Wir sind sehr dankbar, K. Nakagaki, Dr. H. Ishii, Dr. K. Suzuki, A. Yamagata, K. Oofusa für ihre wertvollen Beiträge.

1. Produktion und Aufreinigung von sGMRα <ol><li> Amplify cDNA von sGMRα aus menschlicher Plazenta-cDNA-Bank mittels Polymerase-Kettenreaktion (PCR). </li><li> Dann werden 50 Basis-5&#39;-UTR-Sequenz von Baculovirus-Polyhedrin Ende und 27 Basis-Codierung RGS His-tag an das 3&#39;-Ende der PCR Amplikon von einer anderen PCR. </li><li> Legen Sie die amplifizierte PCR-Produkt in ein Plasmid pMSG1.1MG. </li><li> Inject das Konstrukt psGMR/M1.1MG mit dem Helfer-Vektor pHA3PIG in Eier der Seidenraupe PND-w1 Belastung. </li><li> Hinten die geschlüpften Larven zu Motten G0 bei 25 ° C. Screen-G1 Embryonen, die durch Paarung unter Geschwistern oder mit PND-w1 erhalten für MGFP Ausdruck in den Augen zu erhalten transgenen Seidenraupen mit dem sGMRα Gen. </li><li> Auszug rekombinanten sGMRα aus den Kokons der Seidenraupen transgenen mit Phosphat-Puffer mit 500 mM NaCl und rühren bei 4 ° C für 24 Stunden. </li><li> Centrifuge Probe bei 20000 g für 15 Minuten zu entfernen ausfällt. </li><li> Wenden Sie den Überstand in ein Nickel-Affinitätssäule. </li><li> Die Säule wird mit 10 mM Imidazol, 500 mM NaCl und 20 mM Natriumphosphat, pH6.3, und eluieren mit einem linearen Gradienten von Imidazol von 10mm bis 250mm. Pool der Fraktionen, die das gereinigte sGMRα und dialysieren gegen Phosphat-gepufferte Kochsalzlösung (pH 7,4) (PBS). </li></ol> 2. Biotinylierung von rekombinantem GM-CSF <ol><li> Entfernen Sorbit Vorbereitung durch Dialyse gegen PBS. </li><li> Add 1 ml 20 mM kalten Natrium-meta-Perjodat-Lösung bei 4 ° C, um die Lösung auf Eis. </li><li> Inkubieren Sie die Probe für 30min auf Eis im Dunkeln. </li><li> Add Glycerin, um die Lösung in einer Endkonzentration von 15 mm. </li><li> Dialysieren die Lösung gegen 100 mM Natriumacetat-Puffer (pH 5,5) </li><li> Add Biotinhydrazid die Lösung mit einer Endkonzentration von 5 mM und kontinuierlich rühren die Lösung für 2 h bei Raumtemperatur. </li><li> Entfernen Sie das nicht umgesetzte Material durch Dialyse gegen PBS, pH 7,4. </li></ol> 3. Ein zellfreier GM-CSF-Rezeptor-Ligand-Bindungs-Assay <ol><li> Coat eine Mikrotiterplatte mit 50 ul monoklonalen anti-Polyhistidin Antikörper über Nacht bei 4 ° C. </li><li> Wash fünfmal mit 500μl PBST. </li><li> Add 100 &amp;mgr; einer Blockierungslösung und Inkubation für 3h bei 4 ° C. </li><li> Wash fünfmal mit 500μl PBST. </li><li> Add 50 ul sGMRα in Blocking-Lösung und Inkubation über Nacht bei 4 ° C. </li><li> Wash fünfmal mit 500μl PBST. </li><li> Add 25 &amp;mgr; l der Proben mit GM-CSF Autoantikörper und dann 25 &amp;mgr; l des biotinylierten GM-CSF. Inkubieren 1h bei 4 ° C zu bilden sGMRα-GM-CSF-Komplex. </li><li> Wash fünfmal mit 500μl PBST. </li><li> Add 50 ul 0,4 mM Streptavidin-alkalische Phosphatase für 1 Stunde bei 4 ° C an den biotinylierten GM-CSF gebunden sGMRα erkennen. </li><li> Wash fünfmal mit 500μl PBST. </li><li> Add 50 ul einer Chemilumineszenzsubstrat zur Lösung resuspendiert und für 1h bei Raumtemperatur. </li><li> Mit Hilfe eines Chemilumineszenz-Reader, erkennen die Chemilumineszenz-Aktivität. </li></ol> 4. Repräsentative Ergebnisse: Der erste Schritt in die GM-CSF-Rezeptor-Signalweg ist die Bindung von GM-CSF, GM-CSF-Rezeptor alpha auf der Zelloberfläche. Weil GM-CSF Autoantikörper bindet spezifisch an GM-CSF und blockieren die Bindung an den Rezeptor in vitro 16,17 stellten wir die Hypothese, dass die Autoantikörper dieser ersten Reaktion unmittelbar verbindlich GM-CSF zu hemmen. Wie zuvor beschrieben (Ref. 9-15), wandten wir transgene Seidenraupe Technologie, um eine große Menge an rekombinantem sGMRα mit hoher Reinheit zu erhalten. Wenn die Seidenraupe abgeleitete rekombinante sGMRα auf SDS-PAGE wurde unter nicht-reduzierenden Bedingungen geladen, zeigte die sGMRα sowohl monomere (45 kDa) und dimeren (90 kDa) bildet, während nur die monomere Form unter reduzierenden Bedingungen festgestellt wurde (Abbildung 1B) , was darauf hinweist, dass die rekombinanten sGMRα eine Mischung aus Monomeren und Disulfid-linked-Dimmer 18 Jahre alt war. Mit dem zellfreien System (Abb. 2A), wir die Hemmung der GM-CSF-Bindung an sGMRα von GM-CSF-Autoantikörpern (Abb. 2B und Abb.. 2C) ausgewertet. Diese Methoden wurden in Bezug 19 von Urano et al. Die Wachstumshemmung war eng mit dem Binding-Hemmung (r = 0,988, p = 0,002) bei verschiedenen Konzentrationen der GM-CSF Autoantikörper (Abb. 3A) 19 korreliert. Ebenso wurde die Bindung Hemmung signifikant mit der Wachstumshemmung korreliert. Die Binding-Hemmung in einer dosisabhängigen Weise durch GM-CSF-Autoantikörpern (Abb. 3B) 19 erhöht. Diese beiden Parameter für die Serum-IgG-Fraktionen von verschiedenen Patienten wurden miteinander korreliert (r = 0,589 p = 0,006, Abb.. 3C). Weder Bindungsinhibition noch Wachstumshemmung mit Kd-Werten korreliert, und somit waren beide Parameter unabhängig von Bindungsaffinität 19. Folglich Reproduzierbarkeit der Daten zwischen Bindung und gachstum Hemmung durch drei unabhängige Experimente an drei verschiedenen Proben erhalten wurde, ausgewertet. Der Variationskoeffizient angedeutet, dass die zellfreien System mehr gute als die von Biotest (Tabelle 1) wurde. <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Flussdiagramm des Verfahrens zur Herstellung von sGMRα mit transgenen Seidenraupen. A) Strukturen der Transformationsvektoren 9-11. B) SDS-PAGE und Coomassie Brilliant Blue-Färbung des gereinigten sGMRα unter reduzierenden und nicht reduzierenden Bedingungen. sGMRα, lösliches GM-CSF-Rezeptor alpha; MGFP, Monster grün fluoreszierendes Protein; BmNPVpol5&#39;-UTR, 5&#39;-untranslatierten Region Abfolge von Bombyx mori nuklearen polyhedrosis Virus Polyhedrin; SV40 polyA, SV40 polyA-Signal-Sequenz; P 3xP3, 3xP3 Promotor; P SER1, SER1 Promotor; FibL polyA, Fibroin L-Kette polyA-Signal-Sequenz, hr3, Bombyx mori nuklearen polyhedrosis Virus hr3-Enhancer. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schema der zellfreien Assay-System. A) Ein wettbewerbsfähiger Bindungsassay mit sGMRα von Seidenraupen produziert. B) Wirkung zu neutralisieren und nicht-neutralisierende Antikörper auf die Bindung der Hemmung durch zellfreien System. C) Die Differenz von Bindungsinhibition zwischen verschiedenen Konzentrationen von neutralisierenden Antikörpern und nicht-neutralisierenden Antikörpern. GM-CSF, Granulozyten-Makrophagen-Kolonie-stimulierender Faktor; sGMRα, lösliches GM-CSF-Rezeptor alpha, His, RGS-His-tag, AP, alkalische Phosphatase. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Abbildung 3" /> Abbildung 3. GM-CSF Bindungsinhibition zu sGMRα durch Wirkung von GM-CSF polyklonale Antikörper oder das Serum IgG-Fraktionen von Patienten mit Autoimmun-PAP. A) Beziehung zwischen verbindlichen Hemmung und Wachstumshemmung von GM-CSF Autoantikörper. B) Binding-Hemmung bei verschiedenen Konzentrationen von GM-CSF Autoantikörper. C) Beziehung zwischen IC 50 für Prozent Bindungsinhibition und Prozent Wachstumshemmung durch das Serum IgG-Fraktionen 19. IC 50, 50% ige Hemmkonzentration; GM-CSF, Granulozyten-Makrophagen-Kolonie-stimulierender Faktor. <table border="1"><tbody><tr><td> Probe </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-Assay- </td></tr><tr><td> Anzahl der Bestimmungen </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> Mittelwert (% Bindung Hemmung) </td><td> 61,6 </td><td> 63,8 </td><td> 69,7 </td></tr><tr><td> Mittelwert (% Wachstumshemmung) </td><td> 21,0 </td><td> 73,4 </td><td> 82,8 </td></tr><tr><td> Variationskoeffizient (%) (% Bindung Hemmung) </td><td> 6,0 </td><td> 5,6 </td><td> 8,3 </td></tr><tr><td> Variationskoeffizient (%) (% Wachstumshemmung) </td><td> 64,4 </td><td> 18,3 </td><td> 10,4 </td></tr></tbody></table> Beide Assays wurden bei 5 ng / ml GM-CSF und einer Konzentration von GM-CSF Autoantikörper durchgeführt. Tabelle 1. Vergleich der Koeffizienten von Variationen zwischen Prozent verbindlich und Wachstum Hemmungen durch drei unabhängigen Experimenten.

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Die zellfreien Assay schätzte die neutralisierende Wirkung des GM-CSF-Autoantikörpern mit ausgezeichneter Reproduzierbarkeit und Schnelligkeit. Die Bindung der Hemmung durch GM-CSF Autoantikörper oder Serum des Patienten IgG-Fraktionen wurde durch diesen Test ausgewertet. Die Daten zeigten eine Korrelation zwischen der Bindung Hemmung der zellfreien Assay und die Wachstumshemmung von einem Bioassay mit TF-1 Zellen. Der Bioassay wurde vielfach genutzt, sondern beherbergt Schwierigkeiten beim Vergleich der Daten zwisch...

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

Hintergrund: Bisher haben wir gezeigt, dass neutralisierende Wirkung, nicht aber die Konzentration von GM-CSF Autoantikörper wurde mit dem Schweregrad der Erkrankung bei Patienten mit Autoimmun-pulmonale Alveolarproteinose (PAP) 1-3 korreliert. Als Aufhebung der GM-CSF-Bioaktivität in der Lunge ist die wahrscheinliche Ursache für Autoimmunerkrankungen PAP 4,5, ist es das Versprechen, die neutralisierende Wirkung des GM-CSF Autoantikörper für die Beurteilung der Schwere der Erkrankung bei jedem Patienten mit PAP zu messen. Bis jetzt hat neutralisierende Wirkung von GM-CSF-Autoantikörpern durch die Auswertung der Wachstumshemmung von menschlichen Knochenmarkzellen oder TF-1-Zellen mit GM-CSF stimuliert 6-8 bewertet. Im Tierversuch System, jedoch ist es oft problematisch, verlässliche Daten sowie die Daten aus verschiedenen Laboratorien zu vergleichen, aufgrund der technischen Schwierigkeiten bei der Aufrechterhaltung der Zellen in einem konstanten Zustand zu erhalten. ZIEL: GM-CSF imitieren Bindung an GM-CSF-Rezeptor auf der Zelloberfläche mit zellfreien Rezeptor-Bindungs-Assays. Methoden: Transgene Seidenraupen-Technologie wurde für den Erhalt einer großen Menge für rekombinante, lösliche GM-CSF-Rezeptor alpha (sGMRα) mit hoher Reinheit 9-13 aufgetragen. Das rekombinante sGMRα war in der hydrophilen Sericin Schichten von Seidenfäden, ohne auf die Seidenproteine ​​verschmolzen enthalten, und somit können wir leicht aus den Kokons in guter Reinheit Extrakt mit neutralen wässrigen Lösungen 14,15. Glücklicherweise sind die Oligosaccharid-Strukturen, die entscheidend für die Bindung mit GM-CSF sind, mehr Ähnlichkeit mit den Strukturen des menschlichen sGMRα als die von anderen Insekten oder Hefen hergestellt. ERGEBNISSE: Die zellfreien Testsystem mit sGMRα ergaben die Daten mit hoher Plastizität und Zuverlässigkeit. GM-CSF-Bindung an sGMRα war dosisabhängig gehemmt durch polyklonale GM-CSF Autoantikörper in ähnlicher Weise wie die Biotest mit TF-1-Zellen, was darauf hinweist, dass unser neues zellfreien Testsystem mit sGMRα mehr nützlich für die Messung von neutralisierenden Aktivität ist von GM-CSF-Autoantikörpern als der Bioassay-System mit TF-1 Zelle oder menschliche Knochenmarkszellen. FAZIT: Wir haben einen zellfreien Assay quantifiziert die neutralisierende Kapazität von GM-CSF Autoantikörper.

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

Wir haben eine zellfreie Rezeptorbindungstest, um die Bindung von Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) an die Rezeptoren zu schätzen. Es ermöglicht uns, kompetitive Hemmung des biotinylierten GM-CSF-Bindung an löslichen GM-CSF-Rezeptor alpha von GM-CSF Autoantikörper mit ausgezeichneter Reproduzierbarkeit zu bewerten.

Einem zellfreien Assay System Schätzung der neutralisierende Wirkung von GM-CSF Antikörper mit rekombinanten löslichen GM-CSF-Rezeptor

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

Research

JoVE Journal

Immunology and Infection

12.6K Aufrufe.  Niigata University Medical and Dental Hospital. Wir haben eine zellfreie Rezeptorbindungstest, um die Bindung von Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) an die Rezeptoren zu schätzen. Es ermöglicht uns, kompetitive Hemmung des biotinylierten GM-CSF-Bindung an löslichen GM-CSF-Rezeptor alpha von GM-CSF Autoantikörper mit ausgezeichneter Reproduzierbarkeit zu bewerten.

Serie: 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.

Bioenergetik und die Oxidative Burst: Protokolle für die Isolierung und Bewertung der menschlichen Leukozyten und Thrombozyten

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

Forschung

Immunologie und Infektion

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.

Skalierbare High Throughput Auswahl aus Phagen-Antikörperbibliotheken angezeigt Synthetische

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.

Erzeugung und Identifizierung von GM-CSF Abgeleitet Alveolare ähnlichen Makrophagen und dendritische Zellen aus Knochenmark der Maus

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.

Die Beurteilung Retinal Microglial Phagozytische Funktion<em&gt; In Vivo</em&gt; Mit einem Durchfluss-Zytometrie basierten Assay

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 Lymphocyte Kennzeichnung von<sup&gt; 64</sup&gt; Cu-Antikörper-Rezeptor-Targeting für<em&gt; In Vivo</em&gt; Zellhandel von 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.

Generation von Escape Varianten des Influenza-Virus Monoclonal Antikörper neutralisieren

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.

Messung der Influenza neutralisierenden Antikörperantworten gegen a/H3N2-Viren im menschlichen Serum von Microneutralization Assays mit MDCK-SIAT1 Zellen

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.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen 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 ...

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- Differenzierung von Granulozyten-Makrophagen-Kolonie-stimulierende Faktor (GM-CSF) Maus-Herstellung von T-Helferzellen (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.