作者要感谢过去和现在的金实验室，马克妮兰对斑马鱼的保养和维护，罗伯特·惠勒博士的有益讨论和数据共享的成员，和美国国立卫生研究院资助3RO1GM087308-02S1和1P20RR024475-01A2和缅因州的农业和森林试验站（公开号3303）提供资金。

1。斑马鱼的维护与保养<ol><li>畜牧业：质量产卵成年斑马鱼如前所述13。收集如前所述14衍生的胚胎。 </li><li>显微注射（如果需要）：微导1-4细胞期斑马鱼胚胎与吗啉代寡核苷酸拦截基因产物或mRNA的过表达的基因产物，如前所述15。 <ol><li>保持模拟充足池注入控制（至少48居住，模拟注入控制鱼和48的生活，实验操作的鱼来填补96孔微孔板）。 </li></ol></li><li>维持胚胎：在卵水生长在深培养皿中胚在28℃（60μg/ ml的即时海上盐在蒸馏水中，高压灭菌），直到所需的发育阶段（使用该协议在斑马鱼胚胎呼吸爆发的反应是不可检测年龄超过2天受精后12）。注意：它一直Øbserved，通过任一反应，用1 -苯基-2 -硫脲（PTU）或golden/slc24a5突变斑马鱼防止色素沉着在斑马鱼胚胎不显著改变荧光由PMA（未公布的数据，胚胎在48，72进行测试，和96的感应HPF）。 <ol><li>每天除去死胚用塑料移液管。 </li><li>小心倒出旧鸡蛋水，每天补充新蛋水。 </li></ol></li><li> Dechorionate胚胎：在实验当天，dechorionate胚胎（胚胎是否仍处于绒毛膜）如先前使用（这呼吸爆发测定也可以在从成年斑马鱼12和肾脏详细协议解剖肾脏进行2细镊子14描述从成年斑马鱼解剖先前已经描述16,17）。 </li></ol> 2。溶液的制备<ol><li>准备H2DCFDA的储备液：称取1毫克H2DCFDA的。 <ol><li>迪ssolve 1毫克H2DCFDA在1ml二甲基亚砜（DMSO）中，制成1毫克/毫升的储备溶液。 </li><li>使1.7 ml离心管这种原液的22微升等分。 </li><li>包裹的H2DCFDA原液在铝箔分装，并保持在黑暗中只要有可能，因为H2DCFDA对光敏感。 </li><li>商店H2DCFDA分装于-20℃可保存3个月。 </li></ol></li><li>注意-准备的佛波酯（PMA）的储备液：称取1毫克的PMA使用适当的个人防护设备（ 如手套，护目镜和面罩）。 <ol><li>溶解PMA在1ml的DMSO中，制成1毫克/毫升的储备溶液。 </li><li>使1.7 ml离心管，11微升等份。 </li><li>的PMA原液在-80℃下长达3个月店等分。 </li></ol></li><li>准备H2DCFDA的工作溶液：在实验当天，通过添加1份H2DCFDA原液1做出H2DCFDA工作溶液部分DMSO（500微克/毫升H2DCFDA最终浓度）。例如，加入20微升的H2DCFDA原液和20微升的DMSO的箔纸包裹1.7毫升离心管中。 </li><li>制备PMA的工作溶液：在实验当天，让一个PMA工作液中加入1份的PMA原液到49份无核酸酶的水（20微克/毫升的PMA的终浓度）。例如，稀释10μLPMA原液在490μL无核酸酶的水1.7 ml离心管。 </li><li>准备H2DCFDA的给药溶液：在实验当天，使用1微克/毫升H2DCFDA在蛋水的终浓度一H2DCFDA给药溶液。给药溶液的制备体积可以被修改为需要，但确保最终浓度试剂被保持。代替鸡蛋水为肾脏，用Dulbecco改良的Eagle氏medium/F-12相同体积（50％DMEM，50％的F-12，无酚红）。为了使5毫升H2DCFDA的给药溶液，用5毫升的血清吸管转移5毫升蛋水（或DMEM/F-12）到15毫升锥形离心管包裹在金属箔和标记“H”。 <ol><li>从15毫升锥形管标有“H”和丢弃取出10微升蛋水（或DMEM/F-12）的。 </li><li>加入10μlH2DCFDA工作液到15毫升锥形管标记为&#39;H&#39;和旋涡混合（这个量是足够48胚胎或肾脏样本（半满96孔微孔板）和这些井将提供水平的测量结果的背景荧光）。 </li></ol></li><li>准备H2DCFDA + PMA的给药溶液：在实验当天，请与终浓度的H2DCFDA + PMA给药溶液：H2DCFDA - 1微克/毫升和PMA - 400毫微克/毫升。为了使加入5ml H2DCFDA + PMA给药溶液，用5毫升的血清吸管转移5毫升蛋水（或DMEM/F-12）到一个新的15毫升锥形管标有“H +的P&#39;。 <ol><li>删除110 _6，从15毫升锥形管标有“H +的P&#39;和丢弃升蛋水（或DMEM/F-12）的。 </li><li>加入10μlH2DCFDA工作溶液，再加入100μl的PMA工作液进15毫升锥形管中标记为“H +的P&#39;和涡流混合（此量是足够的48个样本或一个完整的96孔微量培养板的另一半）。 </li></ol></li><li>置于冰上计量解决方案。 </li></ol> 3。酶标仪编程<ol><li>准备工具：上电酶标仪。 <ol><li>热身光源。 </li><li>建立一个程序来读取荧光： 如激励：485纳米;发射：528纳米;光学位置：顶部510纳米;灵敏度：65，与前一个读5秒摇步。 </li></ol></li></ol> 4。 96孔微孔板设置（ 见图2） <ol><li>收集物资：获取菜肴dechorionated胚胎，黑色96孔微孔板，P200移液器和提示，冰桶用H2DCFDA和H2DCFDA + PMA的给药方案，多渠道P200移液器，二无菌水库，铝箔，和剪刀。 </li><li>转移一个胚胎到96孔微孔板的每个孔：用剪刀剪枪头，使得胚胎或肾脏适合通过开口。 <ol><li>设置P200移液器至100μl，并传送一个胚胎随着蛋水（或DMEM/F-12）插入尽可能多的黑色96孔微量培养板的孔中，作为理想的（没有必要改变移液管尖用于每个不同的内的实验条件下胚胎样品，但它可能有必要改变实验条件之间的提示）。一定要避免转移剩余绒毛膜，因为这些往往扭曲收集的数据。对于肾脏的转移，成交量较大移液器（设置为100微升）可以使用和移液器吸头可切割，以获得更大的口径大小，如果需要的话。可能有必要把井无胚胎或肾脏样本，但与给药方案来控制对某些实验操作。 </li></ol></li><li>添加配料解决方案：倾H2DCFDA给药溶液到无菌25毫升水库。 <ol><li>使用多声道的P200移液器和八个技巧，同时吸取100微升的H2DCFDA给药溶液插入96孔微孔板（H2DCFDA 500毫微克/毫升终浓度）一列。 </li><li>作为理想的（通常是六列或48口井，如果填充整个96孔微孔板）为尽可能多的列重复这个（变化的提示是没有必要的）。此溶液加至一半的控制胚胎样品和一半的实验操纵胚胎样品的（一个例子96孔微量培养板设置显示在图2中 ，这些井（着色橙色），将提供背景荧光数据中不诱导用PMA样本） 。 </li><li>倒入H2DCFDA + PMA给药溶液成新，无菌25毫升水库。 </li><li>使用多声道的P200移液器和八个技巧SIMULTaneously吸取100μl的H2DCFDA + PMA给药溶液到剩余的列（ 图2中显示为红色）的96孔微量培养板中（改变的提示是没有必要的，终浓度分别为H2DCFDA-500毫微克/毫升和PMA-200毫微克/毫升） 。 </li></ol></li><li>盖上铝箔微孔板。 </li><li>摇动微孔板为约20秒，在150转均匀化的各溶液很好。 </li><li>孵育微孔板，在28℃时，它不被读取。 </li></ol> 5。荧光定量<ol><li>阅读微孔板时间=使用步骤3.1.2中所述的参数加入PMA后0小时。 <ol><li>继续进行测量，每隔几分钟所需的时间间隔或孵化箔纸包裹微孔板在28°C，直到稍后的时间点，然后取一个端点测量在所需的时间（ 如 4小时，加入PMA后）。 </li></ol></li><li>使用的塑料T转拨移液管来从各孔的胚胎。 </li><li>根据你的动物护理和使用的协议， 如浸泡在三卡因MS222安乐死的胚胎。 </li><li>出售中的生物危险废物容器的微孔板和其他一次性材料。 </li></ol> 6。数据分析<ol><li>决定的时间点，你会在哪个喜欢比较荧光值（加PMA， 表1后如 4小时）。 </li><li>减去个人PMA诱导的对照组的荧光值的平均值未诱导对照组的荧光值。 </li><li>重复此实验组与不PMA。 </li><li>在两列，控+ PMA组和实验+ PMA组（ 表2）存储这些荧光强度值。 </li><li>计算平均值和标准偏差的控制+ PMA石斑鱼的荧光强度值p和实验+ PMA组。 </li><li>使用非配对t检验，以（ 表2）确定统计显着性比较归一化的荧光值。 </li><li>图中控+ PMA组和实验+ PMA组与误差线反映了相应的标准偏差的方法。 </li><li>记录的意义上的图形和在该图中说明的水平（ 图2）。 </li></ol>

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作者什么都没有透露。

吞噬细胞的主要功能是检测，吞噬和消灭病原体。吞噬细胞，以产生足够的呼​​吸爆发的能力是此功能是至关重要的。因此，呼吸爆发反应的量化是一种方法，允许的个人和/或响应于实验操作的组之间的一般先天免疫系统的健康和功能的比较。在这里，我们描述了一个协议来诱导，量化，并比较各个斑马鱼胚胎的组间呼吸爆发的响应。总之，PMA导致ROS的产生的酶NADPH氧化酶。这些活性氧作用于?...

免疫系统是由两个分支：先天免疫和适应性免疫。先天性免疫是进化上比适应性免疫更古老。无脊椎动物是目前认为有只先天免疫，而脊椎动物兼具先天和适应性分支。而适应性免疫赋予具体和持久的免疫力，某些病原体，先天免疫是入侵的细菌，病毒和真菌立即作出反应。先天免疫反应的一个重要方面涉及的细胞因子和趋化因子的释放，从而导致炎症的巨噬细胞（ 如巨噬细胞，中性粒细胞）和招聘吞噬和破坏外来入侵者。 成功的先天免疫反应包括：（1）识别入侵微生物的;（2）感应相应的信号级联反应（ 如释放细胞因子和趋化因子）的;吞噬细胞（3）适当发展/数量充足;（4）吞噬细胞对感染部位迁移;（5）吞噬病原体;和（6）破坏吞噬微生物。在这些步骤中的任何一个A缺乏可能导致被淹没由主机，并屈服于，感染。一个强大的先天免疫反应是生物体的健康是至关重要的，因为它是抵御病原体的所有植物和动物的第一道防线。在脊椎动物中，它也可加强适应性免疫应答1。因此，我们能够评估，以便更好地理解它，并优化它的功能的先天免疫反应的所有方面它是至关重要的。 许多模式生物用于研究先天免疫，从Arabadopsis为C。线虫到果蝇到小鼠培养的人类细胞。使用斑马鱼（ 斑马鱼 ）的模型系统来研究先天免疫系统的优点在于，斑马鱼是脊椎动物，既有先天和适应性即时通讯群落，但先天免疫和适应性免疫的发展在时间上分开。斑马鱼仅仅依靠先天免疫保护，防止感染，直到适应性免疫成为功能齐全，它发生在大约4-6周后受精2。除了 ​​遗传操纵，光学透明性和快速，外部开发工具，先天免疫防御如在斑马鱼胚胎的原理模式提供了在其中以研究在体内的先天免疫反应的复杂性的简化模型。 多个协议已被开发，以评估在斑马鱼胚胎的先天免疫反应的不同方面。微阵列和RNAseq已经证实，通过斑马鱼先天免疫反应引起的细胞因子谱是相似于人类和也建议意想不到基因在先天免疫3,4的参与。斑马鱼胚胎和荧光灯，transgen的透明度病原体和斑马鱼集成电路菌株允许动态宿主-病原体相互作用的体内实时可视化。转基因斑马鱼胚胎中表达绿色荧光蛋白在中性粒细胞特异性髓过氧化物酶启动子5,6或巨噬细胞特异性启动子的mpeg1 7的控制使人们有可能以可视化和量化吞噬细胞迁移到局部感染8的网站以及可视化吞噬和破坏荧光标记的病原体8,9。斑马鱼的胚胎也适合进行高通量测定法和化学画面的生成。因此，最近已经开发了感染10和巨噬细胞迁移到化学诱导损伤11的位点后转录组分析的高通量方法。 上面列出的技术中，没有定量评估吞噬细胞破坏病原体的最后阶段。此最后阶段涉及呼吸爆发（ 即产生的活性氧和其他有毒化合物），其中杀死吞噬病原体。酶的NADPH氧化酶是ROS在吞噬细胞的主要来源。组件的NADPH氧化酶产生的电子转移到氧亚基中，产生超氧阴离子。通过随后的酶促反应，超氧化物可以被转化成过氧化氢 ​​和次氯酸（ 图1A）。它是呼吸爆发吞噬细胞，杀死病原体，因此，斑马鱼胚胎的呼吸爆发潜力的量化指标是总体先天免疫的健康。我们开发了一种基于荧光的测定法来量化在个别斑马鱼胚胎的12个组的呼吸爆发。这个测定法利用市售的，细胞渗透性染料的非荧光，还原形式。这种染料，2&#39;，7&#39;-二氯二乙酸酯（H2DCFDA），被转换成荧光％的化合物，2&#39;，7&#39;-二氯荧光（DCF），在氧化时。由吞噬细胞呼吸爆发产生的活性氧多样可H2DCFDA氧化并产生荧光24。荧光的外观可以被用来量化和比较斑马鱼的组之间的呼吸爆发的反应。蛋白激酶C激动剂佛波醇肉豆蔻酸乙酸酯（PMA），用于化学诱导NADPH氧化酶产生活性氧，从而提高荧光读数（ 图1B）。在此，我们提供的这个斑马鱼胚胎呼吸爆发检测的修改和优化版本的详细协议。此测定法可用于比较在一段时间和/或响应于实验操作（ 例如，吗啉代-介导的蛋白敲低）个别斑马鱼胚胎的基团之间的呼吸爆发。使用这种方法时，在与其他斑马鱼先天免疫测定法结合使用，将提供复杂和关键的更完整的图象先天免疫应答。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Instant Ocean Sea Salt</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>H2DCFDA</td>
			<td>Sigma Aldrich</td>
			<td>35845-1G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PMA</td>
			<td>Fisher</td>
			<td>BP6851</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2438-5X10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tricaine S MS222</td>
			<td>Western Chemical</td>
			<td>100 grams</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM/F-12, No Phenol Red </td>
			<td>Life Technologies</td>
			<td>11039-021</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Deep Petri Dishes</td>
			<td>VWR</td>
			<td>89107-632</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plastic Transfer Pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>#5 Dumont Forceps</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1.7 ml Micro Centrifuge Tubes</td>
			<td>Axygen</td>
			<td>10011-724</td>
			<td></td>
		 </tr>
		 <tr>
			<td>15 ml Conical Centrifuge Tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml Serological Pipettes</td>
			<td>Greiner Bio One</td>
			<td>606180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synergy 2 Multi-Mode Microplate Reader</td>
			<td>BioTek</td>
			<td>Contact BioTek</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 96 Well Microplate</td>
			<td>VWR</td>
			<td>82050-728</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml Sterile Reservoirs</td>
			<td>VistaLab</td>
			<td>3054-2003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>P200 Pipettor</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Multichannel Pipettor</td>
			<td>VWR</td>
			<td>89079-948</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipette Tips</td>
			<td>VWR</td>
			<td>89079-478</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

quantification of the respiratory burst response as an indicator of innate immune health in zebrafish

吞噬细胞呼吸爆发是对病原体感染的先天免疫应答的一部分，并涉及生产活性氧（ROS）。 ROS是有毒和功能，吞噬杀灭微生物。巨噬细胞源性活性氧在体内定量分析提供了关于生物体的挂载一个强大的先天免疫反应的能力的信息。在这里，我们描述了一个协议，在化学诱导的巨噬细胞呼吸爆发的整个斑马鱼胚胎量化和比较活性氧。这种方法使得这成为经氧化活性氧荧光无荧光化合物的用途。个人斑马鱼胚胎移入微孔板的孔中并孵育在此荧光底物有或没有呼吸爆发的化学诱导剂。荧光在各孔用酶标仪定量在期望的时间点。荧光读数调整到消除背景荧光，然后合作mpared使用非配对t检验。这种方法允许对斑马鱼胚胎在不同发育阶段以及响应于实验操作，如蛋白质敲低过表达，或与药理学药剂治疗的呼吸爆发潜力的比较。此方法也可用于监测从成年斑马鱼的肾脏和其他一些鱼类物种整个剖分肾脏或细胞制剂的呼吸爆发的反应。我们认为，这一协议的相对简单性和适应性将补充现有的协议，将感兴趣的研究人员谁寻求更好地理解先天免疫反应。

这里，我们提供48比较在斑马鱼胚胎的呼吸爆发反应（野生型，AB背景）数据和72小时后的受精（HPF）。 48 HPF胚胎担任我们的对照组和72 HPF胚胎作为我们的实验组。所使用的样本量为24未诱导胚胎和24％的发育阶段PMA诱导胚胎。原始荧光读数（以相对荧光单位（RFU））被读出微量的添加PMA的4小时后获得的。在表1中提供的原始荧光值。原始荧光值始终高于在48 HPF未诱导胚胎比72 HPF未诱导?...

Watch this Scientific Journal Video about Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish at JoVE.com

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

先天免疫反应保护免受病原体感染的生物体。先天免疫反应的一个重要组成部分，吞噬细胞呼吸爆发，产生活性氧杀死入侵的微生物。我们描述了量化时所产生的先天免疫反应的化学诱导的活性氧呼吸爆发测定。

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). ...

quantification-respiratory-burst-response-as-an-indicator-innate

Research

JoVE Journal

Immunology and Infection

21.5K 次观看.  University of Maine. 先天免疫反应保护免受病原体感染的生物体。先天免疫反应的一个重要组成部分，吞噬细胞呼吸爆发，产生活性氧杀死入侵的微生物。我们描述了量化时所产生的先天免疫反应的化学诱导的活性氧呼吸爆发测定。 

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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

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. ...

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 ...

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 ...

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 ...

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

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

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 ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...