Name: quantification of the respiratory burst response as an indicator of innate immune health in zebrafish
Uploaded: 2013-09-12T16:45:00
Description: Watch this Scientific Journal Video about Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish at JoVE.com

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...

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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

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

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。

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

Research

JoVE Journal

Immunology and Infection

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

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

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

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

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

Quantitative Measurement of the Immune Response and Sleep in Drosophila

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

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

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

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

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

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

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

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

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

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

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