作者要感谢支持本稿件相关的力士标记E.欧洲委员会第七框架计划（PIOF-GA-2009-255466）和爱尔兰健康研究委员会（HRA_POR/2010/138）。 大肠杆菌是一种礼物科马克·加恩，科克大学博士。

1。诱发肿瘤<ol><li>对于常规的肿瘤诱导，悬浮在200μl的无血清培养基中的细胞的的最低致瘤剂量皮下注射（SC）的到感染的侧翼免费的6-8周龄的雌性Balb / C或无胸腺MF1-nu/nu小鼠的中N = 6（哈伦，英国牛津）（1×10 6 4T1细胞），使用21号注射器针头。用于接种的细胞的存活率大于95％，使用血细胞计数器和台盼蓝染料排除（Gibco）中通过目视计数来确定。 </li><li>肿瘤成立后，肿瘤被允许的成长和发展，并进行了监测，每周两次。根据公式 V =（从头2）Π/ 6， 其中 a为肿瘤的最长直径之和 b是最长的直径垂直的直径为a，计算肿瘤体积。 </li></ol> 2。细菌制剂<ol><li>在此protoco使用的细菌菌株L是E。大肠杆菌K-12 MG1655株，非蛋白毒素表达株，窝藏luxABCDE编码，使所要检测的细菌的质粒BLI。E.大肠杆菌 MG1655含有集成luxABCDE的生长需氧条件下，在37℃下在LB培养基（Sigma-Aldrich公司，爱尔兰）与300微克/毫升红霉素（EM）的补充。的生物发光衍生MG1655创建，使用质粒p16S 勒克斯其中包含组成P的帮助 luxABCDE操纵的。 </li><li>对于准备对小鼠给药，在LB培养基中培养的培养物在37℃下在摇床上以200rpm中期日志相（在600nm处的光密度）。收获细菌，通过离心分离（6,000×g离心5 min），用PBS（Sigma公司）洗涤，并稀释在PBS中1×10 7菌落形成单位（cfu）/ ml的静脉注射，或1×10 10管饲法。 </li></ol> 3。细菌管理员TION <ol><li>将小鼠随机分为实验组，当肿瘤达到约100毫米的体积。对于静脉内给药，内敛小鼠每个收到的10 6个细胞在100μl，直接注射到横向的尾静脉，用28G注射器针头。每个接种活菌计数进行回顾性电镀。 </li><li> GIT殖民研究中，10 9细菌细胞在每100微升鼠标口服灌胃，连续三天。预先存在的共生细菌水平明显下降之前，添加5毫克/毫升链霉素的鼠标饮用水喂养7天前开始口灌胃1。 </li></ol> 4。生物发光成像<ol><li> 2D 体内 BLI成像进行使用IVIS100（钳）。在特定的时间点后细菌性管理，小鼠Caliper的XGI-8气体​​麻醉的麻醉在高灵敏度的2-5分钟，用3％的异氟烷​​，和全身图像分析系统中进行的IVIS 100系统。 </li><li>的三维成像，麻醉小鼠放置在鼠标摄像穿梭背侧成像（IVIS频谱，钳）的光学成像系统的内部。要获得的细菌荧光素酶信号的三维光学重建的图像，发射滤光器的波长范围从500-580纳米，与箱16采集时间3-4分钟每滤波器信噪比最大化使用。该图像采集序列的一部分，结构光图像定义一个高度的地图。本地图的时间输入的漫射光成像体层摄影术（DLIT）重建的算法被用来形成一个三维的光学图像，用一个非负最小二乘优化2。 </li><li>图像分析：利用活体成像软件（钳）地区的利益进行了定性和定量分析。 </li></ol> 5。代表结果在这项研究中，非致病共生细菌的大肠杆菌K-12 MG1655表达luxABCDE操纵子是四轴承SC 4T1异种移植肿瘤的小鼠给药。细菌勒克斯信号，特别是在检测到肿瘤的小鼠IV给药后（ 图2）。从样品小鼠细菌培养回收验证存在的活菌数和检测的光的量（ 图3）之间的线性关系。 在体内成像口服给药的共生细菌在GIT中也取得了使用3D BLI。 <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> 图1。协议的时间轴 。诱导小鼠皮下肿瘤，细菌和管理后，肿瘤发展计划（100毫米）。活体小鼠BLI IM年龄在不同的时间点后细菌性管理（箭头表现出典型的）。 <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="图2" /> 图2。管理E.在MF1 nu / nu小鼠和E. 大肠杆菌 MG1655 luxABCDE荷瘤小鼠皮下 4T1肿瘤诱导大肠杆菌 MG1655 luxABCDE管理后，肿瘤的发展。每个动物接受10 6细胞直接注射到尾静脉。在研究过程中在四个时间点（黑点z轴和图像）与随后的恢复的活菌（菌落形成单位）从样本处死小鼠肿瘤（条形图）对小鼠进行成像。特别是在观察肿瘤随着时间的推移（代表鼠标每个时间点所示）中的细菌数量和质粒基因表达增加。 <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">点击这里查看大图</a> 。<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="图3" /> 图3。肿瘤后，BLI在不同的时间点后静脉给药， 瘤内细菌数量和生物发光的关系。存活的细菌在肿瘤中列举的体外细菌培养。登录值，细菌总数（CFU）与​​体内生物发光单元的绘制。一个强大的细菌的数量和细菌的生物发光信号之间的相关性R 2 = 0.9717 1。 <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="图4" /> 图4。 3D IVIS图片的小鼠胃肠道的殖民地E.大肠杆菌 MG1655的被殖民统治的小鼠口服10 9 cfu的E. GIT 大肠杆菌连续三天。从3D成像的殖民鼠标的样本孤立的形象显示。3D影像显示鼠标数字地图集的骨架，提供解剖学注册。E.大肠杆菌 MG1655的生物发光是在绿色可见在较低的，和紫色在较高的水平。

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没有利益冲突的声明。

在基因治疗的情况下，交付的基因治疗给患者使用生物制剂表现出极大的承诺3-5。如病毒，细菌先天的生物学性质允许高效的DNA传递到细胞或组织，特别是在癌症的上下文中。它已被证明细菌是自然能归巢到肿瘤全身给药时，所得的复制水平高的地方，无论是外部的（非侵入性的物种）或肿瘤细胞（病原体）内。癌特异性的细菌复制最初归因于缺氧的固体肿瘤的性质（低O 2水平）...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 4T1细胞系</td><td> ATCC </td><td> CRL-2539 </td><td>同系来自一个自发​​产生的BALB / c小鼠乳腺肿瘤的乳腺癌模型</td></tr><tr><td> DMEM </td><td> Sigma-Aldrich公司</td><td> D6429 </td><td> Dulbecco改良的Eagle培养基 </td></tr><tr><td> PBS </td><td> Sigma-Aldrich公司</td><td> D8537 </td><td>磷酸盐缓冲液</td></tr><tr><td>精诺真IVIS </td><td>卡尺生命科学</td><td></td><td> IVIS 100的二维成像IVIS为3D的频谱。 </td></tr><tr><td>卢里亚肉汤米勒（LB） </td><td> Sigma-Aldrich公司</td><td> L2542 </td><td>生长培养基E.大肠杆菌 </td></TR&gt; <tr><td>红霉素</td><td> Sigma-Aldrich公司</td><td> E5389 </td><td>抗生素</td></tr><tr><td>链霉素</td><td> Sigma-Aldrich公司</td><td> S9137 </td><td>抗生素</td></tr><tr><td> MF1nu/nu小鼠</td><td>哈伦（英国） </td><td> 069（女）/ 070（NU / +） </td><td> HSD：裸鼠Foxn1nu的 </td></tr><tr><td> Balb / c小鼠</td><td>哈伦（英国） </td><td> 066 </td><td>单倍型：H-2天 </td></tr><tr><td>灌胃针</td><td>兽医技术解决方案（英国） </td><td> DE009 </td><td> 22G×38毫米直灌胃针</td></tr><tr><td>注射器静脉注射</td><td> BD Biosciences公司</td><td> 309309 - 1毫升</td><td>胰岛素注射器与28 G X½英寸的微细四针。 </td></tr><tr><td>肿瘤接种的注射器</td><td>布劳恩</td><td> 9161376V </td><td> Omnifix 26 G X＆frac12寸毫针</td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

本视频介绍了使用全身生物发光成像（BLI）的细菌贩运活体小鼠为研究对象，利用细菌基因和细胞治疗癌症的重点。存在的细菌有吸引力的载体类用于癌症治疗，全身用药后，肿瘤内优先发展拥有一种天生的能力。细菌的设计，以表达勒克斯基因盒许可证BLI检测的细菌和兼肿瘤部位。肿瘤内随着时间的推移的位置和水平的细菌，可以容易地检查，在两维或三维可视化。的方法，是适用于范围广泛的细菌物种和肿瘤异种移植类型。本文介绍了发光细菌在皮下荷瘤小鼠的协议分析。可视化的共生细菌的胃肠道（GIT）中由BLI也被描述。这种功能强大，而且价格便宜，实时成像战略的代表中TS细菌在体内的背景下，特别是基因治疗，癌症研究，传染病研究的一个理想的方法。此视频概述了研究勒克斯标记E.程序大肠杆菌活体小鼠，展示的空间和时间的读出可以实现利用BLI的IVIS系统。

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

本文介绍了管理<em&gt;勒克斯标签</em细菌对小鼠和随后的<em在体内</em分析IVIS生物发光成像。

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K 次观看.  University College Cork. 本文介绍了管理勒克斯标签

视频: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

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

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.