وأيد هذا العمل من قبل مؤسسة العلوم الوطنية في إطار منحة # IOS-1025627 وقبل المعاهد الوطنية للصحة في إطار منحة # 1R21NS078582-01 إلى JAW

<ol>
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الإعلان عن أي تضارب في المصالح.

هذا البروتوكول يحدد هذا النهج للتحقيق في كيفية السلوك، ولا سيما النوم، يرتبط المعلمات الاستجابة المناعية. وتشمل هذه المعايير الحمل البكتيري، نتيجة بقاء، والنشاط كما تقاس NFκB مراسل وسيفيراز في الجسم الحي. معا هذه المعلمات توفير المعلومات حول مدى ذبابة يمكن محار...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم المادة </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td></td><td></td><td></td><td> معدات </td></tr><tr><td> حاضنات </td><td> بيرسيفال العلمية، وشركة </td><td> I30BLLC8 I36VLC8 </td><td> أي حاضنة قادرة على تشغيل ضوء المبرمجة / جداول درجة الحرارة المناسبة. </td></tr><tr><td> ذبابة الفاكهة Activitiy شاشات </td><td> Trikinetics شركة، التايم، MA </td><td> DAM2 </td><td> كما هو موضح في مكان آخر 6، يتطلب هذا النظام واجهة الكمبيوتر، والبرمجيات، وغيرها من الملحقات. </td></tr><tr><td> بيركس زجاج أنابيب </td><td> Trikinetics شركة، التايم، MA </td><td> PGT-5x65 </td><td></td></tr><tr><td> التلألؤ صفيحة ميكروسكوبية ومكافحة التلألؤ </td><td>بيركن إلمر </td><td> TopCount NXT 12 كاشف </td><td> ويمكن استخدام أي قارئ صفيحة ميكروسكوبية قادرة على اكتشاف التلألؤ لهذا النوع من الفحص مراسل. TopCount يحتوي على أجهزة الكشف عن عدة ومكدس الآلي، بل هو قادر على المبرمجة لقراءة لوحات باستمرار من متعددة. </td></tr><tr><td> FluorChem 8900 </td><td> ألفا Innotech </td><td></td><td> التصوير الثقافات البكتيرية اختيارية؛ أي نظام التصوير الرقمي مع القدرة البصرية ضوء كافية. </td></tr><tr><td> Micropipette بولير </td><td> Tritech البحوث، وشركة </td><td> Narishige PC-10 </td><td></td></tr><tr><td></td><td></td><td></td><td> مؤن </td></tr><tr><td> البورسليكات الزجاج الشعيرات </td><td> العالم آلات دقيقة شركة </td><td> 1B100F-4 </td><td></td></tr><tr><td> 3 مل الحقنة </td><td> فيشر العلمية </td><td> BD 305482 </td><td></td></tr><tr><td> حقنة الإبر </td><td> فيشر العلمية </td><td> BD 305196 </td><td> 18 غرام - قطع رأس الإبرة لمنع تلف الأنبوب. </td></tr><tr><td> أنابيب السيليكون، رقم (0.030 &quot;) التطوير التنظيمي (0.065&quot; سمك الجدار) (0.018 &quot;) </td><td> VWR </td><td> 60985-706 </td><td> تستخدم لربط الإبر الزجاج الشعرية لحقنة </td></tr><tr><td> 3 طريقة محبس </td><td> Pharmaseal شركة أمريكية </td><td> K75 </td><td></td></tr><tr><td> Kontes بيليه مدقة للسيارات اللاسلكي </td><td> فيشر العلمية </td><td> K749540-0000 </td><td></td></tr><tr><td> Kontes بيليه مدقة </td><td> فيشر العلمية </td><td> K749521-1590 </td><td></td></tr><tr><td> كرات زجاجية 3MM </td><td> VWR </td><td> 26396-630 </td><td></td></tr><tr><td> صفيحة ميكروسكوبية Microlite 1 + </td><td> الحرارية العلمية </td><td> 7571 </td><td> سلك96-T وحات جيدة تتناسب التلألؤ - يجب أن تكون مبهمة. </td></tr><tr><td> TopSeal :96-A-Microplates جيدا </td><td> PerkinElmer </td><td> 6005185 </td><td> صفيحة ميكروسكوبية الصحافة، وفي فيلم لاصق ختم </td></tr><tr><td> D-وسيفيرين وملح البوتاسيوم </td><td> الذهب التكنولوجيا الحيوية، وشركة </td><td> LUCNA </td><td></td></tr><tr><td></td><td></td><td></td><td> البرمجيات </td></tr><tr><td> Insomniac2 </td><td> متوفرة عند الطلب للمؤلفين </td><td> مخصص؛ أشمور كتبها ليزلي، دكتوراه (وستمنستر كلية) </td><td> MATLAB البرمجيات القائمة التي استخدمت بشكل روتيني لتحليل النوم 2،6،11 </td></tr><tr><td> Drosonex </td><td> متوفرة عند الطلب للمؤلفين </td><td> مخصص؛ Coradetti كتبها توماس (برمجيات الرصيف) </td><td> A PC MSVC6 برنامج يستخدم لتحليل البقاء على قيد الحياة من ملفات البيانات الخام التي تم جمعها معTrikinetics النظام </td></tr><tr><td> فوتوشوب CS3 </td><td> لبن </td><td></td><td> المفيدة للحصول على أرقام لوحة / كفو من الصور الرقمية (اختياري) </td></tr></tbody></table>

quantitative measurement of the immune response and sleep in drosophila

وقد وصفت تفاعل معقد بين الاستجابة المناعية والسلوك المضيفة في مجموعة واسعة من الأنواع. النوم الزائد، على وجه الخصوص، ومن المعروف أن تحدث كاستجابة للإصابة في الثدييات (1) وأيضا في الآونة الأخيرة تم وصفها في ذبابة الفاكهة الدروسوفيلا 2. ومن المسلم به عموما أن النوم هو مفيد إلى المضيف خلال وجود عدوى، وأنه من المهم للحفاظ على جهاز المناعة قويا 3،4. ومع ذلك، فإن الأدلة التجريبية التي تدعم هذه الفرضية هو محدود 4، ووظيفة النوم الزائد خلال استجابة مناعية لا تزال غير واضحة. وقد استخدمنا نهج متعدد التخصصات لمعالجة هذه المشكلة المعقدة، ولقد أجريت دراسات في النظام النموذجي الجينية البسيطة، وذبابة الفاكهة ذبابة الدروسوفيلا. نستخدم الفحص القياسية لقياس السلوك الحركي والنوم في الذباب، وتبين كيفية استخدام هذا الاختبار لقياس السلوك في بالعدوى الذبابد مع سلالة من البكتيريا. هذا الفحص مفيد أيضا لرصد مدة بقاء الفرد في الذباب أثناء العدوى. تدابير إضافية من وظائف المناعة تشمل القدرة من الذباب لمسح وجود عدوى وتفعيل NFκB، وهو عامل النسخ المفتاح الذي تم المركزي للاستجابة المناعية الفطرية في ذبابة الفاكهة. كل نتيجة بقاء وإزالة البكتيريا خلال العدوى معا مؤشرات المقاومة والتسامح للإصابة. المقاومة يشير إلى قدرة الذباب لمسح وجود عدوى، بينما يتم تعريف التسامح وقدرة المضيف للحد من الأضرار الناجمة عن وجود عدوى، وبالتالي البقاء على قيد الحياة على الرغم من مستويات عالية من الممرض داخل النظام 5. في الوقت الحقيقي رصد النشاط NFκB خلال العدوى رؤية ثاقبة آلية الجزيئية للبقاء على قيد الحياة خلال العدوى. استخدام ذبابة الفاكهة في هذه المقايسات مباشرة يسهل التحليل الجيني والجزيئي من النوموتتأثر بالتبادل الاستجابة المناعية وكيفية هذين النظامين المعقدة.

<ol>
	<li>Infection promotes sleep. In this example, Canton-S (CS) wild type flies and mutant flies lacking an NF&kappa;B gene, Relish (RelE20) 14, were loaded into two DAM2 activity monitors (n=32 for each genotype) and infected as described above. Flies were maintained in constant light to eliminate the influence of the circadian clock on behavior and infection2,7,8. The RelE20 mutants were isogenized to CS as described previously11. Both sets of flies were infe...

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Quantitative Measurement of the Immune Response and Sleep in Drosophila

لفهم العلاقة بين الاستجابة المناعية والسلوك، ونحن تصف طريقة لقياس السلوك الحركي في<em&gt; ذبابة الفاكهة</em&gt; خلال العدوى البكتيرية وكذلك قدرة الذباب على شن استجابة مناعية من البقاء على قيد الحياة الرصد والحمل البكتيري، في الوقت الحقيقي ونشاط منظم الرئيسية المناعة الفطرية، NFκB.

قياس كمية الاستجابة المناعية والنوم في<em&gt; ذبابة الفاكهة</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

quantitative-measurement-immune-response-sleep

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Immunology and Infection

Quantitative Measurement of the Immune Response and Sleep in Drosophila

15.3K Views.  University of Pennsylvania Perelman School of Medicine. لفهم العلاقة بين الاستجابة المناعية والسلوك، ونحن تصف طريقة لقياس السلوك الحركي في ذبابة الفاكهة خلال العدوى البكتيرية وكذلك قدرة الذباب على شن استجابة مناعية من البقاء على قيد الحياة الرصد والحمل البكتيري، في الوقت الحقيقي ونشاط منظم الرئيسية المناع...

Video: Quantitative Measurement of the Immune Response and Sleep in Drosophila

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

تحليلات متعددة الألوان من التدفق الخلوي الاستجابة المناعية الخلوية في المكاك وهو قرد اسيوي

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

مقدمة لالدبابير الطفيلية من<em&gt; ذبابة الفاكهة</em&gt; والاستجابة المناعية Antiparasite

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

الكمي للاستجابة انفجر الجهاز التنفسي باعتبارها مؤشر الفطري المناعي الصحة في الزرد

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

باستخدام الحمض النووي الريبي التدخل للتحقيق في الاستجابة المناعية الفطرية في الفأر الضامة

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

والاستفادة من البلعوم الإدارة داخل القصبة الهوائية والقصبات PAMP الغسل لتقييم استجابة المضيف المناعية في الفئران

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

في تحليل المختبر من الاستجابة المناعية الخلوية بوساطة Myd88 لفيروس غرب النيل المسخ سلالة العدوى

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

التصوير غير الغازية من الاستجابة المناعية الفطرية في نموذج الزرد يرقات<em&gt; العقدية iniae</em&gt; العدوى

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

تحليل نوعي وكمي المشبك محصنة في النظام البشري باستخدام التصوير التدفق الخلوي

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

اثنين-فوتون تصوير إينترافيتال من الكريات البيضاء خلال الاستجابة المناعية في الكبد تعامل Lipopolysaccharide الماوس

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...