Nous remercions Hye-Sook Oh, Joanne Morello et Alan Collmer, Département de pathologie végétale et plante-microbe Biologie, Université Cornell, à des discussions utiles. Nous remercions Jesse Munkvold des commentaires sur l&#39;article. Le financement a été fourni par le Programme National Science Foundation génome des plantes, le nombre récompense DBI-0605059 (GBM).

Partie 1: La croissance des plantes et l&#39;entretien Nicotiana benthamiana utilisé dans le dosage devrait être d&#39;environ 7 semaines. Ils doivent être taillés au moins 4-5 jours avant le test d&#39;enlever toutes les branches et les fleurs axillaires. C&#39;est une bonne idée d&#39;enlever les branches axillaires, peu après leur apparition afin de rendre les plantes plus gérable. Partie 2: culture bactérienne JOUR 1: <ol><li> Assiettes ou des cultures de toutes les souches utilisées dans le test sont lancés au même moment. Pour les Pseudomonas spp. qui comprennent deux inducteurs et 3 souches d&#39;épreuve, les bactéries série sur des plaques contenant KBM les antibiotiques appropriés de stocks de glycérol congelé (tableau 2). Une petite quantité de bactéries doivent être répartis sur le centre de la plaque. Incuber la plaque à 30 ° C pendant environ 18-24 heures. Pour Agrobacterium, démarrer une culture liquide en 2 ml LB à partir d&#39;une plaque fraîche et poussent durant la nuit à 250 rpm, 30 ° C. </li></ol> JOUR 2: <ol start="2"><li> Pour Pseudomonas, ajouter 150 à 200 l de liquide à la KBM bactéries et les étaler sur toute la plaque à l&#39;aide d&#39;un épandeur de verre stériles. Mettez la plaque arrière dans l&#39;incubateur et le laisser grandir pour un autre 20-24 heures. Il devrait y avoir une pelouse sur la plaque bactérienne, le lendemain, indiquant une bonne croissance. Pour Agrobacterium, démarrer une culture secondaire dans environ 5-10 ml LB avec 20 acétosyringone pM et laisser pousser durant la nuit de 20 heures à 250 rpm, 30 ° C. </li></ol> Partie 3: Préparation des plantes pour le dosage JOUR 2: <ol><li> Un jour avant le dosage des plantes devrait être transféré dans une chambre à 22-24 ° C, ~ 35-40% de lumière RH et constante. </li><li> Marquer les cercles sur les feuilles qui sont au moins 1,5 cm de diamètre en utilisant un gros marqueur noir. Deux à quatre cercles peuvent être marqués par feuille. Les cercles doivent être bien espacées et de préférence pas traverser une grosse veine. Choisissez bien élargi feuilles. Evitez les feuilles plus âgées ou les feuilles qui sont durs ou épais au toucher et éviter de marquer des cercles sur la partie inférieure de la feuille. </li></ol> Marquage des cercles d&#39;une journée avant l&#39;expérience n&#39;est pas indispensable pour le protocole. Cependant, il fait gagner du temps s&#39;il ya un grand nombre de plantes à détecter, et le rend plus facile à réaliser une synchronisation précise entre l&#39;induction de PTI et inoculations défi. Partie 4: Induction de PTI JOUR 3: <ol><li> Récolter les cellules de la plaque dans 10 mM MgCl 2 pour P. fluorescens et P. putida. Pour Agrobacterium, centrifuger la culture pour recueillir des cellules et de remettre en suspension dans 10 mM MgCl 2 + 10 mM MES pH 5,6. Répétez la centrifugation et la remise en suspension dans le MgCl 2-MES solution. Mesurer la densité optique (DO) des cellules à 600 nm. Réglez le SACO à des valeurs finales nécessaires tel que décrit dans le tableau 2. Pseudomonas devrait être finalement remis en suspension dans 10 mM de MgCl2 et Agrobacterium dans le MgCl 2-MES solution. Un volume total de 25 ml est suffisante pour inoculer environ 40-50 points. </li><li> Infiltrez les inducteurs PTI dans les cercles pré-marqués sur les feuilles à l&#39;aide d&#39;une seringue de 1 ml sans aiguille. Notez l&#39;ordre dans lequel les plantes ont été infiltrés et le moment exact où l&#39;infiltration a été commencé. </li></ol> Partie 5: inoculation d&#39;épreuve <ol><li> Préparer P. syringae pv. tomates DC3000, DC3000 Pst Δ hopQ1-1 et Ps pv. tabaci à relever le défi que décrit dans la partie 4.1 et le tableau 2. </li><li> Sept heures après l&#39;infiltration inducteur, effectuer l&#39;infiltration défi dans le même ordre de plantes que l&#39;induction a été effectué. Généralement, un point sur la périphérie du cercle première inoculation peut être utilisé comme le centre du cercle deuxième inoculation. S&#39;il ya des taches multiples sur une feuille, puis assurez-vous que les infiltrations ne se chevauchent pas. </li><li> Dilutions plaque de série de toutes les cultures utilisées pour l&#39;essai sur des plaques KBM ou LB contenant les antibiotiques appropriés pour déterminer le nombre exact UFC. </li></ol> Partie 6: Notation de la rupture du PTI Jour 5: <ol><li> Cherchez l&#39;apparence de la mort cellulaire due à l&#39;ETI dans les endroits qui ont été contestées avec Pst DC3000. La mort cellulaire à l&#39;intérieur de la zone de chevauchement de l&#39;infiltration indique une panne de PTI et doivent être comptés comme un phénotype positif. </li></ol> Jour 7: <ol start="2"><li> Cherchez l&#39;apparence de la mort cellulaire due à la maladie dans les endroits qui ont été contestées avec Pst DC3000 Δ hopQ1-1 ou Ps pv. Tabaci </em&gt;. Score pour un phénotype positif dans la même manière que décrit dans la partie 6.1. </li></ol> Lorsque les plantes de contrôle (qui ne devrait pas être compromis pour PTI) commencent à montrer la mort cellulaire dans la zone de chevauchement de l&#39;infiltration, arrêter de faire d&#39;autres observations. Partie 7: Les résultats représentatifs La figure 1 montre les résultats d&#39;un essai lorsque P. fluorescens a été utilisé comme inducteur et DC3000 Pst comme le défi. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. P. fluorescens a été infiltré sur N. benthamiana feuilles (cercle noir) pour induire PTI et 7 heures plus tard, le spot a été contestée avec Pst DC3000 (cercle blanc). Plantes silence pour FLS2 a montré une rupture de PTI dans la région où P. fluorescens a été infiltré (A), tel que vu par la mort cellulaire. Contrôle des plantes qui n&#39;ont pas été réduits au silence n&#39;a montré aucune mort cellulaire dans la zone de chevauchement en raison de l&#39;induction de PTI (B). Les flèches rouges indiquent l&#39;absence ou la présence de la mort cellulaire dans la zone de chevauchement de l&#39;infiltration. Les photographies ont été prises 2 jours après l&#39;infiltration. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">cliquez ici</a> pour voir une version agrandie de la figure 1. <table border="1"><tbody><tr><td> Inducteur PTI </td><td> Mort cellulaire provoquant défi </td><td> Nature de la mort cellulaire </td><td> Code de </td></tr><tr><td> Pseudomonas fluorescens 55 </td><td> Pseudomonas syringae pv tomate (Pst). DC3000 6 </td><td> ETI </td><td> Pf / DC </td></tr><tr><td> P. putida KT2440 </td><td> Pst DC3000 </td><td> ETI </td><td> Pp / DC </td></tr><tr><td> P. fluorescens 55 </td><td> Pst DC3000 Δ hopQ1-1 6 </td><td> Maladie </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae pv. tabaci 11528R </td><td> Maladie </td><td> Agro / PTAB </td></tr></tbody></table> Tableau 1: Combinaisons d&#39;induire la mort cellulaire et de PTI-susciter des microbes utilisés dans le dosage de la mort cellulaire par PTI. <table border="1"><tbody><tr><td> Souche bactérienne </td><td> Milieu de sélection </td><td> DO finale utilisée dans l&#39;expérience </td><td> Correspondant UFC / ml </td></tr><tr><td> P. fluorescens 55 </td><td> KBM Ampicilline (100 ug / ml) </td><td> 0.5 </td><td> 1 x 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM Ampicilline (100 ug / ml) </td><td> 0.5 </td><td> 1 x 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampicine (100 ug / ml) </td><td> 0.5 </td><td> 5 x 10 8 </td></tr><tr><td> Pst DC3000 </td><td> KBM rifampicine (100 ug / ml) </td><td> 0,02 </td><td> 2 x 10 7 </td></tr><tr><td> Pst DC3000 Δ hopQ1-1 </td><td> KBM rifampicine (100 ug / ml) </td><td> 100 de dilution de 0,1 fois </td><td> 1 x 10 6 </td></tr><tr><td> P. syringae pv. tabaci 11528R </td><td> KBM rifampicine (100 ug / ml) </td><td> 100 de dilution de 0,1 fois </td><td> 1 x 10 6 </td></tr></tbody></table> Tableau 2: Les conditions de culture et de niveaux d&#39;inoculum utilisé dans le dosage. La DO et les niveaux correspondants UFC peut varier entre différentes marques de spectrophotomètres.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

Dosage de la mort basée sur la cellule peut être utilisée pour déterminer l&#39;implication d&#39;un gène dans le PTI. Par exemple, la perte de fonction des mutants pour un gène d&#39;intérêt peut être testée en utilisant ce dosage. Sur les quatre combinaisons d&#39;inducteur et inoculations défi, étant donné dans le tableau 1, il est possible que seule une combinaison peut entraîner un phénotype dans votre fond végétal. Nous avons observé que les plantes réduit au silence pour FLS2 montrent une répartition de PTI dans le Pf / DC et des combinaisons de Pf/Q1-1 inducteur et inoculations défi (tableau 1). Il est essentiel que les conditions environnementales pour le dosage sont aussi proches que possible de celles décrites dans la partie 3.1. Faible taux d&#39;humidité provoque la mort cellulaire de procéder trop rapidement, ce qui rend le dosage difficile de marquer. Si les conditions décrites dans notre protocole ne fonctionne pas dans votre laboratoire, alors essayez de modifier le niveau d&#39;inducteur ou d&#39;inoculation d&#39;épreuve. Un autre paramètre qui peut être modifié, c&#39;est l&#39;écart de temps entre l&#39;induction et le défi, mais nous avons constaté que les écarts de temps plus courte causé le dosage de briser trop rapidement dans les plantes de contrôle. Nous recommandons que au moins 4-5 plantes être testée dans une expérience et un phénotype positif doit être répété dans au moins 3-4 expériences indépendantes.

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<td>Life Science UV/Vis Spectrophotometer</td>
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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

Pour percevoir des agents pathogènes potentiels dans leur environnement, les plantes utilisent Pattern Recognition Receptors (PRR) présents sur leurs membranes plasmiques. PRR reconnaître les caractéristiques microbiennes conservées appelé pathogène motifs moléculaires associés (PAMP) et cela conduit à la détection PAMP déclenché par l&#39;immunité (PTI), qui empêche efficacement la colonisation des tissus de la plante par des non-pathogènes 1,2. Le système le plus étudié dans PTI est le 3 voie FLS2-dépendante. FLS2 reconnaît le flg22 PAMP qui est une composante de la flagelline bactérienne. Pathogènes réussissent possèdent des facteurs de virulence ou des effecteurs qui peuvent supprimer PTI et permettre à l&#39;agent pathogène à causer la maladie 1. Certaines plantes possèdent à leur tour des gènes de résistance qui permettent de détecter des effecteurs ou leur activité, ce qui conduit à effecteur déclenché par l&#39;immunité (ETI) 2. Nous décrivons une mort cellulaire à base d&#39;analyse pour PTI modifié depuis Oh et Collmer 4. Le test a été normalisé en N. benthamiana, qui est en plus utilisées comme un système modèle pour l&#39;étude des interactions plante-pathogène 5. PTI est induite par l&#39;infiltration d&#39;une souche non pathogène bactérien dans les feuilles. Sept heures plus tard, une souche bactérienne qui cause la maladie ou qui actionne ETI est infiltré dans une zone de chevauchement de la zone d&#39;infiltration d&#39;origine. PTI induite par l&#39;infiltration premier est capable de retarder ou d&#39;empêcher l&#39;apparition de la mort cellulaire due à l&#39;infiltration deuxième défi. Inversement, l&#39;apparition de la mort cellulaire dans la zone de chevauchement de l&#39;inoculation indique une panne de PTI. Quatre combinaisons différentes avec des inducteurs du PTI et inoculations défi ont été normalisées (tableau 1). Le dosage a été testé sur la non-taire N. benthamiana qui a servi de contrôle et de silence pour les plantes qui ont été prédites FLS2 d&#39;être compromises dans leur capacité à développer PTI.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A la mort cellulaire à base d&#39;analyse pour PTI dans les plantes de Nicotiana benthamiana est décrite.

Dosage pour Pathogen-Associated Molecular Motif (PAMP)-Triggered Immunité (PTI) chez les plantes

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

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JoVE Journal

Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K vues.  Boyce Thompson Institute for Plant Research. A la mort cellulaire à base d'analyse pour PTI dans les plantes de Nicotiana benthamiana est décrite.

Vidéo: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Sélection des Plasmodium falciparum Parasites des Cytoadhesion aux cellules du cerveau endothéliales humaines

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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Immunologie et infection

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Étude des effets des probiotiques sur la colonisation pneumococcique l&#39;aide d&#39;un<em&gt; In Vitro</em&gt; Respect de dosage

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un flux Essai d&#39;adhésion à étudier Leucocyte recrutement pour hépatiques humaines sinusoïdales endothélium dans des conditions de contrainte de cisaillement

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield purification de<em&gt; Plasmodium falciparum</em&gt; Mérozoïtes Pour utilisation en opsonisants Anticorps dosages

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

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.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

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 ...

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.

Basées sur la fluorescence neuraminidase Essai d&#39;inhibition pour évaluer la sensibilité des virus de la grippe à la neuraminidase Inhibiteur Classe de Antiviraux

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

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Modèles de souris de l’Infection à Helicobacter et Pathologies gastriques

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Mise en place d’une Infection virale et analyse de l’Interaction hôte-Virus chez Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Imagerie confocale d’ARN bicaténaire et récepteurs de reconnaissance de modèle dans l’infection par le Virus ARN de polarité négative

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;