Name: bioluminescence imaging of nadph oxidase activity in different animal models
Uploaded: 2012-10-22T12:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

这项工作是由NIH RO1 AI079253和退伍军人事务部。

1。动物模型<ol><li>鼠标：使用P47 PHOX-/ -小鼠与年龄和性别匹配的C57BL6/DBA小鼠。获得的实验，从实验动物护理和使用委员会的批准。 </li><li>麻醉：使用连续的异氟醚麻醉诱导系统。汽化器的系统（VetEquip）填充用异氟醚（2-3％）。确认，老鼠是完全麻醉，观察呼吸，运动及角膜反射，在对外部刺激的反应。 </li><li>外科手术：磨砂手术区（台式），用70％的乙醇。戴无菌手套和干净?...

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“实时”测量的活性氧（ROS）可以通过使用荧光和化学发光探针在生活的动物。虽然荧光探针患有具有弱的信号-噪声比12，成像技术描述的是更敏感的检测发光基于鲁米诺-底物L-012的化学反应的活性氧。像所有的生物发光的成像技术，这种方法是有限的器官和组织的依赖于波长的光的吸收和散射。这些实验都集中在建立适当的测量ROS在野生型P47 PHOX-/ -小鼠的...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> L-012 </td><td>光化工美国公司</td><td> 120-04891 </td><td></td></tr><tr><td>酵母多糖</td><td> Sigma公司，圣路易斯，密苏里州</td><td> Z4250 </td><td></td></tr><tr><td>四氯化碳</td><td> Sigma公司，圣路易斯，密苏里州</td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH氧化酶是一种关键的酶介导的抗菌和抗真菌的宿主防御。 NADPH氧化酶除了其抗菌宿主防御中的作用，具有关键的信令功能，调节炎症反应1。因此，发展的方法来衡量“实时”的动力学NADPH氧化酶衍生的活性氧的产生是一个有价值的研究工具，了解相关的宿主防御，炎症和损伤的机制。 慢性肉芽肿病（CGD）是一种遗传性疾病的特点是严重感染和过度炎症反应的NADPH氧化酶。吞噬细胞的NADPH氧化酶的激活需要易位其胞浆亚基（PHOX，P67 PHOX的p47和p40的PHOX）和Rac的膜- ，绑定flavocytochrome（由一个gp91 phox和22页PHOX异二聚体）。损失在任何这些NADPH氧化酶成分结果在CGD的功能的突变。相似患者CGD，gp91 PHOX-缺陷小鼠和的p47 PHOX-缺陷小鼠有缺陷的吞噬细胞的NADPH氧化酶的活性和受损的宿主防御13，14。除了的吞噬细胞，其中含有上述成分的NADPH氧化酶，各种其他类型的细胞表达的NADPH氧化酶的不同的亚型。 在这里，我们描述了一种方法来量化ROS生产的活老鼠，并划定的贡献，NADPH氧化酶的活性氧的产生的炎症和损伤模型中的。这种方法是基于对ROS与L-012（鲁米诺类似物）反应，以发射由一个电荷耦合器件（CCD），被记录的发光。在原始描述的L-012探针，L-012-依赖化学发光完全废除了超氧化物歧化酶，表明在该反应中检测到的主要活性氧是超级超氧阴离子15。随后的研究表明，L-012，可以检测到的其他自由基，包括活性氮物种16，17。 kielland等17表明，局部应用佛波醇肉豆蔻酸乙酸酯，一种强力的NADPH氧化酶的活化剂，导致NADPH氧化酶-依赖性ROS生成使用发光探头L-012的小鼠中可检测到的。在这个模型中，他们表明：，P47 PHOX缺失的小鼠L-012-依赖的发光被取消。 我们比较了野生型小鼠和NADPH氧化酶缺乏P47 PHOX-/ -小鼠在以下三种模式：1）气管内给药的酵母，真菌细胞的促炎症墙衍生产品，可以激活NADPH氧化酶的活性氧的产生; 2）盲肠结扎穿刺（CLP），继发性急性肺部炎症和损伤腹内​​脓毒症模型; 3）口服四氯化碳（CCL），ROS依赖的肝损伤模型。这些模型是专门选定在非感染性炎症，多菌性败血症，毒素诱导的脏器损伤的情况下，分别评估NADPH氧化酶 - 依赖性ROS生成。在野生型小鼠比较，生物发光P47 PHOX-/ -小鼠，使我们能够描出P47 PHOX含NADPH氧化酶的生物发光信号在这些模型中所产生的ROS的具体贡献。 生物发光成像的结果表明增加细胞内ROS水平在野生型小鼠相比，P47 PHOX-/ -小鼠NADPH氧化酶产生的ROS的主要来源是炎症刺激。这种方法提供了一种微创的方法为“实时”监控产生的ROS在体内的炎症。

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH氧化酶在巨噬细胞的活性氧（ROS）的主要来源。由于ROS的短暂性的，它是难以衡量和监测中生活的动物的细胞内ROS水平。 ROS在活小鼠的串行量化的一种微创的方法。

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Research

JoVE Journal

Immunology and Infection

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...