Nous sommes très reconnaissants à K. Nakagaki, le Dr H. Ishii, le Dr K. Suzuki, A. Yamagata, K. Oofusa pour leur précieuse contribution.

1. Production et purification de sGMRα <ol><li> Amplifier l&#39;ADNc d&#39;sGMRα du placenta banque d&#39;ADNc humain en chaîne par polymérase (PCR). </li><li> Ajouter de base 50 5&#39;-UTR séquence de fin de polyhédrine de baculovirus et 27 RGS bases codant His-tag à l&#39;extrémité 3 &#39;de l&#39;amplicon PCR PCR par un autre. </li><li> Insérez le produit amplifié par PCR dans un plasmide pMSG1.1MG. </li><li> Injecter l&#39;psGMR/M1.1MG construire avec le vecteur d&#39;aide pHA3PIG dans des œufs de vers à soie PND-w1 souche. </li><li> Arrière de l&#39;éclosion des larves de mites G0 à 25 ° C. Embryons obtenus par G1 écran d&#39;accouplement entre frères et sœurs ou avec PND-W1 pour l&#39;expression MGFP dans les yeux afin d&#39;obtenir des vers à soie transgénique portant le gène sGMRα. </li><li> Extrait recombinante sGMRα partir des cocons de vers à soie transgénique avec du phosphate-tampon contenant NaCl 500mm et agiter à 4 ° C pendant 24h. </li><li> Centrifuger l&#39;échantillon à 20 000 g pendant 15 min pour enlever un précipités. </li><li> Appliquer le surnageant dans une colonne de nickel-affinité. </li><li> Laver la colonne avec 10 mM imidazole, 500mm de NaCl et de phosphate de sodium 20 mM, pH6.3, et éluer avec un dégradé linéaire d&#39;imidazole de 10mm à 250mm. Piscine des fractions contenant le sGMRα purifié et dialyse contre un tampon phosphate salin (pH 7,4) (PBS). </li></ol> 2. Biotinylation de GM-CSF recombinant <ol><li> Retirez la préparation du sorbitol par dialyse contre du PBS. </li><li> Ajouter 1ml de 20mM de sodium méta-periodate solution froide à 4 ° C à la solution sur la glace. </li><li> Incuber l&#39;échantillon pendant 30 minutes sur la glace dans l&#39;obscurité. </li><li> Ajouter la glycérine à la solution à une concentration finale de 15 mM. </li><li> Dialyser la solution contre le tampon acétate de sodium 100 mM (pH 5,5) </li><li> Ajouter hydrazide de biotine à la solution à une concentration finale de 5 mM et continuellement agiter la solution pendant 2h à température ambiante. </li><li> Retirez les matériaux n&#39;ayant pas réagi par dialyse contre du PBS, pH 7,4. </li></ol> 3. Une cellule sans GM-CSF récepteur-ligand test de liaison <ol><li> Enduire une plaque de microtitration avec 50 pl d&#39;anticorps monoclonaux anti-polyhistidine anticorps nuit à 4 ° C. </li><li> Laver à cinq reprises avec 500μl de PBST. </li><li> Ajouter 100 ul d&#39;une solution de blocage et incuber pendant 3h à 4 ° C. </li><li> Laver à cinq reprises avec 500μl de PBST. </li><li> Ajouter 50 pl d&#39;sGMRα dans la solution de blocage et incuber une nuit à 4 ° C. </li><li> Laver à cinq reprises avec 500μl de PBST. </li><li> Ajouter 25 pi des échantillons contenant du GM-CSF d&#39;autoanticorps et 25 pi de biotinylé GM-CSF. Incuber pendant 1h à 4 ° C pour former sGMRα-GM-CSF complexes. </li><li> Laver à cinq reprises avec 500μl de PBST. </li><li> Ajouter 50 pl de 0.4mm streptavidine phosphatase alcaline pour 1 heure à 4 ° C pour détecter le GM-CSF biotinylé lié sGMRα. </li><li> Laver à cinq reprises avec 500μl de PBST. </li><li> Ajouter 50 pl d&#39;un substrat chimiluminescent à la solution et incubées pendant 1h à température ambiante. </li><li> En utilisant un lecteur de plaques chimiluminescence, de détecter l&#39;activité chimiluminescence. </li></ol> 4. Les résultats représentatifs: La première étape dans la voie du récepteur GM-CSF de transduction du signal est la liaison du GM-CSF à l&#39;alpha du récepteur GM-CSF à la surface cellulaire. Parce que le GM-CSF d&#39;auto-anticorps se lie spécifiquement le GM-CSF et de bloquer sa liaison au récepteur in vitro, 16,17, nous avons supposé que les auto-anticorps inhibent cette première réaction en se liant directement au GM-CSF. Comme décrit précédemment (Réf. 9-15), nous avons appliqué transgéniques magnanerie technologie pour obtenir une grande quantité de sGMRα recombinant avec une grande pureté. Lorsque le ver à soie dérivés sGMRα recombinante a été chargé sur SDS-PAGE sous conditions non réductrices, le sGMRα montré à la fois monomère (45 kDa) et dimères (90 kDa) des formes, tandis que la forme monomérique a été détecté dans des conditions réductrices (figure 1B) , indiquant que le sGMRα recombinante a été un mélange de monomères et de ponts disulfure gradateurs 18. En utilisant le système sans cellule (figure 2A), nous avons évalué l&#39;inhibition de la GM-CSF se liant à sGMRα par le GM-CSF auto-anticorps (figure 2B et fig. 2C). Ces méthodes ont été décrites en référence 19 par Urano et al. L&#39;inhibition de la croissance a été étroitement corrélée avec l&#39;inhibition de la fixation (r = 0,988, p = 0,002) à différentes concentrations de l&#39;auto-anticorps GM-CSF (figure 3A) 19. De même, l&#39;inhibition de la fixation était significativement corrélé avec l&#39;inhibition de la croissance. L&#39;inhibition de la fixation accrue de façon dose-dépendante par le GM-CSF auto-anticorps (figure 3B) 19. Ces deux paramètres pour les fractions IgG sérum provenant de patients différents ont été corrélés entre eux (r = 0,589 p = 0,006, Fig. 3C). Ni d&#39;inhibition de liaison, ni inhibition de la croissance en corrélation avec les valeurs de Kd, et donc, les deux paramètres n&#39;étaient pas affectés par affinité de liaison 19. Par conséquent la reproductibilité des données entre contraignant et gl&#39;inhibition roissance obtenus par trois expériences indépendantes sur trois échantillons différents ont été évaluées. Le coefficient de variation indiqué que le système sans cellule était plus excellent que celui de bioessais (tableau 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Figure 1" /> Figure 1. Organigramme de la procédure pour la production de vers à soie transgénique sGMRα aide. A) Structures de les 9-11 vecteurs de transformation. B) SDS-PAGE et Coomassie Brilliant Blue-coloration de la sGMRα purifiée dans la réduction et non des conditions réductrices. sGMRα, soluble dans le GM-CSF récepteurs alpha; MGFP, monstre protéine fluorescente verte; BmNPVpol5&#39;-UTR, séquence de la région 5 &#39;non traduite de Bombyx mori nucléaires polyédrose polyhédrine du virus; SV40 polyA, séquence signal polyA de SV40; P 3xP3, 3xP3 promoteur; P SER1, SER1 promoteur; FiBL polyA, fibroïne de la chaîne L séquence signal polyA; hr3, Bombyx mori nucléaires polyédrose virus hr3 Enhancer. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Figure 2" /> Figure 2. Schéma du système de dosage sans cellule. A) Un essai de liaison compétitive en utilisant sGMRα produites par des vers à soie. B) Effet des anticorps neutralisants et non neutralisants sur l&#39;inhibition de la fixation par les cellules sans système. C) la différence de l&#39;inhibition de liant entre les différentes concentrations d&#39;anticorps neutralisants et des anticorps non neutralisants. GM-CSF, granulocyte-macrophage colony-stimulating factor; sGMRα, soluble dans le GM-CSF récepteurs alpha; Son, RGS-His-tag, AP, la phosphatase alcaline. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Figure 3" /> Figure 3. GM-CSF d&#39;inhibition de liaison au sGMRα par effet du GM-CSF anticorps polyclonaux ou les fractions IgG sériques de patients atteints de PAP auto-immunes. A) Relation entre l&#39;inhibition de liaison et d&#39;inhibition de croissance par le GM-CSF autoanticorps. B) inhibition de la fixation à différentes concentrations de GM-CSF autoanticorps. C) La relation entre CI 50 pour cent d&#39;inhibition de liaison et d&#39;inhibition pour cent de croissance par les fractions d&#39;IgG sériques 19. IC 50, concentration inhibitrice 50%; le GM-CSF, granulocyte-macrophage colony-stimulating factor. <table border="1"><tbody><tr><td> Exemple </td><td> Une </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-essai </td></tr><tr><td> Nombre de déterminations </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> Valeur moyenne (% d&#39;inhibition de liaison) </td><td> 61,6 </td><td> 63,8 </td><td> 69,7 </td></tr><tr><td> Valeur moyenne (inhibition de la croissance%) </td><td> 21,0 </td><td> 73,4 </td><td> 82,8 </td></tr><tr><td> Le coefficient de variation (%) (% d&#39;inhibition de liaison) </td><td> 6,0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td> Le coefficient de variation (%) (% d&#39;inhibition de croissance) </td><td> 64,4 </td><td> 18,3 </td><td> 10,4 </td></tr></tbody></table> Les deux analyses ont été effectuées à 5 ng / ml de GM-CSF et une concentration égale à la GM-CSF autoanticorps. Tableau 1. Comparaison du coefficient de variations entre contraignante pour cent et la croissance des inhibitions obtenues par trois expériences indépendantes.

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Le test sans cellule estimé la capacité de neutralisation de GM-CSF auto-anticorps avec une excellente reproductibilité et de rapidité. L&#39;inhibition de la fixation par le GM-CSF autoanticorps ou le sérum du patient fractions d&#39;IgG a été évaluée par ce test. Les données ont montré une corrélation entre l&#39;inhibition de la fixation de l&#39;essai sans cellules et l&#39;inhibition de croissance d&#39;un test biologique utilisant cellules TF-1, respectivement. Le bio-essai a été largement utilisé, ...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

Arrière-plans: Auparavant, nous avons démontré que la capacité de neutraliser, mais pas la concentration de GM-CSF d&#39;auto-anticorps a été corrélée avec la sévérité de la maladie chez les patients atteints protéinose alvéolaire auto-immune pulmonaire (PAP) 1-3. Comme l&#39;abrogation de GM-CSF bioactivité dans le poumon est la cause probable pour PAP auto-immunes 4,5, il est prometteur de mesurer la capacité de neutralisation des anticorps GM-CSF pour évaluer la sévérité de la maladie chez chaque patient avec PAP. Jusqu&#39;à présent, la capacité de neutralisation de GM-CSF auto-anticorps a été évaluée en évaluant l&#39;inhibition de la croissance de cellules humaines de la moelle osseuse ou cellules TF-1 stimulées par le GM-CSF 6-8. Dans le système de dosage biologique, cependant, il est souvent problématique pour obtenir des données fiables ainsi que de comparer les données de différents laboratoires, en raison des difficultés techniques dans le maintien des cellules dans un état constant. OBJECTIF: Pour imiter le GM-CSF se liant à des récepteurs du GM-CSF à la surface cellulaire utilisant des cellules sans liaison au récepteur-dosage. Méthodes: soie transgénique technologie a été appliquée pour obtenir une grande quantité d&#39;alpha recombinant soluble du récepteur GM-CSF (sGMRα) avec une grande pureté 9-13. Le sGMRα recombinante a été contenue dans les couches séricine hydrophile de fils de soie sans être fusionnée à la protéine de soie, et donc, nous pouvons facilement extraire à partir des cocons de bonne pureté avec des solutions aqueuses neutres 14,15. Heureusement, les structures d&#39;oligosaccharides, qui sont critiques pour la liaison avec le GM-CSF, sont plus semblables à des structures de sGMRα humaines que celles produites par d&#39;autres insectes ou les levures. RÉSULTATS: Le système de dosage sans cellule en utilisant sGMRα donné les données avec une grande plasticité et leur fiabilité. Contraignant pour sGMRα GM-CSF était dose-dépendante inhibée par le GM-CSF polyclonaux auto-anticorps dans une manière similaire à l&#39;essai biologique utilisant cellules TF-1, indiquant que notre nouveau système de dosage sans cellule en utilisant sGMRα est plus utile pour la mesure de l&#39;activité neutralisante de GM-CSF auto-anticorps que le système d&#39;essai biologique utilisant des cellules TF-1 ou de cellules osseuses humaines. CONCLUSIONS: Nous avons établi un test sans cellule de quantifier la capacité de neutralisation de GM-CSF autoanticorps.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Nous avons conçu un dosage des récepteurs cellulaires sans liant en vue d&#39;estimer la liaison de granulocyte-macrophage colony-stimulating factor (GM-CSF) pour les récepteurs. Il nous permet d&#39;évaluer l&#39;inhibition compétitive de biotinylé GM-CSF se liant à des récepteurs solubles de GM-CSF alpha par le GM-CSF d&#39;auto-anticorps avec une excellente reproductibilité.

Un système de dosage Free Cell Estimation de la capacité de neutralisation de GM-CSF recombinant soluble à l&#39;aide d&#39;anticorps GM-CSF récepteur

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

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JoVE Journal

Immunology and Infection

12.6K vues.  Niigata University Medical and Dental Hospital. Nous avons conçu un dosage des récepteurs cellulaires sans liant en vue d'estimer la liaison de granulocyte-macrophage colony-stimulating factor (GM-CSF) pour les récepteurs. Il nous permet d'évaluer l'inhibition compétitive de biotinylé GM-CSF se liant à des récepteurs solubles de GM-CSF alpha par le GM-CSF d'auto-anticorps avec une excellente reproductibilité.

Vidéo: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioénergétique et les oxydatif: Protocoles pour l&#39;isolement et l&#39;évaluation des leucocytes humains et plaquettes

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

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Immunologie et infection

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalable High Throughput sélection à partir de bibliothèques d&#39;anticorps synthétiques phage

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Génération et identification de GM-CSF dérivé alvéolaires comme Macrophages et cellules dendritiques à partir de moelle osseuse de souris

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Évaluation Retinal microglial phagocytaire Fonction<em&gt; In Vivo</em&gt; L&#39;utilisation d&#39;un test basé sur la cytométrie de flux

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murine lymphocytaire par étiquetage<sup&gt; 64</sup&gt; Cu-anticorps du récepteur de ciblage pour<em&gt; In Vivo</em&gt; Le trafic cellulaire par PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Génération des variantes d’évasion de neutraliser les anticorps monoclonaux Influenza Virus

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Grippe neutralisante d’anticorps aux virus a (H3N2) dans le sérum humain par micro-neutralisation de mesure des analyses utilisant des cellules de MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Différenciation des souris Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-production de cellules T auxiliaires (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.