我们感谢博士戴安娜Baronas洛厄尔的手稿的批判性阅读。这项工作得到了科学种子格兰特的FAU查尔斯·E·施密特学院，并从埃利森医学基金会的老化奖学金KJ支持

1，XLD（木糖赖氨酸去氧胆酸盐）琼脂平板 XLD琼脂是沙门氏菌，这似乎是在XLD琼脂平板上菌落黑色选择性生长培养基。然而，如果有污染的担忧，一个普通的LB板可以被取代。 <ol><li>称取5毫升去离子水5.5克XLD琼脂和重悬。 </li><li>调匀，直到所有的琼脂是湿的。添加95毫升去离子水，直到所有肿块消失，介质完全悬浮。 </li><li>熬中期至完全溶解（不要高压灭菌）。 </li><li>冷却介质在室温至50℃。 </li><li>倒25毫升琼脂中的每个95×15毫米（直径×高度）板（用封口膜密封板可以储存在4℃可保存1个月）。 </li></ol> 2，线虫生长培养基（NGM）的RNAi喂养板 C.编制线虫 NGM板先前已描述19，这里的过程简要描述以添加抗生素氨苄青霉素和RNAi的化学诱导物异丙基-β-D-1-硫代半乳糖苷（IPTG）到NGM媒体，使RNAi的馈送板。 <ol><li>将3克氯化钠和2.5克细菌蛋白胨于1升去离子水。 </li><li>添加17克细菌用琼脂到媒体。 </li><li>高压灭菌的培养基45分钟，并在水浴中冷却介质到50°C。 </li><li>添加下列溶液:1毫升胆固醇（5毫克/毫升的95％乙醇），将1ml的1M的CaCl 2，加入1ml 1M的MgSO 4干燥，和25ml的1M磷酸钾缓冲液（pH 6.0）。拌匀。 </li><li>加入1 ml 1M的IPTG和500μl的氨苄青霉素（100 mg / ml的无菌水）。 </li><li>摇匀瓶内的溶液倒入60×15毫米（直径×高度）培养皿用吸管援助和25ml血清移液管下面的无菌程序。用12ml琼脂填满每块板。板可被储存在4℃下长达1个月。 </li></ol>必需的自噬基因BEC-1作为一个例子，研究宿主基因在抵御沙门氏菌感染的功能。实验步骤示于图1和表1。协议制备的RNAi治疗的动物进行感染如下，括号中给出各实验步骤的日子。 <ol><li>接种BEC-1 RNAi的喂养和放置-80℃冷冻的细菌片状变成补充100毫克/毫升氨苄青霉素（ 第1天 ）2毫升LB培养基介质控制空载体L4440的RNAi喂养细菌。在整个实验过程中重复此步骤，每周一次有新鲜的RNAi细菌。保存文化在4℃的冰箱时，不使用。 </li><li>种子上的RNAi板百毫升过夜RNAi的细菌培养。准备三BEC-1 RNA干扰和三个控制空v埃克特的RNAi板。培养板在37℃培养过夜（ 第2天 ）。 </li><li>从37℃培养箱中取出的RNAi板，让他们冷却至室温，在板凳上。拿起吃得饱L4野生型N2雌雄同体和他们转移到BEC-1 RNA干扰和控制空载体的RNAi板。将每盘三虫，一式三份板。在同一天，如在步骤3.2（3日 ）中所述制备的RNAi板。 </li><li>孵育的RNAi板的蠕虫在20℃培养箱中培养36〜40小时，传输蠕虫在步骤3.3准备新鲜相应的RNAi板。蠕虫被转移后，将培养板在20℃培养箱中培养64小时（4天 ）。 </li></ol> 4，准备为沙门氏菌感染<ol><li>连胜沙门氏菌 -80℃冷冻股票于1 XLD琼脂平板上孵育板在37℃过夜（5天 </st荣&gt;）。 </li><li>选择一个良好的孤立的黑色沙门氏菌的殖民地，振荡培养过夜（ 第6天 ），其接种于2毫升LB培养基，于37℃。 </li><li>种子80毫升沙门氏菌 1 C.过夜培养线虫 60×15毫米（直径x高度）NGM琼脂平板上并在总准备6片。培养板在室温下搅拌6小时。细菌培养应该干的，并形成对板（7天 ）草坪。 </li></ol> 5，传染的RNAi治疗的蠕虫沙门氏菌 <ol><li>转让BEC-1的RNAi治疗和控制空载体的RNAi治疗L4 N2雌雄同体（设立在步骤3中的蠕虫后代） 沙门氏菌板。将每板40蠕虫的3片为每个组。孵育虫板在20℃下48小时（7天 ）。 </li><li>准备一组新鲜的RNAi板如步骤3.1和3.2（7天及说明<stroNG&gt; 8天）。 </li><li> 48小时后感染，转移沙门氏菌感染蠕虫在步骤5.2准备相应的RNAi板和孵化在20℃（9日 ）。 </li></ol> 6，存活分析<ol><li>每日分数蠕虫的生存，并在产蛋时间蠕虫转移到新鲜对应RNAi板。准备一套新的RNAi板之前，每个蠕虫传输如步骤3.1和3.2中所述。触摸蠕虫身体（头部，中部和尾部）轻轻地用一个终端扁平铂丝。蠕虫的得分为死了，如果该蠕虫体内也没有动静观察。 </li><li>分数蠕虫每日或隔日的生存，并每周两次传送蠕虫新鲜对应RNAi板后，蠕虫停止产卵。 </li><li>毕竟虫死，汇集来自一式三份板作为一个数据集的生存数据。每组输入的生存数据到相应的统计软件，如格拉夫派得PRISM生成生存曲线，并进行Kaplan-Meier生存分析。整个实验被重复至少一次，以确认结论。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

线虫是一种简单的遗传模型有机体，吃细菌的营养源。因此，很容易与肠道病原体替代它的正常细菌的食物来调查C之间的相互作用线虫和所选择的病原体。在此协议被描述为沙门氏菌感染和C结合线虫喂食的RNAi治疗研究宿主基因在抵御沙门氏菌感染的作用。曾患协议公开C.线虫蠕虫致病菌他们的一生20时，包括沙门氏菌 。在本?...

自由生活的土壤线虫是用于研究许多生物学问题的简单和基因听话的模式生物。C.线虫显性存在自我施肥雌雄同体。雄性自发地由X染色体的过程中配子1,2非分离产生。在丰富的食物，C的存在线虫通过四个幼虫到成虫不断发展。温度也影响C。线虫的发展;更快的发展在较高温度下观察。在实验室中，C.线虫是培养在20℃下在琼脂平板上的标准温度与晶种菌大肠杆菌 （菌株OP50）作为食物1,2。 在过去的十年中，C.线虫已经成为一个无脊椎生物，研究宿主-病原体相互作用3-5。在自然界中，C.线虫吃细菌的养分吨源1,2。它的正常细菌实验室食品，OP50，可以很容易地被其他病原体检查C之间的相互作用线虫和任何选定的病原体。在这些条件下，肠道是感染的主要部位。事实上，广泛的细菌病原体已经显示出致死性感染C.线虫 3-5。 对革兰阴性菌沙门氏菌是一种胃肠道病原体引起人类食源性疾病的全球6,7。C.线虫是一种很好的模式主机沙门氏菌为这种细菌复制和呈现持续肠道感染8-10。C。线虫已经被用于识别新的和先前已知的沙门氏菌毒力因子11。有趣的是，C。线虫的免疫系统成功地限制了沙门氏菌复制。据此前报道，Inhib所自噬基因银行足球比赛呈现在C增加沙门氏菌复制线虫 ，导致受感染蠕虫10的过早死亡。巨自噬（本文中被称为自噬）是一个涉及亚细胞膜的重排螯合细胞质和细胞器运送到溶酶体降解12动态过程。自噬作用已有报道，以限制沙门氏菌复制在C线虫和哺乳动物10,13。 该三线虫基因组测序的第一个多细胞真核生物的基因组;它是响应于RNAi的治疗14-16。此外，RNA干扰可以被有效地通过使蠕虫摄取含有靶基因，被称为RNA干扰喂食16,17的双链RNA的细菌给药。已经生成的全基因组RNAi筛选16,18全基因组RNAi的喂养库。在本文中， 沙门氏菌感染亲母育酚结合的RNAi喂养，让测试C。感兴趣的线虫基因的防止沙门氏菌感染的能力。

<table><tbody><tr><td>LB Broth</td><td>Fisher</td><td>BP9723-500</td><td></td></tr><tr><td>XLD agar</td><td>EMD Chemicals</td><td>1.05287.0500</td><td></td></tr><tr><td>Bacto Agar</td><td>Fisher</td><td>DF0140-01-0</td><td></td></tr><tr><td>Peptone</td><td>Fisher</td><td>BP1420-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>S671-500</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher</td><td>C69-500</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher</td><td>M65-500</td><td></td></tr><tr><td>IPTG</td><td>Gold Biotechnology</td><td>12481C50</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-25</td><td></td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt;</td><td>ATCC</td><td>ATCC14028</td><td></td></tr><tr><td>Petri Dish 95 x 15 mm</td><td>Fisher</td><td>FB0875714G</td><td></td></tr><tr><td>Petri Dish 60 x 15 mm&amp;nbsp;</td><td>Fisher</td><td>08-757-13A</td><td></td></tr><tr><td>Falcon Serological pipet</td><td>Fisher</td><td>13-668-2</td><td></td></tr><tr><td>Falcon Express Pipet-Aid</td><td>Fisher</td><td>13-675-42</td><td></td></tr><tr><td>MaxQ6000 shaking incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>SHKE6000-7</td><td></td></tr><tr><td>Incubator</td><td>Percival</td><td>I-36DL</td><td></td></tr></tbody></table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

在过去的十年中，C.线虫已经成为一个无脊椎生物，研究宿主和病原体之间的相互作用，包括对革兰阴性菌鼠伤寒沙门氏菌的宿主防御。沙门氏菌建立持续性感染C.肠道线虫和结果在受感染动物的过早死亡。许多免疫机制C.已经确定对线虫对沙门氏菌感染捍卫。吞噬，进化上保守的溶酶体降解途径，已经示出，以限制沙门氏菌复制在C线虫和哺乳动物。这里，一个协议描述来感染C.线虫与鼠伤寒沙门氏菌 ，其中蠕虫暴露在沙门氏菌在有限的时间，类似沙门氏菌感染的人类。 沙门氏菌感染显著缩短C的寿命线虫&lt;/ em&gt;的。使用必需的自噬基因BEC-1作为一个例子，我们结合这种感染的方法与C线虫 RNAi的喂养方法，并表明该协议可以用于检查C的功能线虫的宿主基因在抵御沙门氏菌感染。由于C。线虫全基因组的RNAi文库的情况下，该协议使得它可以全面筛查C。线虫基因，对沙门氏菌等肠道致病菌保护利用全基因组的RNAi文库。

在20°C时，野生型N2蠕虫的寿命中位数为17天（ 图2A和表2）。 沙门氏菌感染显著降低N2蠕虫的寿命中位数天至10.5天（P = 0.0002，log-rank检验）（ 图2A ）。 如果C。线虫基因在抵御沙门氏菌感染中起重要作用，据预测，其抑制将传授易患沙门氏菌感染。事实上，相对于沙门氏菌感染控制的RNAi治疗N2动物的沙...

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A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

线虫已成为研究宿主-病原体相互作用的新遗传模型。在这里，我们描述了一个协议，感染C。线虫与鼠伤寒沙门氏菌加上双链RNA干扰干扰技术研究宿主基因在抵御沙门氏菌感染的作用。

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 ...

a-protocol-to-infect-caenorhabditis-elegans-with-salmonella

Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K 次观看.  Florida Atlantic University.  线虫已成为研究宿主-病原体相互作用的新遗传模型。在这里，我们描述了一个协议，感染C。线虫与鼠伤寒沙门氏菌加上双链RNA干扰干扰技术研究宿主基因在抵御沙门氏菌感染的作用。 

视频: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

科研

遗传学

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

免疫与感染

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 ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

用原生动物给鱼作动物感染的载体

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

慢性沙门氏菌感染诱发肠纤维化

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

秀 丽隐杆线虫 感染模型中的遗传免疫研究

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

生物学

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

一个协议感染

摘要

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此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

自动分离

高通量检测到的表型

一种高通量、高含量、液态的 Pathosystem线虫

用原生动物给鱼作动物感染的载体

慢性沙门氏菌感染诱发肠纤维化

秀丽隐杆线虫感染模型中的遗传免疫研究

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

自动化秀丽隐杆线虫的聚集定量

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

一个协议感染

摘要

探索更多视频

此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

自动分离

高通量检测到的表型

一种高通量、高含量、液态的 Pathosystem线虫

用原生动物给鱼作动物感染的载体

慢性沙门氏菌感染诱发肠纤维化

秀 丽隐杆线虫 感染模型中的遗传免疫研究

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

自动化秀丽隐杆线虫的聚集定量

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

秀丽隐杆线虫感染模型中的遗传免疫研究

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响