定量测定的免疫反应和睡眠模式<em>果蝇</em
Summary
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>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。
Abstract
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。
Protocol
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已安装用于测量运动及睡觉的苍蝇，其中包括果蝇</em…
Representative Results
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?…
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Material Name | Company | Catalogue number | Comments |
| Equipment | |||
| Incubators | Percival Scientific, Inc. | I30BLLC8 I36VLC8 |
Any incubator capable of running programmed light/temperature schedules is appropriate. |
| Drosophila Activitiy Monitors | Trikinetics Inc., Waltham, MA | DAM2 | As described elsewhere6, this system requires a computer interface, software, and other accessories. |
| Pyrex Glass Tubes | Trikinetics Inc., Waltham, MA | PGT-5×65 | |
| Microplate scintillation and luminescence counter | Perkin Elmer | TopCount NXT 12 detector |
Any microplate reader capable of detecting luminescence can be used for this type of reporter assay. TopCount contains multiple detectors and an automated stacker; it is capable of being programmed to read continuously from multiple plates. |
| FluorChem 8900 | Alpha Innotech | Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient. | |
| Micropipette Puller | Tritech Research, Inc. | Narishige PC-10 | |
| Supplies | |||
| Borosilicate Glass Capillaries | World Precision Instrument Inc. | 1B100F-4 | |
| 3 ml Syringe | Fisher Scientific | BD 305482 | |
| Syringe Needles | Fisher Scientific | BD 305196 | 18 G – cut off the tip of the needle to prevent damage to the tubing. |
| Silicone Tubing, i.d. (0.030″) o.d. (0.065″) Wall Thickness (0.018″) | VWR | 60985-706 | Used for attaching glass capillary needles to a syringe |
| 3 Way Stopcock | American Pharmaseal Company | K75 | |
| Kontes Pellet Pestle Cordless Motor | Fisher Scientific | K749540-0000 | |
| Kontes Pellet Pestle | Fisher Scientific | K749521-1590 | |
| Glass balls 3mm | VWR | 26396-630 | |
| Microplate Microlite 1+ | Thermo Scientific | 7571 | Select 96-well plates that are appropriate for luminescence – they must be opaque. |
| TopSeal-A:96-well Microplates | PerkinElmer | 6005185 | Microplate Press-On Adhesive Sealing Film |
| D-Luciferin, Potassium Salt | Gold BioTechnology, Inc. | LUCNA | |
| Software | |||
| Insomniac2 | Available upon request to the authors | custom; written by Lesley Ashmore, Ph.D. (Westminster College) | Matlab based software that has been used routinely for analysis of sleep2,6,11 |
| Drosonex | Available upon request to the authors | custom; written by Thomas Coradetti (Sidewalk Software) | A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system |
| Photoshop CS3 | Adobe | Useful for obtaining numbers of cfu/plate from digital images (optional) |
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Tags
Quantitative MeasurementImmune ResponseSleepDrosophilaInfectionExcess SleepMammalsDrosophila MelanogasterRobust Immune SystemMultidisciplinary ApproachLocomotor BehaviorPathogenic Strain Of BacteriaDuration Of SurvivalImmune FunctionNFκBTranscription FactorInnate Immune ResponseBacterial ClearanceResistance To InfectionTolerance To Infection
Citer Cet Article
Kuo, T., Handa, A., Williams, J. A. Quantitative Measurement of the Immune Response and Sleep in Drosophila. J. Vis. Exp. (70), e4355, doi:10.3791/4355 (2012).