这项工作是由美国国家科学基金会的支持下授予＃IOS-1025627，由美国国立卫生研究院资助项目＃1R21NS078582-01 JAW

该协议使用的设置（ 图1）获得4个不同的读数收集苍蝇的细菌感染。这些输出包括：1）睡眠/唤醒的行为; 2）生存结果; 3）细菌负荷在飞; 4）实时测量体内的的NFκB记者活动。结合，也可在果蝇的遗传工具，这些测量提供机械洞察分子免疫功能和行为之间的联系。 1。测量运动及睡眠苍蝇<ol><li>已安装用于测量运动及睡觉的苍蝇，其中包括果蝇活动监测的的系统（DAM2，Trikinetics），孵化器，黑暗的房间，并编写的动物实验和活动管，以前6。同样的方法用在这里，有一些小的修改。 <ol class="lalpha"><li&gt;照明的培养箱培养箱本身控制。因此，重要的是计算机和孵化器之间同步的时间设置。 </li><li>活动管清洗再利用，通过在去离子水的热板上煮沸。少量的洗涤剂中加入第一煮沸，管以及漂洗和煮沸两次以上。如果是用来堵塞的管纱，用于活动管（含食品，蜡，苍蝇和纱线），可清洗，恕不另行清除的纱。 </li></ol></li><li>在此之前启动一个实验，后期蛹上演的地方文化苍蝇孵化器为三到四天，以适应实验的照明和其他环境条件。光：暗或不变的条件下，已被广泛用于衡量睡眠行为。在这个例子中，苍蝇驯化恒定的光，消除影响生物钟的免疫反应和行为2,7,8。如在图1A中描述的</strong&gt;时，负载1-4天到Trikinetics DAM2活动的老苍蝇显示器6，记录前至少三天的感染。 </li></ol> 2。感染与致病性细菌的苍蝇<ol><li>这里所描述的协议是特定为 S。 粘质沙雷氏菌 ，这是很容易的成长和维护。协议的长期存储和培养条件的其他细菌物种会有所不同。S.粘质沙雷氏菌在15％至50％的甘油在-80℃下存储要准备长期储存，混合2卷1体积的高压灭菌50％的甘油过夜培养的菌液（OD 600 = 0.5 - 1.0）在1.5ml离心管中。这将导致在最终的甘油浓度为17％。存储管在-80℃下为了制备一个短期中使用的细菌源，刮去无菌200μl的移液管尖，冷冻的股票的LB琼脂平板上进行三通条纹。下孵育过夜，37＆D例如，C分离菌落。该板在4℃下存放 排定感染的前一天，从LB琼脂平板挑取单个菌落，无菌200μl的移液管尖和淹没的前端到含有5毫升LB培养基中培养管中。培养细菌过夜，或最多16小时的孵化器内振动筛，在37℃和250 rpm，直到它达到在指数生长期。的浓度进行测定，用分光光度计在OD 600。在这个阶段的浓度范围从0.5至1。如果浓度过高，继代培养的细菌和生长另一个几个小时，以得到一个最佳浓度。火源附近执行的程序。向培养基中加入某些细菌的抗生素耐药性菌株的设计和生长，并选择适当的抗生素，S.粘质沙雷氏菌 （ATCC＃8100）是对抗生素有抗药性，因此，生长在不含抗生素的无菌介质中。为了验证无菌技术，做一个模拟的丘尔再没有细菌作为对照。 感染苍蝇的解决方案包含细菌（稀释至OD 600 = 0.1）和1％的食用色素（亮蓝FCF），在PBS中。准备感染溶液和1％在PBS中的蓝色的食品着色中使用的LB培养基中加入等当量的溶液的喷射控制。在冰上的存储解决方案。 </li><li>准备玻璃针注射苍蝇。拉玻璃毛细管（OD = 1毫米，ID = 0.58毫米，WPI）以精尖，用微量拉出器（成茂）。在解剖显微镜下，用细钳子折断的针的前端，使该开口大到足以填满与注射流体用注射器通过施加抽吸，但足够小，以尽量减少在注射过程中损坏对苍蝇。打破了头，烙铁头尺寸应该大约为40-50微米。玻璃针与管的长度为连接到一个3毫升的塑料注射器。手动施加正压或s注射介质的流动的控制，减税与注射器。避免污染的橡胶管，注射器装置与注射流体被用于感染和控制注射。 </li><li>麻醉苍蝇，把它们在CO 2焊垫。 CO 2通过一个密封的容器中的水加湿垫和减少静电，可以操纵的苍蝇复杂的焊盘上。注入苍蝇进入该地区的背胸盾片以上的玻璃针戳。成飞注射介质通过验证由食品着色 - 苍蝇变成蓝色作为食品着色通过系统传播。细菌，苍蝇接受的剂量可以量化为第3节所述，在下面。一些实验性的设计，包括接收，对照组注射PBS和食用色素，但没有细菌的苍蝇无菌损伤。 </li></ol> 3。确定细菌负荷方法之一evaluatin克的抗细菌感染的免疫应答是确定的细菌负荷感染后。D。果蝇是一个很好的模型，以确定此参数，因为整个飞可以匀浆估算总细菌数在个别。此协议的背后的基本原理是，如Luria液体培养基（LB）上的陪替氏培养皿的琼脂（LB平板）在固体培养基上生长时，一个单一的细菌形成可见的区别的菌落。因此，通过均质化感染苍蝇在LB液体培养基中，生成的匀浆物的系列稀释液，并传播到L​​B平板稀释的匀浆，感染苍蝇的细菌细胞的数量可以被确定。应该用来验证，没有与其他细菌物种的污染，感染的对照组注射PBS和食用色素，但没有细菌的苍蝇。不应该有任何在此条件下的LB琼脂平板上的菌落。 <ol><li>高压灭菌，将用于此的所有材料程序，然后再进行实际的同质化步骤。这包括200微升吸头切剪刀，LB培养基，用于同质化和杵。准备板，通过蒸压LB /琼脂培养基浇成10厘米的无菌培养皿中至少有1天前均质苍蝇。 </li><li>麻醉和收集苍蝇的1.5 ml微量离心管和存储管上的冰。 </li><li>附近的火焰，以防止污染进行同质化。尤其是当包含苍蝇的控制同质化，无感染，建议使用的细菌菌株，如S.粘质沙雷氏菌 ，这是不对抗生素有抗药性。均质的最小的2组10苍蝇每实验条件。的数目来确定用于每匀浆苍蝇由实验者。有些团体已使用每一个飞匀浆，这是有用的，以评估细菌负荷变化之间的苍蝇8，而另一些则使用3-10苍蝇每匀浆比较不同基因型或实验条件9-12。 <ol class="lalpha"><li>加入400μlLB培养基中，每个离心管中含有苍蝇，并用一个小电机和杵（Kontes）的同质化。 </li><li>使用灭菌切割枪头，串行1:10 1.5微升的微量离心管中，含有180微升的LB培养基中加入20μl匀浆的LB /细菌培养基稀释匀浆。切吸头飞的碎片和防止堵塞，确保适当的音量被转移。 </li><li>稀释因子​​是凭经验确定，取决于基因型的飞，所使用的细菌菌株，和实验条件。对于野生型果蝇感染S.粘质沙雷氏菌 ，使用稀释液1:10 2或1:10 3苍蝇匀浆感染后立即，和苍蝇匀浆稀释1:10 4或1:10 5 24小时后感染。 </li><li>加入100μlLB /细菌培养基从话筒rocentrifuge，管中含有的最终稀释度和传播介质上使用玻璃球（VWR）的LB平板上，以确保均匀分布。 </li><li>丢弃到100％的乙醇溶液的玻璃球。放置的LB板中，在37℃培养箱中培养过夜。 </li><li>作为一个可选步骤，使用可见光的成像系统，以获得图像的LB平板上（电泳凝胶成像8900的Alpha Innotech公司）。计数的菌落的数量无论是在板本身或从Photoshop软件（Adobe Photoshop CS3中）使用的计数工具板的图像。 </li><li>计算的数目的菌落形成单位每飞使用下面的公式：[π/（N * D * v时）] * V，其中n = LB平板上生长的菌落数; N =苍蝇的数目汇集在一微量离心管中，D =稀释因子;和v =溶液的体积分散到每块板; V =初始体积的匀浆。 </li></ol></li></ol> 4。评价睡眠和感染后的生存期<ol><li>为测量细菌负荷的苍蝇，没有收获，持续监测行为再过7-10天。生存的持续时间由基因型的苍蝇，环境条件的影响，并用于感染的细菌种类。 </li><li> 〜10天之后，终止实验，下载和处理行为数据，如前面描述的6。 Insomniac2定制软件，它是基于在Matlab中，用来分析睡眠参数。重要的是要消除死苍蝇从行为分析。通常情况下，这限制了分析，在感染后的第一天之内，取决于感染的类型和苍蝇的基因型。在测定中的死亡表示由所有活动计数达到零的时间。为了便于分析求生存，Drosonex，自定义编写的软件在Microsoft Visual C + + 6.0，用于处理从的Trikinetics DAM系统的原始数据文件。软件Excel文件，每个飞的存活时间（小时），编译报告活动DAT一个从所有的显示器到一个单一的电子表格，报告的百分比飞存活时间的推移由用户设定。 Excel文件的设计融入其他统计软件包进行进一步的分析。 </li></ol> 5。使用荧光素酶报告基因在感染过程中，测量NFκB活动苍蝇在本实验中使用转基因κB-luc的产生Kuo 等人所描述的，2010年2。简言之，κB-luc的记者包含8个重复的NFκB的结合序列，启动子插入荧光素酶的开放阅读框的上游。 <ol><li>调整苍蝇在1.2节中描述的实验的照明条件。在这个例子中，1-4日龄κB-luc的苍蝇都装在小瓶含5％蔗糖，2％琼脂食物的媒介，并放置在恒定的光（LL）2天达到同年龄的其他果蝇在感染过程中。 </li><li>准备一个96孔微孔板苍蝇。在各孔中的食物介质含有2层，（ 图2）;顶端层中含有荧光素，荧光素酶的基板。加入300微升的5％的蔗糖，2％琼脂溶液，向每个孔中，并允许固化。接下来，添加50微升的顶层每个孔中含有5％的蔗糖，1％琼脂，和2mM萤光素（金生物技术公司）。在文献中用于测量的记者活动在果蝇中有各种不同的荧光素浓度，并已低至100μM13。所需的浓度可以凭经验确定。在分配食品层之间，用细网布，以促进干燥，同时保持无菌覆盖板。板应彻底干燥，长达一小时，以防止苍蝇停留在冷凝水滴。由于萤光素是光敏感，避免露出光板超过必要的。 </li><li>应用透明的粘合剂膜（上密封-A，Perkin Elmer公司制），以一个96孔的微孔板上和多孔的2的孔，每孔的量使用的微细针。这些孔将不仅使空气交换，每孔，而且还提供了一种以个人为基础麻醉苍蝇。使用锋利的刀片和直边，引进削减各列之间。这将提供一个简单的方法，8个一组的加载/卸载苍蝇的一次。 要加载到微孔板的苍蝇，从孵化器和麻醉苍蝇在CO 2焊垫取出小瓶。负载过得一个 - 酮，每个孔中的柱列。如果飞在不经意间被卡住的粘接密封，独自离开苍蝇，因为他们往往​​能够释放自己没有干预。微孔板的孵化器，LL为8-24小时，以适应新的环境和消耗的萤光素底物。 </li><li>培养细菌，S.粘质沙雷氏菌 ，以及感染如上所述制备溶液。 </li><li>麻醉吨他通过使用微量吸移管连接到一个低压CO 2行的小费苍蝇。将直接上面的为每个孔的通风孔的微量吸移管前端。注意，以确保CO 2压力是足够高，麻醉的苍蝇，但低到足以避免损伤对苍蝇。在八组，单独传送每个飞的CO 2焊盘，并感染如上所述。感染后，返回到原来的每个飞和重新密封微孔板。 </li><li>测量发光（TOPCOUNT-NXT的发光及闪烁计数器; Perkin-Elmer公司）。的TOPCOUNT发光计数器包含堆叠盒板，它允许编程和自动读数连续递增任何长度的时间（通常长达五天）。此功能并不适用于所有的仪器和数据收集与其他仪器进行实验，因此可能不那么频繁。发光计数器装在一个RO嗡与受控的温度和照明时刻表。当加载到堆叠带板，堆叠板之间的苍蝇清晰的空白板，以确保苍蝇接收光。计划光度计，根据制造商的规格，收集读数，每隔一小时至少24小时。在此示例中，检测器被编程，读取各孔，持续10秒，并表达的结果作为每秒的计数（任意单位）。这个值是平均每口井在3个读数。导出到电子表格中的数据文件和执行标准的分析，图表发光的结果。 </li></ol>

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M., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+caspase+Dredd+is+required+to+resist+gram-negative+bacterial+infection.">The Drosophila caspase Dredd is required to resist gram-negative bacterial infection.</a> EMBO Rep. 1, 353-358 (2000).</li><li>Wu, L. P., Choe, K. -. M., Lu, Y., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Immunity:+Genes+on+the+Third+Chromosome+Required+for+the+Response+to+Bacterial+Infection.">Drosophila Immunity: Genes on the Third Chromosome Required for the Response to Bacterial Infection.</a> Genetics. 159, 189-199 (2001).</li><li>Dionne, M. S., Ghori, N., Schneider, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+is+a+genetically+tractable+model+host+for+Mycobacterium+marinum.">Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum.</a> Infect. Immun. 71, 3540-3550 (2003).</li><li>Romeo, Y., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+immunity:+methods+for+monitoring+the+activity+of+Toll+and+Imd+signaling+pathways.">Drosophila immunity: methods for monitoring the activity of Toll and Imd signaling pathways.</a> Methods Mol. Biol. 415, 379-394 (2008).</li><li>Lu, Y., Wu, L. P., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+arm+of+the+Drosophila+innate+immune+response+requires+an+I{kappa}B+kinase.">The antibacterial arm of the Drosophila innate immune response requires an I{kappa}B kinase.</a> Genes Dev. 15, 104-110 (2001).</li><li>Koh, K., Evans, J. M., Hendricks, J. C., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+age-associated+changes+in+sleep:wake+cycles.">A Drosophila model for age-associated changes in sleep:wake cycles.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 13843-13847 (2006).</li><li>Bushey, D., Hughes, K. A., Tononi, G., Cirelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep,+aging,+and+lifespan+in+Drosophila.">Sleep, aging, and lifespan in Drosophila.</a> BMC Neurosci. 11, 1471-2202 (2010).</li><li>Allee, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The social life of animals. , (1938).</li><li>Stanewsky, R., Jamison, C. F., Plautz, J. D., Kay, S. A., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+circadian-regulated+elements+contribute+to+cycling+period+gene+expression+in+Drosophila.">Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila.</a> Embo. J. 16, 5006-5018 (1997).</li><li>Levine, J. D., Funes, P., Dowse, H. B., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+analysis+of+behavioral+and+molecular+cycles.">Signal analysis of behavioral and molecular cycles.</a> BMC Neurosci. 3, 1 (2002).</li><li>Ayres, J. S., Schneider, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anorexia+in+resistance+and+tolerance+to+infections+in+Drosophila.">The role of anorexia in resistance and tolerance to infections in Drosophila.</a> PLoS Biol. 7, e1000150 (2009).</li><li>Plautz, J. D., Kaneko, M., Hall, J. C., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+photoreceptive+circadian+clocks+throughout+Drosophila.">Independent photoreceptive circadian clocks throughout Drosophila.</a> Science. 278, 1632-1635 (1997).</li><li>Huber, 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=Sleep+homeostasis+in+Drosophila+melanogaster.">Sleep homeostasis in Drosophila melanogaster.</a> Sleep. 27, 628-639 (2004).</li><li>Zimmerman, J. E., Raizen, D. M., Maycock, M. H., Maislin, G., Pack, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+video+method+to+study+Drosophila+sleep.">A video method to study Drosophila sleep.</a> Sleep. 31, 1587-1598 (2008).</li><li>Toth, L. A., Rehg, J. E., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+differences+in+sleep+and+other+pathophysiological+sequelae+of+influenza+virus+infection+in+naive+and+immunized+mice.">Strain differences in sleep and other pathophysiological sequelae of influenza virus infection in naive and immunized mice.</a> J. Neuroimmunol. 58, 89-99 (1995).</li><li>Hendricks, J. C., 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=Rest+in+Drosophila+is+a+sleep-like+state.">Rest in Drosophila is a sleep-like state.</a> Neuron. 25, 129-138 (2000).</li><li>Wu, M. N., Koh, K., Yue, Z., Joiner, W. J., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+for+sleep+and+circadian+mutants+reveals+mechanisms+underlying+regulation+of+sleep+in+Drosophila.">A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila.</a> Sleep. 31, 465-472 (2008).</li></ol>

没有利益冲突的声明。

该协议概述的方法来研究如何行为，特别是睡眠，是与免疫反应参数。这些参数包括细菌量，存活的结果，和NFκB活性在体内荧光素酶报告。这些参数提供了一只苍蝇可以对抗感染的信息。细菌和生存结果是涉及一个简单的测量果蝇的免疫反应参数。 相对E20突变体，缺乏NFκB转录因子，是中央的免疫反应，屈服于迅速细菌感染。遗传性或其他操作的行为也可能影响这些...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>孵化器</td><td> ，波斯富街科学公司</td><td> I30BLLC8 I36VLC8 </td><td>任何孵化器的运行程序化光/温度时间表是适当的。 </td></tr><tr><td> 果蝇酶活性的监视器</td><td> Trikinetics公司，马萨诸塞州沃尔瑟姆</td><td> DAM2 </td><td>如其他地方6，本系统需要计算机接口，软件和其他配件。 </td></tr><tr><td>高硼硅玻璃管</td><td> Trikinetics公司，马萨诸塞州沃尔瑟姆</td><td> PGT-5x65 </td><td></td></tr><tr><td>微孔板闪烁和发光计数器</td><td>Perkin Elmer公司</td><td> TOPCOUNT NXT 12探测器</td><td>可以使用任何能够检测发光酶标仪为这种类型的报告基因分析。 TOPCOUNT包含多个检测器和一个自动化堆垛机，它是能够被编程，以连续读出从多个板。 </td></tr><tr><td>电泳凝胶成像8900 </td><td>阿尔法INNOTECH公司</td><td></td><td>细菌培养成像是可选的，任何一个数字成像系统与可视光的能力是足够的。 </td></tr><tr><td>微管牵引机</td><td> ，Tritech公司研究公司</td><td>成茂PC-10 </td><td></td></tr><tr><td></td><td></td><td></td><td> 耗材 </td></tr><tr><td>高硼硅玻璃毛细管</td><td>世界精密仪器有限公司</td><td> 1B100F-4 </td><td></td></tr><tr><td> 3毫升的注射器</td><td> Fisher Scientific则</td><td> BD 305482 </td><td></td></tr><tr><td>注射器针头</td><td> Fisher Scientific则</td><td> BD 305196 </td><td> 18 G - 切断的针的前端，以防止管子损坏。 </td></tr><tr><td>硅胶管，ID（0.030“）外径（0.065”）壁厚（0.018“） </td><td> VWR </td><td> 60985-706 </td><td>用于安装玻璃毛细管针注射器</td></tr><tr><td> 3通阀</td><td>美国Pharmaseal公司</td><td> K75 </td><td></td></tr><tr><td> Kontes颗粒杵无绳电机</td><td> Fisher Scientific则</td><td> K749540-0000 </td><td></td></tr><tr><td> Kontes颗粒杵</td><td> Fisher Scientific则</td><td> K749521-1590 </td><td></td></tr><tr><td>玻璃球3毫米</td><td> VWR </td><td> 26396-630 </td><td></td></tr><tr><td>微孔板微晶1 + </td><td> Thermo Scientific的</td><td> 7571 </td><td>范围吨96 - 孔板是适当的用于发光的 - 它们必须是不透明的。 </td></tr><tr><td> TopSeal-A :96井的微孔板</td><td>珀金埃尔默</td><td> 6005185 </td><td>微孔板按在粘接密封膜</td></tr><tr><td> D-荧光素钾盐</td><td>金生物技术公司</td><td> LUCNA </td><td></td></tr><tr><td></td><td></td><td></td><td> 软件 </td></tr><tr><td> Insomniac2 </td><td>可根据要求作者</td><td>定制编写的莱斯利阿什莫尔，博士（威斯敏斯特大学） </td><td>基于Matlab软件已被用于常规分析睡眠2,6,11 </td></tr><tr><td> Drosonex </td><td>可根据要求作者</td><td>自定义;写的托马斯·Coradetti（人行道软件） </td><td> A PC MSVC6计划，从原始数据文件，用于生存分析，收集与Trikinetics系统</td></tr><tr><td> Photoshop CS3的</td><td>土砖</td><td></td><td>可用于从数字图像获得号码的菌落形成单位/板（可选） </td></tr></tbody></table>

quantitative measurement of the immune response and sleep in drosophila

甲之间复杂的相互作用的免疫反应和主机行为已经描述了在很宽的范围内的物种。过量的睡眠，特别是，已知作为响应于在哺乳动物中1感染发生，最近也被描述在果蝇 2。人们普遍接受，睡眠在感染的主机是有益的，并且是很重要的一个强有力的免疫系统3,4的维护。然而，支持这一假说的实验证据表明，是有限的4，和多余的睡眠期间的免疫应答的功能尚不清楚。我们使用了多学科的方法来解决这个复杂的问题，果蝇果蝇的简单遗传模型系统，进行研究。我们使用标准的分析测量在果蝇的自发行为，睡眠，并演示了如何使用该法是衡量行为的苍蝇infecteD与细菌的致病性菌株的。该试剂盒也可以用于监测感染的持续时间期间在个别蝇的生存。免疫功能的其他措施包括清除感染，并是在果蝇的天然免疫反应的核心，一个关键的转录因子NFκB的激活能力的苍蝇。生存结果，并在感染细菌清除感染性和耐受性的指标。抵抗是指苍蝇清除感染的能力，而容差定义为主机的能力的限制感染造成的损害，从而尽管有很高水平的系统5内的病原体生存。在感染过程中的实时的NFκB活动的监测提供了洞察生存在感染过程中的分子机制。利用果蝇在这些简单的分析，有利于睡眠的遗传和分子生物学分析和免疫反应，这两个复杂的系统如何往复影响。

<ol><li>感染促进睡眠。在这个例子中，广交会-S（CS）的野生型苍蝇和突变果蝇缺乏NFκB基因，Relish的 （ 相对E20）14，装入两个DAM2活动监视器（每组32为每个基因型）和感染如上所述。苍蝇保持在恒定的光，消除影响生物钟的行为和感染2,7,8。 相对E20的突变体isogenized CS如前所述，11。这两组的苍蝇感染S.粘质沙雷氏菌 ，其结果如图3中所示。在?...

Watch this Scientific Journal Video about Quantitative Measurement of the Immune Response and Sleep in Drosophila at JoVE.com

Quantitative Measurement of the Immune Response and Sleep in Drosophila

要了解免疫反应和行为之间的联系，我们描述了一种方法来测量运动的行为中<em&gt;果蝇</em以及细菌感染的免疫反应能力的苍蝇安装监控求生存，细菌负荷，并实时活动的一个关键的调节先天免疫，NFκB。

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 ...

quantitative-measurement-immune-response-sleep

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Immunology and Infection

Quantitative Measurement of the Immune Response and Sleep in Drosophila

15.3K 次观看.  University of Pennsylvania Perelman School of Medicine. 要了解免疫反应和行为之间的联系，我们描述了一种方法来测量运动的行为中果蝇

视频: Quantitative Measurement of the Immune Response and Sleep in Drosophila

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

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. ...

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). ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

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 ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...