这项工作是由过敏和美国国立传染病授予P01 A1078894到CAG

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作者宣称，他们没有竞争的经济利益。

巴斯德牙龈口腔感染模型提供了病原体引起的慢性炎症的局部和全身的网站学习的重要工具。这种独特的模式使得促进慢性炎症和免疫病理学宿主和病原体的具体机制的表征。此外，该模型可用于筛选新的治疗策略，包括免疫和药物干预。在这个协议中列出的步骤描述了成功运用这种模式和细节的方法来评估P的发生，发展和结局牙龈诱导的慢性炎症。 有...

慢性炎症的病理组织损伤和许多慢性疾病的人的统一特性的主要驱动力。这些疾病包括肿瘤，自身免疫和慢性炎症性疾病1。许多慢性疾病的病因仍不清楚，但可以理解为是复杂和多方面的，既涉及遗传易感性和开征环境因素。而炎症的perpetuators仍然难以捉摸，免疫激活的细胞和分子分布重叠相当与那些在宿主反应病原体2观察到的模式。 越来越多的证据暗示感染的研发微生物病原体和慢性炎症的进展及其临床表现多样2,3。病原体可诱导并通过颠覆了宿主的免疫系统，建立持续性感染，直接维持慢性炎症<sup&gt; 4。在不存在的微生物的持久性的，感染可能会沉淀从由分子模拟对自身抗原触发的免疫反应，改变自身抗原使得它们的免疫原性，或能够释放先前被屏蔽宿主抗原破坏的慢性炎症。然而，很少有具体的病原体被确定为一个特定的慢性疾病的普遍原因。相反，大多数现有的数据表明，病原体使用不同的机制来引起慢性炎症的临床表现及疾病预后的遗传易感宿主3来自各行各业。因此，由特定的病原体引起的慢性炎症可能对公共健康产生重大影响，以及治疗许多慢性疾病和预防的机制进行详细的了解。 尽管参与了慢性炎症的诱导和维持宿主和病原体的具体机制是了解甚少，在病原体引起的慢性炎症模型的进步已经开始促进我们这些过程的理解。 巴斯德牙龈口腔感染模型是病原体引起的慢性炎症，允许主机和病原体在局部（口腔骨质流失）促进慢性炎症的具体机制和系统网站（动脉粥样硬化），5,6分析了独特的，良好的特点小鼠模型。 牙龈是一种革兰氏阴性厌氧口腔病原体与人类牙周疾病，感染驱动的慢性炎症性疾病，其特征牙齿支持组织7的破坏。除了 ​​病理感染的初始点，越来越多的证据暗示P。牙龈诱导的开发和全身性疾病，包括动脉粥样硬化5的进展慢性炎症，这种疾病的特点是慢性inflammation中的动脉血管壁。特定病原体小鼠P的口腔感染牙龈诱导局部炎症反应而导致的破坏牙齿的支撑牙槽骨8 P。牙龈可以从感染小鼠的嘴被恢复42天感染后8和小鼠发展水平高的病原体特异性血清抗体滴度9。在动脉粥样硬化的使用载脂蛋白-E建立的小鼠模型- / -小鼠（的ApoE - / - ），口腔感染与体育牙龈引起慢性炎症，其驱动主动脉窦10和无名动脉11内炎性斑块沉积。 P的无名动脉内渐进性炎症牙龈感染的小鼠能够使用体内的MRI活的动物进行监测。从体育组织学，动脉病变牙龈感染的小鼠表现出的脂质陪伴积累增加尼德由活化的血管内皮细胞的，增加的免疫细胞浸润，以及升高的表达的炎性介质12。使用该模型在基因敲除小鼠的已阐明主机信令组件和炎症介质的作用，以及细胞驱动P.特异性相互作用牙龈诱导免疫病理学12 - 14。此外，利用定义的细菌的突变体的实验已经确定了临界P.牙龈毒力因子的局部和全身的网站15有助于慢性炎症。 本文详细介绍了方法学体育的评价诱发牙龈慢性炎症的局部和全身的网站。我们提供的牙槽骨骨量丢失的微型电脑使用阿米拉软件分析的详细协议。此外，我们定义序列在体内活的动物MRI检查的效用逐步在评估无名动脉内flammation。我们有方法的可视化和炎性斑块在动脉病变量化，并描述它们的组织学特征。利用转基因小鼠和定义的细菌的突变体，使该模型尤其适合用于识别主机和参与起始，进展，以及疾病的预后的微生物因子。此外，该模型可用于筛选新的治疗策略，包括疫苗和药物干预。

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			<td style="width:196px;">Company</td>
			<td style="width:132px;">Catalogue Number</td>
			<td style="width:88px;">Comments&#160;</td>
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			<td height="21" style="height:21px;">Anaerobic chamber DW Scientific Model MG500&#160;</td>
			<td>Microbiology International</td>
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			<td>Becton-Dickinson&#160;</td>
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			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>M5625-25G</td>
			<td></td>
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			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Becton-Dickinson&#160;</td>
			<td>212750</td>
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			<td height="21" style="height:21px;">Carboxymethyl cellulose (medium viscocity)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C-4888</td>
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			<td height="21" style="height:21px;">Sulfamethoxazole and Trimethoprim Oral Suspension 200 mg/40 mg per 5ml</td>
			<td>Hi-Tech Pharmacal</td>
			<td>NDC 50383-823-16</td>
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			<td height="21" style="height:21px;">&#956;CT 40&#160;</td>
			<td>Scanco</td>
			<td></td>
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			<td height="21" style="height:21px;">HistoChoice Tissue Fixative</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H2904</td>
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			<td height="21" style="height:21px;">Sudan IV</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S4261-25G</td>
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			<td height="21" style="height:21px;">vertical-bore 11.7T Avance spectrometer&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Paravision</td>
			<td>Paravision</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">rat anti-mouse F4/80</td>
			<td>Serotec</td>
			<td>MCA497R</td>
			<td></td>
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			<td height="21" style="height:21px;">rat anti-mouse TLR2</td>
			<td>eBioscience</td>
			<td>13-9021-80</td>
			<td></td>
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			<td height="21" style="height:21px;">Leica S4 Dissecting Scope</td>
			<td>Leica</td>
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			<td></td>
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			<td height="21" style="height:21px;">Microm HM 550 Cryostat</td>
			<td>Microm</td>
			<td></td>
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a mouse model for pathogen-induced chronic inflammation at local and systemic sites

慢性炎症的病理组织损伤和许多慢性疾病的人一个统一的特征，包括肿瘤，自身免疫，慢性炎症性疾病的主要驱动力。新出现的证据牵连的开发和慢性疾病的进展有多种临床表现的病原体引起的慢性炎症。由于慢性疾病的复杂和多因素病因学，设计性实验的因果关系的证明，并建立了机械的联系是人类几乎是不可能的。使用动物模型的一个优点是，既可能影响特定疾病的过程中遗传和环境因素可被控制。因此，在设计感染的相关动物模型表示在确定有助于慢性炎症主机和病原体的特定机制的关键步骤。 在这里，我们描述的病原体引起的慢性infla小鼠模型mmation在局部和全身部位感染后的口腔致病菌牙龈卟啉菌 ，与人类牙周疾病密切相关的细菌。特定病原体小鼠经口感染诱导产生破坏牙齿的支撑牙槽骨，牙周疾病的一个标志局部炎症反应。在动脉粥样硬化的建立小鼠模型中，感染与体育牙龈加速动脉窦和无名动脉内的炎性斑块沉积，伴随着激活血管内皮，增加免疫细胞浸润，并表达升高病灶内炎性介质。我们详细方法炎症的局部和全身部位的评估。利用转基因小鼠和定义的细菌的突变体，使该模型尤其适合用于识别主机和参与起始，进展，以及疾病的预后的微生物因子。 Additionally，则模型可用于筛选新的治疗策略，包括疫苗和药物干预。

使用适当的小鼠模型和口腔感染治疗方案， 第牙龈诱导慢性炎症和免疫病理学，在本地（口腔）和全身性位点（血管）（ 图1）。 在小鼠中，口腔感染与体育引起牙龈驱动牙齿的破坏支撑牙槽骨8局部炎症反应。 体育牙龈感染的小鼠发生血清抗体反应，此有机体，主要是IgG同种型9。在图4中示出的结果是代?...

Watch this Scientific Journal Video about A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites at JoVE.com

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

动物模型中已被证明是在界定有助于慢性炎症的发展宿主和病原体的具体机制的宝贵工具。在这里，我们描述口腔感染的小鼠模型与人类病原体牙龈卟啉和细节的方法，以评估炎症的进展，在局部和全身的部位。

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

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Immunology and Infection

17.4K 次观看.  Boston University School of Medicine. 动物模型中已被证明是在界定有助于慢性炎症的发展宿主和病原体的具体机制的宝贵工具。在这里，我们描述口腔感染的小鼠模型与人类病原体牙龈卟啉和细节的方法，以评估炎症的进展，在局部和全身的部位。 

视频: A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

科研

免疫与感染

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

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 ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.