:239px;">Fisher brand</td>
			<td style="width:177px;">22-456-883</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">2L flask</td>
			<td style="width:239px;">VWR international</td>
			<td>89000-370</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Petri Dishes with Clear Lids, Raised Ridge; 100 . x 15 mm;</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">25384-302</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">LB Agar, Miller</td>
			<td style="width:239px;">Difco</td>
			<td style="width:177px;">244520</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Innoculing Loop</td>
			<td style="width:239px;">VWR international</td>
			<td>80094-488</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:399px;">Rainin Clasic Pipettes in various sizes 
			0.1 &#181;l to 2 &#181;l, 
			2 &#181;l to 20 &#181;l, 
			20 &#181;l to 200 &#181;l, 
			100 &#181;l to 1000</td>
			<td style="width:239px;">Rainin</td>
			<td style="width:177px;">PR-2 
			PR-20 
			PR-200 
			PR-1000</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:399px;">Micropipette tips (assorted sizes)</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">30128-376 
			53503-810 
			16466-008</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Broth Base, Miller</td>
			<td style="width:239px;">Difco</td>
			<td>241420</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Disposable Culture Tubes Borosilicate Glass</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">47729-576</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">S-500 Orbital Shaker</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">14005-830</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">centrifuge</td>
			<td style="width:239px;">VWR international</td>
			<td style="width:177px;">37001-300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PBS pH 7.4 10x</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:177px;">70011044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">170-2501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Semimicrovolume Cuvettes</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">223-9955</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Vertical Capillary Puller</td>
			<td style="width:239px;">Kopf Needle Pipette Puller</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">3.5&#39;&#39; Replacement glass Capillaries for Nanojet II</td>
			<td style="width:239px;">Drummond Sientific Company</td>
			<td style="width:177px;">3-000-203-G/X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Nanoject II</td>
			<td style="width:239px;">Drummond Sientific Company</td>
			<td style="width:177px;">3-000-204</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Forceps</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:177px;">11255-20</td>
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			<td height="21" style="height:21px;width:399px;">10mL Syringe</td>
			<td style="width:239px;">BD</td>
			<td style="width:177px;">309604</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mineral Oil, White, light</td>
			<td>Macron Fine Chemicals</td>
			<td>6358-10</td>
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			<td height="21" style="height:21px;width:399px;">minutein pins</td>
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			<td style="width:177px;">26002-10</td>
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			<td height="21" style="height:21px;width:399px;">1.5mL&#160; Microcentrifuge tubes; Seal Rite</td>
			<td style="width:239px;">USA Scientific Inc.</td>
			<td style="width:177px;">1615-5500</td>
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			<td height="21" style="height:21px;width:399px;">Motorized Pestle; Talboys Laboratory Stirrer</td>
			<td style="width:239px;">Troemner</td>
			<td style="width:177px;">103</td>
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			<td height="21" style="height:21px;width:399px;">Talboys High Throughput Homogenizer</td>
			<td style="width:239px;">OPS Diagnostics</td>
			<td style="width:177px;">930145</td>
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			<td height="21" style="height:21px;width:399px;">5/32&#39;&#39; Grinding Balls</td>
			<td>OPS Diagnostics</td>
			<td style="width:177px;">GBSS 156-5000-01</td>
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			<td height="21" style="height:21px;width:399px;">Vortex Genie</td>
			<td style="width:239px;">Scientific indurstries inc.</td>
			<td style="width:177px;">G560</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Multichannel Pipettor (10uL-300uL)</td>
			<td style="width:239px;">Sartorius</td>
			<td>730360</td>
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			<td></td>
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			<td height="21" style="height:21px;width:399px;">WASP2 Whitley Automated Spiral Plater</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
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			<td height="42" style="height:42px;width:399px;">ProtoCOL automated colony counter / plate counter/ plate reader&#160;</td>
			<td style="width:239px;">Microbiology International</td>
			<td></td>
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			<td height="21" style="height:21px;width:399px;">TRIzol</td>
			<td style="width:239px;">Life Technologies</td>
			<td style="width:177px;">15596-026</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR tubes; Low-Profile 0.2 ml 8-Tube Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TLS0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">qPCR caps; Optical Flat 8-Cap Strips</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">TCS0803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">RQ1 RNase-Free DNase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m610a</td>
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			<td height="21" style="height:21px;width:399px;">M-MLV Reverse Transcriptase</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">m170b</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">dNTPs</td>
			<td style="width:239px;">Promega</td>
			<td style="width:177px;">U1240</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Oligo-dT</td>
			<td style="width:239px;">IDT</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">&#160;SsoAdvanced SYBR Green Supermix</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">172-5260</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX Connect Real-Time PCR Detection System</td>
			<td style="width:239px;">Bio-Rad</td>
			<td style="width:177px;">185-5200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2115</td>
		</tr>
	</tbody>
</table>

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

在果蝇是首屈一指的模式生物用于研究免疫防御的功能和进化中的一个。先天免疫的许多方面都昆虫和哺乳动物之间是保守，并且因为果蝇可容易进行遗传和实验操作，它们是强有力的用于研究免疫系统功能和疾病的生理后果。这里展示的程序允许苍蝇通过引入细菌感染直接进入体腔，绕过上皮障碍和更被动形式防御，并允许焦点对全身感染。该程序包括用于主机死亡率，系统性病原体负荷，并诱导宿主免疫系统的程度的测量速率协议。这种感染过程是便宜，耐用和定量重复的，并且可以在功能性遗传学，进化生活史，和生理学研究中。

本节中示出了可以果蝇的细菌感染后获得的结果， 图 1显示，感染剂量与细菌悬浮液用于注射的光密度变化，并且该递送可通过均质化和电镀可靠地估计剂量立即蝇注射后。如示于图2中 ，不同的病原体可引起不同程度的宿主死亡（ 图2A）和主机死亡率可以是剂量依赖性的（ 图2B）。重要的是，此协议允许在要达到的不同类型的感染： 雷氏普罗威登斯菌可导致慢性，亚致死性感染持续20天或更长时间（ 图3A）。然而，其他细菌一样Escerichia大肠杆菌将主要由主机蝇清零6小时感染后（ 图3B）内。诱导免疫SYS的统可通过抗菌肽转录（ 图4A和B）RNA分离和随后的qRT-PCR来估计。类似但不定量，苍蝇表达GFP的抗微生物肽基因的启动子的控制下，可用于可视化诱导免疫系统（ 图4C）的。 <img alt="图1" src="/files/ftp_upload/52613/52613fig1.jpg" /> 图1：确定感染剂量苍蝇注射50 NL菌悬液覆盖范围的光学密度（0.0001 - 0.05）的。苍蝇是立即匀浆并镀以确定由喷射引入的细菌数。最初的细菌量强烈与相关初始OD注射（R 2 = 0.96） <img alt="图2" src="/files/ftp_upload/52613/52613fig2.jpg" /> <s仲&gt;图2：注射用细菌病原体后存活5-7天龄的男性注射50 NL的任一细菌悬浮液或无菌培养基并监测存活。 （A）野生型果蝇注射约5000细菌从三个不同的品种之一。主机死亡率很大程度上取决于用于感染的细菌种类。 （B）野生型果蝇注射三种不同剂量Enterococcous粪 - 50，500，每飞5000细菌。苍蝇死时更快速地以更高感染剂量（C）的感染的免疫系统突变体和野生型同基因控制线分别注射大约500 洋葱伯克霍尔德菌 。进行了配对数秩比较表明，野生型苍蝇的生存比感染突变显著更好（χ2 = 59.02，自由度= 1，P &lt;0.0001）。 图3：注射蝇后细菌负荷注射约（A） 的 5000 P.雷氏普罗威登斯菌 （B）3400 E.大肠杆菌 。细菌负荷被注射后，并在各种后续时间点立即确定。每个数据点代表单个苍蝇的细菌负荷。E.大肠杆菌是迅速从主机的挑战，同时清除P.雷氏普罗威登斯菌持续了主机的余生，。 <img alt="图4" src="/files/ftp_upload/52613/52613fig4.jpg" /> 图4：诱导免疫基因表达注射后 。 （ 一 ）果蝇注射50 NL P的雷氏普罗威登斯菌或无菌PBS中任腹部或胸部， 还是留给未经处理除了CO <sUB&gt; 2麻醉。感染后六小时后，收集为Diptericin A基因 ，使用qRT-PCR测定的RNA分离和表达苍蝇。 （A） 的表达水平进行作图，Diptericin的比率A转录到RP49转录和缩放，使得CO 2的控制被定义为具有1的表达水平条形代表比率的平均值和标准误差从每个状态（正= 4）。 （B）的表达水平进行作图，一个2ΔΔCT与用作标准未诱导状态的CO 2感知控制苍蝇的平均C 的T值。条形代表从每个条件ΔΔCT中的平均值和标准误差（4例）。虽然在转录物诱导无差异，由于注射部位，面板A和B的比较表明如何ΔΔCT法可以潜在高估诱导水平。 （C）苍蝇表达GFP下Diptericin控制的发起人注射50 NL的P.雷氏普罗威登斯菌 （OD = 0.1），然后成像后7天。 GFP的面板显示绿色荧光蛋白在感染苍蝇的腹部脂肪体的表达，表明激活Diptericin的细菌感染的果蝇启动子，但不是媒体注入控制。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 基因 </td><td> 前锋 </td><td> 反 </td></tr><tr><td> RP49（也称为RPL32） </td><td> 5&#39;AGGCCCAAGATCGTGAAGAA 3&#39; </td><td> 5&#39;GACGCACTCTGTTGTCGATACC 3&#39; </td></tr><tr><td> Diptericin一个 </td><td> 5&#39;GCGGCGATGGTTTTGG 3&#39; </td><td> 5&#39;CGCTGGTCCACACCTTCTG 3&#39; </td></tr><tr><td> Drosomycin </td><td> 5&#39;CTGCCTGTCCGGAAGATACAA 3&#39; </td><td> 5&#39;TCCCTCCTCCTTGCACACA 3&#39; </td></tr><tr><td> 防御 </td><td> 5&#39;GAGGATCATGTCCTGGTGCAT 3&#39; </td><td> 5&#39;TCGCTTCTGGCGGCTATG 3&#39; </td></tr><tr><td> 家蝇Attacin一个 </td><td> 5&#39;CGTTTGGATCTGACCAAGG 3&#39; </td><td> 5&#39;AAAGTTCCGCCAGGTGTGAC 3&#39; </td></tr><tr><td> Metchnikowan </td><td> 5&#39;AACTTAATCTTGGAGCGATTTTTCTG 3&#39; </td><td> 5&#39;ACGGCCTCGTATCGAAAATG 3&#39; </td></tr></tbody></table> 表1：引物定量RT-PCR。

Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

systemic-bacterial-infection-immune-defense-phenotypes-drosophila

Research

JoVE Journal

Immunology and Infection

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

24.4K 次观看.  Cornell University. Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

视频: Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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

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

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

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

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

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

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...