笔者想感谢实验室成员斑马鱼的保养和维护。这项工作是由美国国立卫生研究院，美国国家研究服务奖A155397到EA哈维和NIH R01GM074827安娜Huttenlocher支持。

成人和胚胎斑马鱼维持在按照威斯康星大学麦迪逊分校的研究动物资源中心的大学。 1.准备显微注射针<ol><li>在拉5，空气时开始的空气压力200，热量502，拉90，速度80，时间70，广播时间：准备使用微量牵引装置具有以下设置的薄壁玻璃毛细管注射针晶（1.0 OD / 0.75的ID）在拉5月底。 </li><li>使用细镊子，断绝拉到针的尖端，以使顶端开口的直径为大约10微米。 </li></ol> 2.准备幼虫注射菜<ol><li>冲洗凝胶梳子约为4-5毫米的无菌水车道宽度并晾干。 </li><li>准备在E3的介质19和微波炉的1.5-2％的高熔体琼脂糖溶液，直至溶液澄清。一旦冷却，倒入一些琼脂糖到培养皿（100×15毫米）和漩涡，加入足够的琼脂糖完全覆盖培养皿底部。 </li><li>一旦琼脂糖层已经凝固，将漂洗并干燥凝胶梳子上顶，以使非精梳端只是搁置在培养皿的顶部和梳理端正在触摸琼脂糖。确保梳子是尽可能的水平，创造了30°角相对于所述底部琼脂糖层。 </li><li>倾额外少量琼脂糖在梳子和底部琼脂糖层之间的界面，以使新鲜的琼脂糖层覆盖所述梳状的孔中。允许取出梳子之前完全冷却。用枪头，从水井中删除任何悬垂件琼脂糖。 </li><li>倒在4℃下在注塑模具及商店的顶部E3介质。 </li><li>每次使用前，更换新鲜培养基和温暖注入坡道在28.5°C，至少注射前一小时。 </li><li>立即注射之前，取代E3上的注射用斜含有200微克/ ml的3-氨基苯甲酸甲酯（三卡因）的E3介质。 </li></ol> 3.准备S. iniae接种<ol><li>准备并高压灭菌补充有0.2％酵母提取物和2％蛋白胨（THY + P）托德Hewitt肉汤培养基：30g / L的托德休伊特，2克/升酵母提取物，将20g / L的蛋白胨。对琼脂平板，加14克/ L琼脂。 </li><li>夜感染吹打冷冻细菌库存100微升等分试样到10ml，THY + P肉汤在密封的15ml试管之前准备的细菌培养物。孵育过夜不用搅拌，在37℃。经过14-16小时的增长，使用过夜培养既可以使冷冻股票或在注射使用做准备。 </li><li>通过将1毫升在500μl80％的甘油的过夜培养物在1.7毫升离心管使冷冻储存。为了避免反复冻融，从该混合并储存在-80℃下使100μl的一次性使用的等分试样。 </li><li>对于S. iniae用于infecti通过加入0.1过夜培养毫升100 10毫升培养的总体积为9.9，TH​​Y + P肉汤毫升：项，稀释过夜培养1。成长在37°C时不搅拌约4-5小时。监测使用NanoDrop分光光度计的光密度（OD）在600nm和收获细菌在中期对数生长期时，OD 600 nm的到达0.250-0.500。为0.250的OD 600毫微米相当于约10 8个菌落形成单位（CFU）/毫升。 </li><li>沉淀将1ml细菌培养物在1.7毫升离心管中，在1500×g离心5分钟。重悬在1ml新鲜的PBS并重复。测量细菌在PBS的OD 600纳米，沉淀，重悬在PBS中以达到所需的浓度。 </li><li>为了帮助在显微注射的可视化，事先添加酚红对细菌悬浮液以注射为0.1％的最终浓度。 </li><li>对于涉及在注射热灭活细菌的实验中，加热该细菌在PBS中在95℃下进行30分钟。确认热杀死过程由固体，T​​HY + P琼脂板上电镀的注射体积（大约1 NL）中并在37℃温育过夜减压活菌到检测不到的水平的数目。 </li></ol> 4.标签S. ËINIA用CellTracker红色荧光染料<ol><li>标记活细菌细胞，制备CellTracker荧光染料或等效的储液。作为所使用的染料来在20×50微克等份粉末，需要在DMSO中进行重新悬浮，微升DMSO中添加7.3到管以得到10mM的储备浓度。 </li><li>测试一系列浓度的染料（ 例如，0.5-25μM）上的细菌，以确定污渍的细胞的最低最佳浓度。快速分裂的细胞和更长的实验可能需要染料的浓度较高。 <ol><li>上述（第3节）所描述的颗粒1.0毫升细菌培养在1.7毫升管离心。悬浮在1球毫升新鲜的PBS，并添加染料的适当体积的细菌培养物。 </li><li>孵育不搅拌，在37℃下进行30分钟。降速的细菌重悬在1ml预热THY + P肉汤孵育不搅拌另外30分钟，在37℃。 </li><li>降速的细菌和测量OD600纳米及以上的（第3节）详见稀释细菌注射前，在PBS洗两次。我们通常注入约100 CFU 1 NL注射量为白细胞招聘和吞噬的研究。 </li></ol></li></ol> 5.准备斑马鱼幼虫的感染<ol><li>之前设置了繁殖对夜间和所描述的罗森等27孵育胚胎在E3中在28.5°C，直到准备感染收集胚胎。 </li><li>对于斑马鱼，将被成像，防止颜料（黑化）的开发中加入N-二苯基硫脲（PTU）的E3的培养基中于24小时受精后（HPF）为0.2纳米的最终浓度。 </li><li>对于涉及胚胎感染2岁旦，dechorionate胚胎一双细镊子手动。可替代地，dechorionate胚胎通过去除E3和用链霉蛋白酶（2毫克/毫升）约5分钟，或直至轻柔吹打替换破坏的胚胎从绒毛膜。大部分幼虫自然应该由3 DPF孵化。 </li><li>通过将dechorionated幼虫到含有200微克/毫升三卡因E3介质麻醉斑马鱼几分钟之前感染。 </li></ol> 6.耳泡注射液S. iniae进入第三天高龄幼虫<ol><li>打开微量，并设定​​时间范围为“毫秒”。打开对二氧化碳罐阀让气体进入线。该微量的压力应为大约20 PSI。由黑色圆形旋钮为增量的顺时针转动调节在微量单元中的压力缓解压力或逆时针旋转的下降压力。 </li><li>涡准备S.在酚红和PBS，并使用微加载提示iniae文化装载2-3微升培养成一把拉住毛细管注射针头。 </li><li>安装在连接到立体显微镜下一个磁性支架并将其定位显微加载针，使针是在约一45-65°的角度相对于所述显微镜的基极。 </li><li>按微量的脚踏上以分配一滴接种到针的尖端。测量使用在显微镜的目镜比例尺下降的直径。 <ol><li>可替代地，通过注射体积为矿物油对带有标尺条的玻璃显微镜载玻片一滴估算滴的直径。液滴的直径应为约0.10mm，大约是一个1 NL体积。 </li><li>通过调整时间设定在调整墨滴大小的微量或通过剪切掉更多针尖细镊子。需要注意的是喷射时间应在20 - 35毫秒，以避免造成过多的组织损伤。 </li></ol></li><li>使用塑料移液管，移12麻醉幼虫放入注射模具的每个孔中。 </li><li>使用玻璃棒，毛发循环，或塑料尖端轻轻地定位幼虫，使磁头指向朝向显微镜和卵黄囊的背面是对井的左侧。点朝天花板幼虫了左耳。 </li><li>期待通过立体显微镜的目镜中，使用在显微旋钮排队装入针与耳泡，使得两者都在同一视场。皮尔斯的耳泡与针尖的外皮层，以使针尖仅仅是囊泡内部。 </li><li>按下脚踏上注入1 NL S的所需剂量的iniae。请务必使用足够低的压力，以便不破裂空腔。如果注射是成功的，耳泡，但不是周围组织，应填写与酚红接种（ 图1艾 ）。立即从注射板删除任何错误注入鱼。 </li><li>仔细缩回针从幼虫和用手移动喷射板，以使下一个幼虫是图。这样做在放大5倍。 注意：在针的回缩可能导致耳泡的外一些细菌，其可以歪斜存活和白细胞募集的结果的沉积。为了避免这种情况，建议注入荧光色素标记的细菌和视觉扫描荧光显微镜下注射的幼虫以除去其中这已发生的任何幼虫。一个正确的注射幼虫所示（ 图1碧 ）。 </li><li>为了确保注射体积/接种物保持不变在实验的过程中，注入一滴细菌的Suspension到含有100微升无菌PBS的每48 个胚胎后1.7毫升离心管中。板中的100μl的上，THY + P琼脂平板在37℃下过夜，以确定在注射体积的CFU。 </li><li>当12幼虫在注射坡道的整个集团已经注入，小心使用塑料移液管从井中取出幼虫，并放置到一个新的培养皿（35×10 平方毫米）。取下三卡因溶液，并用约新鲜E3媒体2毫升取代，让幼虫恢复。 </li><li>吸管注入幼虫成单个孔96孔板的孵育在28.5℃。监测幼虫随着时间的推移生存在这些板块或更高版本的成像或CFU计数删除。 </li></ol>中性粒细胞7.苏丹黑染色注意：下面的步骤可以在室温下在定轨振荡器，除非OT进行在小培养皿（35×10mm的2）herwise说明。对于每个步骤，试剂的使用量是约每皿2毫升，使用刚好足够的液体，以完全覆盖幼虫。 <ol><li>对受感染的幼虫的​​固定，使用玻璃巴斯德吸管除去E3介质，并替换为在PBS溶液中的冰冷却的4％多聚甲醛。立即将鱼在4℃安乐死。修复一夜之间在4℃。 </li><li>在PBS，每次5分钟洗涤3次。 </li><li>染色用苏丹黑（0.18％库存1:5稀释在70％的乙醇，0.1％苯酚）处理30分钟至5小时。使用立体显微镜来检查，如果中性粒细胞颗粒扛起了污点。 </li><li>执行一系列的5分钟洗涤开始在70％乙醇中的单次洗涤之后的1的单一洗涤：1的比例70％乙醇和PBS，随后在PBS中进行最后的清洗。 </li><li>再水合到PBS加0.1％吐温（PBT）。 </li><li>以清除颜料从幼虫，洗净，以1％的氢氧化钾/ 1％的过氧化氢为约6-10分钟。监测样本closely和大约5分钟后，检查在立体显微镜下的幼虫，看看颜料多已被清除。如果溶液被留在过长，卵黄囊会膨胀和爆裂。 </li><li>在PBS中洗涤3次，每次5分钟。 </li><li>储存在PBS或PBT样品在4℃下长达1周，直到准备图像和计数。 </li><li>图像苏丹黑染色幼虫，鱼转移到PBT，然后到80％的甘油，并放置到一个单腔抑郁症的幻灯片。位置固定下使用移液管尖端的立体显微镜幼虫。图像使用彩色数码相机立体显微镜（ 图1 AII-IV）。 </li></ol> 8.枚举从受感染的幼虫活菌<ol><li>安乐死幼虫在所需时间感染后在E3的介质冰冷200-300毫克/升三卡因溶液。 </li><li>吸管安乐死幼虫进入1.7毫升离心管。除去三卡因溶液和替换用100μl的0.2％TRITON X-100的PBS（PBSTx）。 </li><li>通过一个27G的针头上下传球10次均质幼虫。 </li><li>准备在无菌PBS匀浆的系列稀释。例如，如果感染剂量为100 CFU，吸管100微升匀浆入900微升无菌PBS中。使用新的枪头，转移100微升稀释的匀浆到900微升无菌PBS。 </li><li>板100微升哥伦比亚CNA琼脂为革兰氏阳性菌的选择性分离的稀释液，孵育在37℃下48小时。此介质将提供更大的选择比，THY + P琼脂板上，这将同时支持革兰氏阴性和革兰氏阳性细菌的生长。 </li><li>在平板上少于500单个菌落，计数菌落数并乘以稀释因子相乘以确定CFU数量。 </li></ol> 9.固定幼虫成像<ol><li>修正的幼虫在PBS溶液作为去冰 - 冷的4％多聚甲醛划在第7。 </li><li>在室温下，在成像前洗3次，每次5分钟的PBS。 </li></ol> 10.准备幼虫的实时成像<ol><li>制备1.5％低融点琼脂糖中的E3通过加热溶液在微波中，直至溶液澄清。 </li><li>将在55℃水浴琼脂糖溶液，使其冷却，但不会变硬。 </li><li>添加三卡因到琼脂糖的0.016％的最终浓度。 </li><li>用塑料移液管，放置4-5麻醉幼虫在一个立体的舞台玻璃底菜。 </li><li>除去从55℃水浴中的三卡因和琼脂糖溶液，让它冷却，在室温下搅拌1-2分钟。而琼脂糖冷却，从被麻醉的幼虫尽可能除去尽可能多的液体。 </li><li>倒入冷却的琼脂糖放入盘中，直到大约一半的表面被水覆盖。摇动盘传播的琼脂糖。需要注意的是，如果琼脂糖太热，它会杀死幼虫。此外，如果过多琼脂糖加入到皿中，在显微镜的物镜可以通过琼脂糖聚焦时击中培养皿的底部。 </li><li>用移液管，拿起那些漂浮在盘子的两侧的幼虫和吸管它们放回中心。 </li><li>根据体视显微镜，根据需要用吹风环路，长枪头或玻璃棒轻轻放置幼虫。用于耳泡的成像，定位幼虫，使得左耳泡是平贴在培养皿的底部。 </li><li>让琼脂糖冷却约10分钟，移动盘之前。幼虫可移仓，如果琼脂糖并不牢固。轻轻吸管一些三卡因和E3溶液到琼脂糖层的顶部，以保持湿润。 </li></ol>感染11.共聚焦成像<ol><li>放置玻璃底皿幼虫到倒置显微镜具有FV-1000激光扫描confoca的阶段升系统。 </li><li>针孔设定为200-300微米，使用数值孔径0.75 / 20X物镜，设置Z轴堆叠具有3-6微米的切片。 </li><li>使用连续扫描线来调整激光功率和检测器的增益为每个通道。 </li><li>使用顺序逐行扫描每个荧光通道（ 例如，488和543纳米）和微分干涉对比（DIC），进行左侧耳泡的时间间隔电影每3分钟2-6小时，观察初始招聘和吞噬中性粒细胞和巨噬细胞。对于较长的时间进程，放置在培养皿的盖以防止蒸发和干燥从琼脂糖。 </li><li>收购仍是固定的（ 图1B）的图像或生活（ 图2），在幼虫或20X 40X的放大倍率。 </li></ol> 12.光转化Dendra2标记的白细胞在耳泡注：Dendra2可以从绿色将光转换为红色荧光通过聚焦405nm激光（50-70％的激光功率应足以）上的感兴趣区域（ROI）的区域，持续1分钟。下面是一步一步协议用于FV-1000激光扫描共聚焦系统： <ol><li>使用Z堆栈扫描与488 nm和543 nm激光可视化的样本。使用连续扫描线来调整激光功率和检测器的增益。检查以确保没有不小心光转换红色荧光。 </li><li>在“图像采集控制”窗口，“刺激设置”下，选中“使用扫描仪”工具，然后选择“主”。选择405 nm激光，并定于70％的电力。使用循环选项，定义投资回报率在耳泡。 </li><li>在“刺激开始设置”中选择1帧的预激活和60000毫秒的激活时间“激活串联”。就在“获取设置”窗口中的“时间扫描标题”，选择2的0时01分00秒（一个用于前和一个用于光转化后）tervals。 注意：时间经过一系列扫描完成后，将Dendra2应已光转换到其红色荧光的状态。 </li><li>通过使用488纳米和543纳米激光器的可视化的光转换的红色荧光以及任何剩余的绿色荧光（ 图3）的Z堆叠扫描样品。 </li></ol>

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J., Mar&#237;n-Juez, R., 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=Establishment+and+Optimization+of+a+High+Throughput+Setup+to+Study+Staphylococcus+epidermidis+and+Mycobacterium+marinum+Infection+as+a+Model+for+Drug+Discovery.">Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery.</a> J. Vis. Exp. (88), (2014).</li><li>Deng, Q., Harvie, E. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+signaling+mechanisms+mediate+neutrophil+attraction+to+bacterial+infection+and+tissue+injury.">Distinct signaling mechanisms mediate neutrophil attraction to bacterial infection and tissue injury.</a> Cell. Microbiol. , (2012).</li><li>Sar, A. M., 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=Zebrafish+embryos+as+a+model+host+for+the+real+time+analysis+of+Salmonella+typhimurium+infections.">Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.</a> Cell. Microbiol. 5 (9), 601-611 (2003).</li><li>Lin, A., Loughman, J. A., Zinselmeyer, B. H., Miller, M. J., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+S+Inhibits+Neutrophil+Recruitment+during+the+Early+Stages+of+Streptococcus+pyogenes+Infection.">Streptolysin S Inhibits Neutrophil Recruitment during the Early Stages of Streptococcus pyogenes Infection.</a> Infect. Immun. 77 (11), 5190-5201 (2009).</li><li>Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</li><li>He, S., 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=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J. Pathol. 227 (4), 431-445 (2012).</li><li>Haldi, M., Ton, C., Seng, W. 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Immun. 81 (1), 110-121 (2013).</li><li>Haddon, C., Lewis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Ear+Development+in+the+Embryo+of+the+Zebrafish,+Danio+rerio.">Early Ear Development in the Embryo of the Zebrafish, Danio rerio.</a> J. Comp. Neurol. 365, 113-128 (1996).</li><li>Whitfield, T. T., Riley, B. B., Chiang, M. -. Y., Phillips, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+zebrafish+inner+ear.">Development of the zebrafish inner ear.</a> Dev. Dyn. 223 (4), 427-458 (2002).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J. Vis. Exp. (25), (2009).</li><li>Colucci-Guyon, E., Tinevez, J. Y., Renshaw, S. A., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+professional+phagocytes+in+vivo:+unlike+macrophages,+neutrophils+engulf+only+surface-associated+microbes.">Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes.</a> J. Cell Sci. 124 (18), 3053-3059 (2011).</li><li>Gurskaya, N. 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The authors declare that they have no competing financial interests.

此处所用的感染的方法是用于在2-3旦胚胎和幼虫的初始局部感染宿主的免疫应答的研究是有用的。炎症刺激，如感染，在一个封闭的腔体的焦点，如耳泡允许对嗜中性粒细胞和巨噬细胞趋化及吞噬的研究。注入细菌进入耳泡的一个需要注意的是中性粒细胞的能力，以有效地吞噬细菌在填充流体的腔可以依赖于特定的微生物。尽管大肠杆菌和枯草芽孢杆菌 ，不容易通过在耳泡28</sup...

链球菌iniae是一个主要的水生病原体，其能够引起全身性疾病在两个鱼和人1。而S. iniae负责的水产养殖业巨大损失，这也是一个潜在的人畜共患的病原体，能够与类似的其他引起链球菌人类病原体的临床病症引起的疾病免疫功能低下的人类宿主。鉴于其相似性与人类的病原体，它来研究S.是非常重要的在天然宿主的上下文iniae疾病的发病机制。 S的 ​​成年斑马鱼模型iniae感染揭示宿主白细胞向感染的局部部位，以及一个快速时间来承载死亡健壮浸润，时间太短，涉及适应性免疫系统7。为了获得一个深入的研究，以S.先天性免疫应答iniae感染在体内 ，有必要使用一个模型，该模型是更适合为n对创实时成像。 幼虫斑马鱼具有许多优点，使得它越来越具有吸引力脊椎动物模型用于研究宿主 - 病原体相互作用。斑马鱼是相对便宜且易于使用和维护相比哺乳动物模型。适应性免疫并不功能成熟之前4-6周受精后，但幼虫具有高度保守的脊椎动物先天免疫系统与补体，Toll样受体，细胞因子，和嗜中性粒细胞和巨噬细胞具有抗微生物功能，包括吞噬作用和呼吸爆发2-6 8-11。此外，遗传易处理性和发展的胚胎和幼虫阶段的光学透明性允许对稳定的转基因品系的产生与荧光标记的免疫细胞使得能够检查实时体内宿主-病原体相互作用。使用photoconvertible蛋白质，这些转基因品系，如书房的产生dra2允许单个宿主细胞的起源和命运的超过12感染的过程中的跟踪。 当开发一个斑马鱼幼体感染模型，显微注射的选址将确定是否感染最初是局部或全身。全身血液感染入尾静脉或居维叶的导管是最常用的研究微生物病原体在斑马鱼和可用于研究在病原体菌株之间的毒力的主机和微生物细胞，细胞因子应答，和差之间的相互作用是有用的。对于生长较慢的微生物，早期注入的胚胎在16-1,000细胞阶段的卵黄囊可用于产生全身感染13,14，具有最佳的发育阶段对发现是间生长缓慢的微生物的显微注射在16到128细胞阶段15。但是，蛋黄许多微生物囊注射，以后主机的发展阶段往往是致命的吨他主持由于对白细胞浸润16-18的微生物和缺乏营养丰富的环境。 本地化感染通常会导致对感染的部位，可以很容易量化以非侵入性成像的白细胞的定向迁移。这种类型的感染可以允许介导白细胞迁移以及各种白细胞群体的不同迁移和吞噬能力调查机制的解剖。局部感染也是有用的研究中的细菌菌株的毒力之间的差异时，以及研究微生物入侵机制，因为物理主机的障碍必须越过为一个局部感染，成为全身。斑马鱼通常升高的温度下25-31℃的19，但它们也可以被保持在温度高达34-35℃的某些人类病原体侵袭的严格的温度要求的研究毒力20，21。 许多不同的位点已被用来产生一个初始局部细菌感染包括后脑心室22，背侧尾肌18，心包腔23和耳泡（耳）5，16，24。然而，已经发现，注射细菌进入尾肌可造成组织损伤的细菌中，当调查白细胞响应13可能扭曲的结果的和炎症独立。虽然少损伤与注入到相关的后脑，虽然它最初是在缺乏幼胚白细胞，后脑脑室稳步随着时间的推移获得更多的免疫细胞为小胶质细胞定居。后脑心室也是比较困难的位置到图像。耳囊是一个封闭的空腔，没有直接进入脉管25,26。它通常是缺乏列伊kocytes，但白细胞可以响应于炎症刺激如感染被招募到耳泡。它也是细菌微量注射在斑马鱼老化2-3天受精后，因为成像的易用性和喷射的可视化（dpf）的一个优选的位点。因此，我们选择了耳泡作为我们本地化细菌感染的部位。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.7 ml eppendorfs</td>
			<td>MidSci</td>
			<td>AVSS1700</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml falcon tube</td>
			<td>BD Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 in. needle</td>
			<td>BD Biosciences</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning Incorporated</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Biosciences</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA agar</td>
			<td>Dot Scientific, Inc</td>
			<td>7126A</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-7m</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>MDS</td>
			<td>2292</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel comb</td>
			<td>VWR</td>
			<td>27372-482</td>
			<td>4.2 mm width, 1.5 mm thick</td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td></td>
			<td></td>
			<td>Custom made by drilling a 16&#8211;18 mm hole in the center of a 35-mm tissue culture dish bottom and placing a 22-mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>High melt agarose</td>
			<td>Denville Scientific, Inc.</td>
			<td>CA3510-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>H325</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Olympus</td>
			<td>with FV-1000 system</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Fisher</td>
			<td>BP165-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Tritech (Narishige)</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection system</td>
			<td>Parker</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipet tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Tritech (Narishige)</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>Flaming/Brown P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotmeter</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldheyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>100 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma Aldrich</td>
			<td>P-4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Ricca Chemoical Company</td>
			<td>572516</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>BP665-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>P-6310</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche</td>
			<td>165921</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease peptone</td>
			<td>Fluka Biochemika</td>
			<td>29185</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cell culture dish</td>
			<td>Corning Incorporated</td>
			<td>430165</td>
			<td>35 mm x 10 mm</td>
		</tr>
		<tr>
			<td>Sudan Black</td>
			<td>Sigma Aldrich</td>
			<td>S2380</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall glass capillary injection needles</td>
			<td>World Precision Instruments, Inc.</td>
			<td>TW100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Todd Hewitt</td>
			<td>Sigma Aldrich/Fluka Analytical</td>
			<td>T1438</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzoate)</td>
			<td>Argent Chemical Laboratory/Finquel</td>
			<td>C-FINQ-UE-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fluka Biochemika</td>
			<td>92144</td>
			<td></td>
		</tr>
	</tbody>
</table>

non-invasive imaging of the innate immune response in a zebrafish larval model of streptococcus iniae infection

水生病原体， 链球菌iniae，负责超过100亿美元的损失每年用于水产养殖业和能够引起全身性疾病在鱼类和人类。更好地理解S. iniae疾病的发病机制，需要一个合适的模型系统。遗传易处理性和斑马鱼的早期发育阶段的光学透明度允许对转基因品系的产生和非侵入性的成像用荧光标记的免疫细胞。适应性免疫系统不完全功能，直到数周后受精，但斑马鱼幼体有一个保守的脊椎动物先天性免疫系统与两个中性粒细胞和巨噬细胞。因此，幼虫感染模型的生成允许的先天免疫控制S.的具体贡献的研究iniae感染。 注射的部位将决定感染是否全身或局部开始。在这里，我们提出我们的协议耳泡注射的斑马鱼时效2-3天受精后，以及我们的技术感染荧光共焦成像。本地化感染部位可观察初始微生物入侵，招募宿主细胞和传播感染。使用S的斑马鱼幼体模型我们的研究结果iniae感染表明斑马鱼可用于检查主机嗜中性粒细胞和巨噬细胞在局部细菌感染的不同的贡献。此外，我们将描述如何免疫细胞photolabeling可用于感染过程期间跟踪单个宿主细胞的命运。

S的 ​​显微注射iniae进入耳泡（ 图1和图2）的结果的初始局部宿主反应。当正确地注入，细菌只应被看作在耳泡而不是在周围组织或血液。这可显微注射过程中使用的酚红染料（ 图1A）被可视化。另外，如果标记的细菌注射，感染幼虫的快速扫描立即注射后可以确认菌仅在耳泡而不是周围的组织（ 图1B）。虽然低至10 CFU野生型S....

Watch this Scientific Journal Video about Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection at JoVE.com

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

non-invasive-imaging-innate-immune-response-zebrafish-larval-model

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

10.7K 次观看.  University of Wisconsin-Madison. Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle. 

视频: Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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 ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...