我们感谢H.弗莱明批判性阅读和编辑的稿件。这项工作是由Misrock博士后奖学金（TD）和NDSEG研究生奖学金（AP）的支持。 SNB是一个霍华德休斯医学研究所研究员。

1。细胞的制备<ol><li>使用标准的细胞培养技术的传代细胞系。在这个实验中，我们使用OVCAR-8细胞（NCI DCTD肿瘤库，冯检基，MD）。细胞生长到目标汇合的80 - 100％。 </li><li>用5ml胰蛋白酶消化5分钟孵育细胞，然后加入5 ml RPMI培养基+ FBS的胰蛋白酶失活。 </li><li>收获和血球计数细胞。重悬于无酚红的DMEM中，在目标浓度为5×10 7个细胞/ ml，或约200微升每烧瓶。 </li><li>添加15％降低生长因子基底膜（BD Biosciences公司），并保持细胞悬液植入直到冰。 </li></ol> 2。肿瘤植入<ol><li> 4周龄雌性NCR /怒江小鼠的使用3％的异氟醚麻醉并等待大约5分钟，以确保麻醉过深。兽医软膏，防止肌肤干燥，而在麻醉状态下对眼睛。 </li><li>负载的细胞（100微升每个肿瘤）的1毫升注射器，附加27 1/ 2压力表针。 </li><li>对于每两个双边的后侧翼注射部位，用钳子轻轻提起皮肤做一个帐篷，并注入细胞基底部。小心不能刺入过深，产生血液。使用镊子轻轻取出注射器没有飞溅。 </li><li> 2 - 4毫米的肿瘤直径为10 - 20天，直到达到每天监视肿瘤生长。 </li></ol> 3。活菌制剂<ol><li>过夜培养的细菌在3毫升LB培养基+氨苄青霉素-80℃冰柜股票的或冷冻板。在这个实验中，我们使用了S。鼠伤寒沙门氏菌株ELH430（SL1344 PhoPQ AROA）。 </li><li>早晨，培养1:100稀释过滤LB和成长OD600 0.4 - 0.6。 </li><li>旋转，并洗涤3次，在磷酸盐缓冲盐水（PBS），重悬在终OD 600为0.1（约1×10 7个细胞/ ml）。 </li></ol> 4。菌素注射液<ol><li>麻醉动物D位置在其一侧用尾巴指向你的惯用手。兽医软膏，防止肌肤干燥，而在麻醉状态下对眼睛。 </li><li>尾静脉扩张，用温水或热灯。 </li><li>负载的细菌（每只小鼠100微升），在1毫升注射器，只是小于90度的弯曲的前端。 </li><li>尖尾静脉注射器对准，穿透深度浅（应该感到无阻力），并注入细菌。对于一个成功的注射观察血流量位移。 </li></ol> 5。鼠标成像<ol><li>麻醉动物感应室，然后将每个鼠标成像平台上。兽医软膏，防止肌肤干燥，而在麻醉状态下对眼睛。保持精确的定位，以确保时间点之间的定量结果。 </li><li>图片动物使用IVIS频谱成像系统（卡尺生命科学）。如果为超过1动物，地方光他们之间的障碍，以防止交叉亮起的税款。动物应首先成像于腹面，确认本地化细菌。 </li><li>图片动物背侧使用IVIS每隔2小时的实验的持续时间（36小时）。生成一个时间过程，对于一个给定的投资回报率。 </li></ol> 6。量化细菌体内的分布<ol><li>使用CO 2的吸收性尿布的背面上，并将其置于动物安乐死。 </li><li>将动物暴露两后肢的侧翼皮下肿瘤。剪切周围的皮肤组织，为了分离，除去肿瘤。控股肿瘤用镊子去除皮肤使用一个反相剪刀运动。当大部分的皮肤已经分开，完全切除肿瘤，用小镊子。 </li><li>放置在预先称重的微量离心管中的器官。由于这些管的质量可以显着不同，重要的是要预先称重定量结果。 </li></ol> 7。量化细菌体内的分布<ol><li>对于每一个时间点，消费税和重的肿瘤。 </li><li>加入500μlPBS + 15％甘油和均质使用一个组织（BioSpec）的Tearor的。冲洗3次，用乙醇样品之间的均化器。 </li><li>生成序列稀释和菌落计数板。板多单盘上使用预先切片板稀释。 </li></ol>

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没有利益冲突的声明。

使用此过程中，我们是能细菌的肿瘤的生长和基因表达的动态课程生成时间。虽然这些测量的常规在体外进行分批培养或微流体装置中，它们是更难以执行在体内 。 有几种修改，可以应用这些方法。虽然我们使用OVCAR-8细胞系，以产生我们的小鼠模型，也可以使用一些其他的细胞系等价。例如，可用于luciferized细胞系，允许肿瘤和细菌荧光素酶通道的三维共定位?...

合成生物学进展迅速，在过去的十年中，现在的定位是影响能源和健康的重要问题。然而， 体内遗传电路开发设计标准的安全问题，并没有扩展到临床上已放缓。加快高影响医疗应用将需要利用接口直接与医疗基础设施，遗传受控的实验室环境以外的电路，功能，安全和临床公认的微生物宿主的方法。 优先在肿瘤生长的能力，由于癌症治疗研究已进行了一些菌株。这些都包含C. novyi，大肠杆菌，V. cholorae，长双歧杆菌 ，和S。鼠伤寒沙门氏菌S. 3-8。 鼠伤寒沙门氏菌产生了特别令人感兴趣的，因为他们已经在数9-12的人体临床试验显示出安全性和耐受性。这些细菌学<html>一个最初创建的抗肿瘤效果，通过刺激宿主的免疫系统和癌细胞代谢所需的营养物质的耗竭。通过基因修改，后来又增加治疗货物的生产。虽然这些研究的重要进展，在肿瘤的治疗中使用的细菌，多数依靠现有努力的高水平表达，通常会产生高剂量的交付，脱靶效应，与主机发展的阻力13-16 。 现在，合成生物学可能会增加可编程的货物生产，利用设计计算遗传电路，可以执行先进的传感和交付17-20。这些电路可被设计为作为检测肿瘤特异性刺激自律和货物的生产必要的运输系统。然而，研究这些电路在体内的功能迄今一直具有挑战性。 由于质粒ve_content"&gt;合成电路的共同框架中，我们描述了一种基于质粒的基因表达在体内使用的小鼠模型的动态特性的方法，这些方法利用时间推移发光成像和定量测量的生物分布。一起这些方法提供了一个框架， 在体内的临床应用研究基于质粒网络。

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>BD Biosciences</td>
 <td>309602</td>
 <td></td>
 </tr>
 <tr>
 <td>3/10 cc Insulin Syringe</td>
 <td>BD Biosciences</td>
 <td>309301</td>
 <td></td>
 </tr>
 <tr>
 <td>PrecisionGlide Needle </td>
 <td>BD Biosciences </td>
 <td>301629</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 (1X), 
 liquid, with L-glutamine</td>
 <td>Invitrogen</td>
 <td>11875-119 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sigma Aldrich</td>
 <td>A0166-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>LB Agar Broth</td>
 <td>Sigma Aldrich</td>
 <td>L2897</td>
 <td></td>
 </tr>
 <tr>
 <td>Luria-Bertani broth</td>
 <td>BD Biosciences</td>
 <td>244610</td>
 <td></td>
 </tr>
 </tbody>
</table>

measuring growth and gene expression dynamics of tumor-targeted s. typhimurium bacteria

这些实验的目的是产生定量的时间-过程数据的生长和基因表达动力学的减毒S.鼠伤寒沙门氏菌的细菌菌落生长肿瘤内。 我们生成的模型移植瘤小鼠皮下注射人类卵巢癌细胞株OVCAR-8（NCI DCTD肿瘤仓库，弗雷德里克，MD）。 我们转化减毒菌株的鼠伤寒沙门氏菌细菌（ELH430:SL1344 phoPQ 1）组成性表达的荧光素酶（luxCDABE）质粒的可视化2。这些菌株专门殖民肿瘤基本上是无毒的，而其余的鼠标1。 一旦建立可测量的肿瘤，细菌具有不同的剂量经尾静脉静脉注射。肿瘤局部，细菌基因表达的实时监控，60小时使用的过程在体内成像系统（IVIS）。在每个时间点，肿瘤被切除，匀浆，接种相关基因表达数据的定量细菌菌落。 总之，这种数据产生在体内生长和生长肿瘤内的细菌的基因的表达动力学的定量测量。

使用这个协议中，我们能够产生的肿瘤靶向细菌在体内生长和基因表达的动力学数据。在图1中概括的全部工作流程。 在第一阶段中，我们注入细菌（绿色）和图像的动物使用IVIS发光（蓝色）作为一个记者来衡量基因表达。 然后，在第二阶段中，我们的消费税，均质化，和菌落计数板肿瘤的数量来确定在肿瘤生长的细菌。 <p class="jo...

Watch this Scientific Journal Video about Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria at JoVE.com

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

这些实验的目的是产生定量的时间 - 过程的减毒的生长和基因表达的动力学数据<em&gt; S。鼠伤寒沙门氏菌</em&gt;内肿瘤生长的细菌菌落。此视频覆盖肿瘤细胞制备和植入，活菌制剂和注射，整个动物发光成像，肿瘤切除，及菌落计数。

肿瘤靶向性生长和基因表达的动力学测量<em&gt; S。鼠伤寒沙门氏菌</em&gt;细菌

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

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Immunology and Infection

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

12.3K 次观看.  Massachusetts Institute of Technology. 这些实验的目的是产生定量的时间 - 过程的减毒的生长和基因表达的动力学数据 S。鼠伤寒沙门氏菌内肿瘤生长的细菌菌落。此视频覆盖肿瘤细胞制备和植入，活菌制剂和注射，整个动物发光成像，肿瘤切除，及菌落计数。

视频: Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

科研

免疫与感染

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

一个协议感染<em&gt;秀丽隐杆线虫</em&gt;与<em&gt;鼠伤寒沙门氏菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.