Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (MSH-95368, 89773 e-MOP MOP-106623 a CZ) e la Fondazione canadese per l&#39;innovazione. SCM è il destinatario di un CIHR / Canadian Association of Gastroenterology borsa post-dottorato. CZ è un investigatore CIHR Nuovo.

1. Moltiplicazione Trichuris uova Muris <ol><li> Per generare nuovi lotti di uova di Trichuris Muris, infettare 20-30 topi immunodeficienti (es. NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) o 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - / -)) o topi geneticamente suscettibili ( ad es. AKR / J), che sono 6-8 settimane di età con circa 300 uova di Trichuris Muris mediante sonda gastrica. </li><li> Dopo 32-35 giorni sacrificare i topi da CO 2 asfissia. </li><li> Esporre la parte ventrale del mouse e bagnato l&#39;addome con il 70% di etanolo. </li><li> Utilizzando pinze afferrare la cute addominale e fare una piccola incisione con le forbici. Tagliare la pelle per esporre il contenuto addominale. </li><li> Identificare il cieco e l&#39;intestino prossimale di grandi dimensioni, e rimuoverli. </li><li> Posizionare il cieco e colon prossimale in un piatto da 10 cm petri contenenti Dulbecco modificato (DMEM) con 500 U / ml penicillina e 500 mg / ml di streptomicina (P / S). Tagliare il cieco aperto per esporre il lume e il worm. Utilizzando posto forcipe cieco in un bicchiere di tampone fosfato (PBS) e agitare delicatamente per rimuovere le feci. </li><li> Ritorna cieco a capsula di Petri e con pinze curve lisce (Roboz RS-5047) delicatamente estrarre vermi e metterli in un pozzetto di una piastra ben 6 contenente 5 ml di DMEM + P / S. </li><li> Ripetere questo processo per tutti i topi. </li><li> Mettere 6 pozzetti in un contenitore Tupperware con un tovagliolo di carta umido per mantenere l&#39;umidità e incubare a 37 ° C per 4 ore. </li><li> Dopo 4 ore rimuovere worm utilizza pinze curve lisce e dividerli in 2 nuovi pozzi della piastra 6 pozzetti contenenti 5 ml DMEM + P / S. Raccolto il contenuto del pozzo originario e porlo in una provetta da 15 ml falco. In provetta a 3000 giri e rimuovere e prenotare surnatante come antigene 4 ore (può essere utilizzato per restimolazione cellulare dopo PBS dialisi, dove i rendimenti delle proteine ​​tipiche sono 10-20 mg per 20 topi) e risospendere uova contenenti pellet in acqua di rubinetto in autoclave. </li><li> Mettete i 6 piastra ben indietro a 37 ° C durante la notte. Rimuovere e smaltire i vermi. Raccolto il contenuto dei pozzetti in un tubo falco e far girare a 3000 rpm per 5 min. Rimuovere il surnatante e riserva come antigene durante la notte (può essere utilizzato per Trichuris ELISA specifico anticorpo isotipo Ag dopo PBS dialisi, dove i rendimenti delle proteine ​​tipiche sono 10-20 mg per 20 topi) e di uova contenenti risospendere pellet in acqua di rubinetto in autoclave. </li><li> Combina 4 ore e uova notte e filtrare le uova attraverso il filtro 70μm di togliere qualunque vermi rimasto poi trasferire in un centimetro 75 2 pallone. Tenere pallone in un luogo buio coperto in lamina per 6 settimane a temperatura ambiente (RT). </li><li> Lavare le uova in acqua di rubinetto in autoclave e risospendere a 50 uova vitali in 50 microlitri. Per fare questo, uova centrifuga a 3000 rpm per 5 minuti e risospendere in ~ 50ml acqua sterile. Togliere 50 microlitri uova e posto su un vetrino. Utilizzando l&#39;ingrandimento 40x contare il numero di valide uova embrionate in microlitri 50. Diluire le uova per dare 50 uova vitali in 50 microlitri. Per le immagini di uova vitali si veda lo studio di Kopper e Mansfield 16. </li></ol> 2. Infezione acuta dei topi sperimentali con Trichuris Muris uova (200 uova / mouse) <ol><li> Risospendere completamente le uova e rimuovere il volume sufficiente per trattare il numero di topi X 2 (es. per 10 topi prendere 200 x 10 microlitri topi x 2 = 4 ml di uova) e metterli in un tubo 14 ml tappo a scatto. </li><li> Invertire il tubo a scatto 14 ml tappo a mescolare. Portate fino a 200 ml di uova in siringa da 1 ml su un ago appropriato alimentazione dimensioni (per i mouse peso di 25 grammi abbiamo utilizzato 18 gauge 1 1 / 2 &quot;aghi). Collottola In una mano il mouse e utilizzando invece amministrare uova mediante sonda gastrica . </li><li> Aspettare 21 giorni. </li></ol> 3. Vendemmia sperimentale <ol><li> Il giorno 21 sacrificare i topi utilizzando CO 2. </li><li> Raccogliere il sangue dai topi da puntura cardiaca, in modo non eparinizzata siringa, e conservare a 4 ° C. Il giorno seguente isolare siero e congelare a -20 ° C. Siero può essere utilizzato per il rilevamento di Trichuris-IgG e IgE specifiche con il metodo ELISA. </li><li> Esporre la parte ventrale del mouse e bagnato l&#39;addome con il 70% di etanolo. </li><li> Utilizzando pinze afferrare la cute addominale e fare una piccola incisione con le forbici. Tagliare la pelle e membrana sottostante per esporre il contenuto addominale. </li><li> Identificare il piccolo intestino, cieco e del colon. Afferra il cieco con una pinza e tirare fuori della cavità addominale. Con un altro paio di pinze spingere tenue verso destra esporre i linfonodi mesenterici (mln). Rimuovere la mln facendo attenzione a non tagliare negli intestini e posto in un falcon da 15 ml contenente 2 ml di media. Se lo si desidera possono essere elaborati e restimolati in vitro (4x10 6 / ml a 1 ml in 24 piastre con 1 mg / ml o con αCD3/CD28 50μg/ml 4h Trichuris Ag) per 72 ore per l&#39;analisi di citochine da parte ELISA ed f intracellularecitometria basso. </li><li> Tagliare il cieco e del colon. </li><li> Utilizzando pinze, spingere fuori pellet fecali da colon distale e posto in un tubo da 2 ml Axygen. Congelare a -20 ° C. Pellet fecali possono essere analizzati mediante western blot per l&#39;espressione di resistina-come molecola-β (RELM-β). </li><li> Rimuovere la punta del cieco (~ 0.5 cm). Per rimuovere le feci tenere punta cieco con una pinza e filo con PBS usando una siringa da 3 ml e 22-gauge in una capsula di Petri. Mettere in fresco paraformaldeide 4% in una provetta da 5 ml tappo a scatto Falcon. Conservare a 4 ° C. Questi possono essere inclusi in paraffina, sezionati e montati su vetrini per l&#39;analisi istologica. </li><li> Rimuovere un pezzo di colon prossimale (~ 1 cm) per RNA. Mettere campione in 2 ml di tubo Axygen RNAlater con 500 microlitri. Conservare a 4 ° C. Questi campioni possono essere analizzati per l&#39;espressione di mRNA da qPCR. </li><li> Mettere il resto del cieco in una capsula di Petri etichettati e congelare a -20 ° C. </li></ol> 4. Enumerazione di Trichuris vermi Muris nel cieco <ol><li> Rimuovere cieco dal freezer e metterlo in una capsula di Petri contenente ~ 5 ml di acqua. </li><li> Utilizzando le forbici, tagliare aperto cieco per esporre il lume. </li><li> Tenere il cieco con una pinza e scuotere vigorosamente il cieco per rimuovere la maggior parte delle feci. </li><li> Trasferimento ad un altro cieco capsula di Petri contenente acqua e usando liscia pinze curve dolcemente raschiare il IXC il tessuto sottostante. </li><li> Trasferimento cieco per un nuovo piatto di Petri contenente acqua e vigorosamente raschiare restante tessuto con una pinza, strappa il tessuto in piccoli pezzi. </li><li> Utilizzare un microscopio da dissezione per contare tutti i vermi in tutti e 3 i piatti di Petri. Come si trova un verme rimuoverlo dal piatto per assicurare il worm stesso non viene contato due volte. Se vermi sono danneggiato (rotto a metà) contare solo finisce spesso o sottile per ottenere un conteggio accurato. </li></ol> 5. Rappresentante Risultati L&#39;infezione Trichuris Muris modello permette ai ricercatori di quantificare carico parassita e risposta immunitaria sistemica, come pure per esaminare istologicamente la risposta infiammatoria all&#39;interno del cieco distale. Quando il lume del cieco raccolto è esposto, worm Trichuris possono essere enumerati utilizzando un microscopio da dissezione (Figura 1A-B). Parametri di immunità ai Trichuris anche isotipo anticorpale passaggio a IgG1 (associata con l&#39;immunità di tipo Th2) negli animali resistenti e IgG2a (associata a immunità di tipo Th1) di animali sensibili (Figura 1C). Espressione del calice cellule specifiche proteine-resistina come molecola-β (RELM-β) è associato con una clearance di Trichuris ed è rilevabile dall&#39;analisi Western blot (Figura 2). La porzione distale del cieco possono essere colorati con acido periodico di Schiff (PAS) per valutare le risposte delle cellule caliciformi e il grado di infiammazione intestinale in risposta alle infezioni (Figura 3). <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Figura 1"/> Figura 1. Quantificazione di immunità ai Trichuris Muris. (A) Resistente (B6) topi espellere Muris Trichuris dopo 21 giorni post-infezione. Al contrario, suscettibile (AKR / J) i topi diventano con infezione cronica da Trichuris, con conseguente istituzione parassita. Ogni barra rappresenta la media ± SEM di 4-8 animali per ciascun gruppo. (B) Worms vengono conteggiati utilizzando un microscopio da dissezione. Pannello superiore mostra i vermi in isolamento, pannello inferiore mostra i vermi in presenza di IXC e le feci. (C) Trichuris specifici titoli IgG2a sono determinati da ELISA di siero diluito (1:2 di diluizione, a partire dalla diluizione 1:20 e termina a 1:2560) su piastre rivestite di antigene escretorio Trichuris (O / N Ag, 5 mg / ml). Suscettibili AKR / J topi hanno livelli notevolmente superiori di Trichuris specifici IgG2a siero di topi resistenti B6. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Figura 2"/> Figura 2. Liquidazione di Trichuris Muris è associata con l&#39;espressione del calice cellule specifiche proteine ​​RELM β-7. RELM-β di produzione possono essere rilevati da pellet fecali da Trichuris infetti resistenti (B6), ma non sensibili (AKR / J) nei topi mediante analisi Western Blot. Ogni corsia è rappresentativo di un animale unico, (N) = Naive, (Tm) = 21 giorni Trichuris topi infettati. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Figura 3"/> Figura 3. L&#39;esame istologico della distale infezione Trichuris cieco segue. Micron sezioni 5-7 della distale cieco macchiato di PAS. Iperplasia delle cellule caliciformi e la produzione di muco sono evidenti in resistente (B6), ma non sensibili (AKR / J) nei topi dopo l&#39;infezione Trichuris.

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Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti da The University of British Columbia.

Questo protocollo dettagli di un elevato standard dose acuta di infezione Trichuris Muris, che può essere modificato come richiesto dallo sperimentatore. Per esempio, i topi possono essere sacrificati e tessuti raccolti in giorni diversi. Per determinare che i topi hanno stabilito con successo pieno carico vermi possono essere sacrificati al giorno 14, momento in cui tutti i mouse dovrebbe portare un peso di circa 200 vermi. I topi possono anche essere infettati per 32 giorni in cui ogni verme rilevato avrà r...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Aghi di alimentazione animale (18 x 1 ½ &quot;) </td><td> Bottone automatico </td><td> 7912 </td><td></td></tr><tr><td> Liscio Pinza curva </td><td> Roboz </td><td> RS-5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg-Prkdc scid IL2r gtm1Wjl / SzJ (NSG) </td><td> Jackson Laboratories </td><td> 005557 </td><td> Questi sono i topi che abbiamo usato, comunque, ogni topi immunodeficienti o ceppo suscettibile dovrebbe funzionare. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76104 </td><td></td></tr><tr><td> 2 ml tubi </td><td> Axygen </td><td> MCT-200-C </td><td></td></tr><tr><td> Provette da 15 ml </td><td> Falco </td><td> 352096 </td><td></td></tr><tr><td> 6 piatti ben </td><td> Falco </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldeide </td><td> Microscopia Elettronica Scienza </td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ anticorpi </td><td> Peprotech Inc </td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris Muris è un patogeno naturale di topi ed è biologicamente e antigenicamente simili alle specie di Trichuris che infettano gli esseri umani e animali 1. Uova infette sono dati mediante sonda gastrica, schiudono nel piccolo intestino distale, invadono le cellule epiteliali intestinali (IXC) che la linea di cripte del cieco e del colon prossimale e alla maturazione dei worm rilasciare le uova nell&#39;ambiente 1. Questo modello è uno strumento potente per esaminare i fattori che controllo CD4 + T helper (Th) l&#39;attivazione delle cellule così come i cambiamenti a livello dell&#39;epitelio intestinale. La risposta immunitaria che si verifica in ceppi inbred, come C57BL / 6 e BALB / c, è caratterizzato da citochine Th2 polarizzata (IL-4, IL-5 e IL-13) e l&#39;espulsione di vermi, mentre associata citochine Th1 (IL -12, IL-18, IFN-γ) promuovere le infezioni croniche in AKR geneticamente suscettibili / topi J 2-6. Citochine Th2 promuovere cambiamenti fisiologici nel microambiente intestinale compreso rapido turnover di IXC, la differenziazione delle cellule caliciformi, di reclutamento e cambiamenti della permeabilità epiteliale e la contrazione della muscolatura liscia, che sono stati implicati in espulsione verme 7-15. Qui dettaglio un protocollo per la propagazione Trichuris uova Muris, che possono essere utilizzati in esperimenti successivi. Forniamo anche un raccolto sperimentale campione con suggerimenti per l&#39;analisi post-infezione. Nel complesso, questo protocollo fornirà ai ricercatori gli strumenti di base per eseguire un mouse Trichuris Muris modello di infezione che può essere utilizzata per affrontare questioni relative alla propensione Th nel tratto gastrointestinale e immunitario funzioni effettrici delle IXC.

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

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

Trichuris infezione Muris è un modello di immunità intestinale Th2 dove topi resistenti generare una risposta protettiva Th2 e topi suscettibili generare una patologica risposta Th1.

Trichuris Muris Infezione: un modello di tipo 2 Immunità e infiammazione nell&#39;intestino

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

trichuris-muris-infection-model-type-2-immunity-inflammation

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Immunology and Infection

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

16.2K Views.  University of British Columbia. Trichuris infezione Muris è un modello di immunità intestinale Th2 dove topi resistenti generare una risposta protettiva Th2 e topi suscettibili generare una patologica risposta Th1.

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

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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Immunologia e infezioni

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Applicando Fluorescence Resonance Energy Transfer (FRET) per esaminare Effector Translocation efficienza da<em&gt; Burnetii Coxiella</em&gt; Durante siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Taglio di precisione polmone mouse Fette per la visualizzazione in diretta cellule dendritiche polmonare

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Misurazione di High-throughput di membrana plasmatica risigillazione efficienza in cellule di mammifero

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Effetti del gusto segnalazione proteina Abolishment su infiammazione dell'intestino in un modello murino di malattia infiammatoria intestinale

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Il modello di infezione del Waxworm di Galleria mellonella per candidosi disseminata

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...