作者要感谢王超崔，为图4中，乌尔里克Nehrdich和戴维·威特的图像鱼照顾，和其他实验室成员的有益讨论楼埃丝特·科尔特，俯下身去，拉尔夫·卡瓦略。支持这项工作是由荷兰经济事务部的智能组合方案和教育，文化和科学，欧洲委员会第七框架项目ZF健康（健康F4-2010-242048），欧洲玛丽·居里部初始培训网络FishForPharma（PITN-GA-2011-289209），由澳大利亚NHMRC（637394）。澳大利亚的再生医学研究所是由维多利亚州政府和澳大利亚政府的拨款支持。

<ol>
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作者没有透露。

在这篇文章中所描述的感染方法经常被用来研究宿主先天免疫基因或细菌致病基因的功能。静脉微量注射方法（5和6.1协议）是最常用的此类研究。在尾静脉注射后血岛，是最方便的地点为静脉注射1日龄胚胎。作为尾静脉变得更加难以渗透在稍后阶段，我们更喜欢静脉注射部位的胚胎在2-3 DPF居维叶管道。在所有情况下，关键是一致的细菌数，应检查电镀注射接种菌落计数注入胚胎。必须考虑以下几个方面来实现可重复注射。首先，它必须有高品质的玻璃微毛细管注射针头。如果针尖过大，注入将创建足够大的穿刺孔出血发生和注入的细菌就会流出来的血液循环。其次，细菌必须直接注射进入血液循环。如果不是内静脉或注射液利差如果卵黄囊扩展到针尖，胚胎必须被丢弃的实验。第三，作为一个载体，注入研究 PVP40 海鱼将有助于防止细菌在玻璃针下沉。 PVP40提高了悬挂的同质化，导致整个注射时间更重现接种。 PVP40也可以用来为 S 鼠伤寒杆菌注射，但在这里是不那么重要，因为较大的规模和明亮的荧光细菌可以在实验过程中的视觉控制较注射接种。对于执业注射，我们建议使用酚红染料（1％V / V）或1微米（Invitrogen公司）不同颜色的荧光领域。一旦掌握技术，它需要大约30 minutES注入50-100胚胎，包括琼脂糖板对齐的胚胎和调整细菌的注射量所需的时间。 虽然是最常用的静脉注射进入血液岛（协议）或到居维叶（6.1协议）的管道，感染这个视频文章中所描述的替代路线已经证明有用的研究巨噬细胞和中性粒细胞趋化（协议6.2，6.3和6.4），研究减毒菌株（6.5协议）的增长，以适应高吞吐量的应用（6.6协议）4-8斑马鱼感染模型。所述的微注射方法也可用于注射病毒18，19，真菌孢子14，20或原虫（玛丽亚Forlenza，个人通讯）。注射在这部影片的文章中描述的过程是一个有益的补充皮下的细菌注入zebr最近描述的过程afish胚胎21。此过程被应用于研究组织表面发现有效的吞噬细菌，但由嗜中性粒细胞，吞噬细菌，在巨噬细胞，吞噬后的血液注入到充满液体的体腔的细菌或几乎无法。皮下注射胚胎定位相似，但为这个视频文章中所描述的尾部肌肉注射针插入皮肤下注入了体节的细菌。 重要的是要考虑微量注射本质上是一种人工感染途径。然而，早期胚胎，具有很高的耐外部暴露于细菌病原体。事实上，胚胎沐浴在菌悬液，浸泡实验，不能在我们手中22重现。虽然一些胚胎可能成为感染后浸泡，我们已经观察到的死亡率和中明确一个大的变化离子之间的个别胚胎在这种检测炎症的标记基因。微注射在这篇文章中介绍的方法实现重复性感染和研究细菌与宿主先天免疫细胞相互作用特别有用。量化个别胚胎细菌感染的有效途径是感染胚胎的荧光图像分析与定制，专用像素量化软件17，23。这种方法已被证明关联电镀感染胚胎后菌落计数结果。同样，每胚胎白细胞数量相对变化已在转基因白细胞荧光记者的数字图像分析量化，这种方法是适用于量化主机leukopoietic的响应感染24。 主机先天免疫基因的功能，可以在体内有效地调查相结合的描述感染模型吗啉击倒。吗啉代是人工合成的反义寡核苷酸，针对特定的RNA和基因产物的表达减少到1 -细胞期斑马鱼的胚胎注入其他视频文章中，有25本杂志10所示。在啉击倒研究，这是所有胚胎组相比上演正确细菌注射前非常重要的。这一点尤其重要，因为胚胎有一个发展中的免疫系统，随着时间的推移变得越来越能干。 目前可用来区分的主要先天免疫亚群，巨噬细胞和中性粒细胞在斑马鱼的胚胎中，有几个转的记者线。 MPX和LYZ发起人已用于生成记者行中性粒细胞16，26，27，并在csf1r（FMS）和MPEG1发起人被用来产生巨噬细胞的记者，14，28。虽然CSF此外在xanthophores表达1R（FMS），MPEG1表达完全是在巨噬细胞。在这部影片的文章显示，最近开发的MPEG1记者线条非常有用的成像巨噬细胞吞噬细菌。

<table border="1"><tbody><tr><td> 试剂 </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td>酚红</td><td>西格玛</td><td> P0290 </td></tr><tr><td>甘油</td><td> Sigma-Aldrich公司</td><td> 228210 </td></tr><tr><td> DIFCO米德尔7H10琼脂</td><td>流式细胞，迪肯森和公司</td><td> 262710 </td></tr><tr><td>边界层米德尔OADC富集</td><td>流式细胞，迪肯森和公司</td><td> 211886 </td></tr><tr><td> DIFCO米德尔7H9肉汤</td><td>流式细胞，迪肯森和公司</td><td> 271310 </td></tr><tr><td>边界层米德尔ADC的富集</td><td>流式细胞，迪肯森和公司</td><td> 211887 </td></tr><tr><td>吐温80 </td><td> Sigma-Aldrich公司</td><td> P1754 </td></tr><tr><td>聚乙烯吡咯烷酮（PVP40） </td><td> Calbiochem公司，默克</td><td> 529504 </td></tr><tr><td> N &lt;/ em&gt;的苯基（警察机动部队） </td><td> Sigma-Aldrich公司</td><td> P7629 </td></tr><tr><td>甲酸乙酯3 - 氨基磺酸盐（Tricaine，） </td><td> Sigma-Aldrich公司</td><td> A5040 </td></tr><tr><td>甲基纤维素</td><td> Sigma-Aldrich公司</td><td> M0387 </td></tr><tr><td> SeaPlaque琼脂糖（低熔点琼脂糖） </td><td>龙沙公司</td><td> 50100 </td></tr><tr><td> FluoSpheres（1.0微米，红色荧光（580/605）） </td><td> Invitrogen公司</td><td> F8821 </td></tr><tr><td> FluoSpheres（1.0微米，黄绿色荧光（505/515）） </td><td> Invitrogen公司</td><td> F8823 </td></tr></tbody></table>

infection of zebrafish embryos with intracellular bacterial pathogens

斑马鱼（ 斑马鱼 ）胚胎越来越多地用于研究在宿主-病原体相互作用1脊椎动物先天免疫系统的功能模型。先天免疫系统的主要类型的细胞，巨噬细胞和中性粒细胞，在胚胎发育的第一天前发展的适应性免疫反应的淋巴细胞的成熟。轻松获得大量的胚胎，其无障碍由于外部发展，胚胎和幼虫的光学透明度，广泛的遗传工具，广泛的突变资源和转基因记者线的集合，所有添加到多功能的斑马鱼沙门伤寒（伤寒沙门氏菌）和分枝杆菌模型。可以驻留细胞巨噬细胞，并经常被用来研究在斑马鱼胚胎的宿主-病原体相互作用。这两种感染过程细菌病原体是有趣的比较，因为S。鼠伤寒沙门氏菌感染是急性和致命的，在一天之内，而研究海鱼感染是慢性的，并可以成像幼虫第2阶段，3。微注射到胚胎（ 图1）细菌的网站，确定是否感染将迅速成为全身或将保持最初的本地化。建立一个快速的全身性感染，可通过微注射直接进入血液循环，通过尾静脉注射后血岛，或通过居维叶，卵黄囊上广泛流通渠道的心连接到主干血管导管的细菌。 1 DPF，在这个阶段的胚胎有phagocytically活跃的巨噬细胞，但尚未成熟的中性粒细胞，注射进入血液岛是首选。居维叶管道为2-3 DPF时，胚胎也已经开发出功能（过氧化物酶产）中性粒细胞，注射优先为t他注射部位。研究，对局部感染的骨髓细胞的定向迁移，细菌可注入尾肌，耳泡，或后脑心室4-6。此外，脊索，这似乎是一个结构通常无法髓细胞，是高度敏感的局部感染7。高吞吐量应用的一个有用的替代方法是细菌注射到胚胎在受精后8小时的蛋黄。结合荧光的巨噬细胞或中性粒细胞的荧光细菌和转基因斑马鱼线创建理想的情况下，宿主 - 病原体相互作用的多色成像。这部影片的文章将描述与S.静脉和局部感染斑马鱼胚胎的详细协议鼠伤寒或M。海鱼细菌和随后与先天免疫系统的细胞相互作用的荧光成像。

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

透明斑马鱼的胚胎已经证明有用的模型可视化和主机之间的先天免疫细胞和细胞内细菌病原体的功能，如研究的相互作用，<em&gt;鼠伤寒沙门氏菌</em&gt;和<em&gt;分枝杆菌</em&gt;。微注射细菌和多色荧光成像是在斑马鱼胚胎感染模型中的应用，所涉及的基本技术。

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Research

JoVE Journal

Immunology and Infection

43.5K 次观看.  Leiden University. 透明斑马鱼的胚胎已经证明有用的模型可视化和主机之间的先天免疫细胞和细胞内细菌病原体的功能，如研究的相互作用，鼠伤寒沙门氏菌和分枝杆菌。微注射细菌和多色荧光成像是在斑马鱼胚胎感染模型中的应用，所涉及的基本技术。

视频: Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

'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. ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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 ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...