El proyecto fue apoyado por el NIH NIAID Contrato HHSN2662000500021C N º. Damos las gracias a Ming Chen por su asistencia técnica.

1. El programa de la lanzadera de 96 Amaxa Nucleofector <ol><li> Abra un archivo nuevo parámetro. </li><li> Seleccione el número de pozos que va a utilizar para la transfección estándar arrastrando el cursor sobre el diagrama de placa de 96 pocillos. Use un mínimo de tres pozos a la piscina para cada muestra experimental. </li><li> Introduzca el código del programa: en part1 seleccione &#39;FF&#39; y seleccione part2 &#39;168 &#39;de los menús desplegables </li><li> De &#39;los monocitos, humanos &quot;Solución caja de selección </li><li> En la Opción de control, seleccione &quot;estándar&quot;. </li><li> Haga clic en Aplicar. </li><li> Para incluir un control sin transfección, seleccione los pozos más en el diagrama de como se requiere y seleccione &#39;No Control de programa &quot;de la Opción de Control y haga clic en Aplicar. </li><li> Seleccione cualquier pozos restantes no utilizados en el diagrama de la placa y haga clic en Anule. </li></ol> 2. Prepare los países en desarrollo para la transfección <ol><li> Todo el trabajo debe realizarse en condiciones estériles en una campana de cultivo celular que sea posible. Prepare la solución nucleofection mediante la adición de 96 y complemento de los monocitos humanos de 96 pozos de solución Nucleofector en la proporción de 450 a 2025. Mezclar y dejar a temperatura ambiente. Usted tendrá que 20μl de solución nucleofection por pozo. Crea un exceso de 10% para permitir la pipeta de error. </li><li> Coloque el número de módulos nucleocuvette que necesita en la placa nucleocuvette en la orientación correcta, es decir la inserción de la primera de las filas 1 y 2. </li><li> Determinar el número de centros de datos necesarios para el experimento en el cálculo de 500.000 células por pocillo y el precipitado por centrifugación a 400 g durante 10 minutos. Retire con cuidado el sobrenadante. </li><li> Resuspender las células en la solución nucleofection pipeteando suavemente hacia arriba y abajo varias veces. </li><li> Divida el volumen correcto de las células se resuspendieron en eppendorfs etiquetados para el tratamiento en particular por ejemplo, GLO y RIG-I. </li><li> Añadir siRNA 0.25μg por cada 500.000 células y mezclar con la pipeta. No uso de metas de siRNA en su muestra de control no de la transfección. </li><li> Pipeta de 20μl de la mezcla por encima a los módulos de nucleocuvette, de acuerdo a su diseño experimental, lo que garantiza que el líquido se envía al fondo del pozo. </li><li> Cubrir la placa nucleocuvette con la tapa y llave de la placa sobre una superficie dura un par de veces para ayudar a asegurar la eliminación de burbujas de aire. </li></ol> 3. Transfectar los países en desarrollo <ol><li> Inserte la placa en la bandeja de transporte Nucleofector de 96 pozos, haga clic en Upload y luego comenzar. </li><li> Seguir el progreso del proceso de transfección en la pantalla del ordenador portátil, una cruz de color negro sobre un fondo verde significa un éxito en la transfección de que así, mientras que una barra de color negro sobre un fondo rojo significa que no tuvo éxito. </li><li> Al finalizar el proceso de transfección, retire el plato y añadir 80μl de medio de cultivo DC a cada pocillo utilizando una pipeta multicanal. </li><li> Incubar la placa durante 10 minutos a 37 ° C y 5% de CO 2. </li><li> Transferir el volumen de 100μl de la nucleocuvettes en tubos matriz que contiene 100μl de pre-calentado medio de cultivo DC, mantener la orientación correcta. </li><li> Retire y deseche los tubos donde la transfección no ocurrió. </li><li> Se incuba a 37 ° C y 5% de CO 2 para el intervalo de tiempo de 24 horas o de otro tipo deseado. </li></ol> 4. Infectar a las células con NDV <ol><li> Retire los tubos de la matriz de la incubadora a la campana y los tubos de cultivo de células piscina para cada muestra experimental en eppendorfs </li><li> Que sedimenten las células suavemente al girar en una centrífuga de sobremesa durante 5 minutos y retirar el sobrenadante. </li><li> Resuspender las células en medio de cultivo libre de suero que contiene NDV en una MOI de 1 y se incuba a 37 ° C y 5% de CO 2 durante 45 minutos, con eppendorfs forrados de una manera aséptica. </li><li> Agregar 900μl de medio de cultivo DC, y volver a incubar por 80-10 h. </li></ol> 5. Las células de la cosecha <ol><li> Pellet células girando eppendorfs en una centrífuga de sobremesa y eliminar el sobrenadante. </li><li> Las células de la cosecha para el ARN o la extracción de proteínas de acuerdo con el protocolo. </li></ol> 6. Los resultados representativos: Utilizando el protocolo optimizado transfectadas países en desarrollo con RIGI de metas de siRNA durante 24 horas y luego se infectaron las células con NDV (un paramixovirus detectado por RIG-I) que estimulan la vía de la respuesta del interferón. De QRT-PCR análisis demostró que derribar del gen en un 75% en el nivel de transcripción. También se observó una reducción similar en la expresión de IFNβ, que es un efector corriente abajo de RIG-I en la cascada de señalización de IFN. Asimismo, se observó que la expresión de IFNβ en los no infectados, el control de las células transfectadas no fue detectable mientras que la de MxA, un gen IFNβ respuesta aguas abajo, fue mínima (Figura 1). Una segunda transfección de países en desarrollo con RIGI de metas de siRNA se realizó con células suficiente para incluir el análisis de Western blot. Cuando elRT-PCR resultados fueron similares a lo que vimos anteriormente (62% y 66% tumbar de RIG-I y la expresión IFNβ respectivamente) (fig. 1B), el Western blot probaron para RIG-I reveló que la expresión de este gen ha sido completamente bloqueado (fig. 1C). <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Figura 1A"/> Figura 1. (A) MoDCs fueron transfectadas con siRNA dirigidos RIG-I o siRNA GLO no específica y el efecto sobre la expresión de RIG-I, IFNβ y MxA determinado por QRT-PCR. El protocolo de transfección utiliza un buffer de monocitos y un programa específico nucleoporation FF168 (Lonza Walkersville Inc.) Después de la incubación durante 24 h, las células transfectadas fueron infectadas, ya sea con NDV (+) o hacia la izquierda no infectados (-). Después de una incubación de 10 h, se recogieron las células y los niveles de transcripción de ARN extraído representan los resultados de dos experimentos de replicar. Tomado de Bowles et al 16. (B) MoDCs obtenida a partir de una diferente capa leucocitaria que se utiliza para la Figura 1 se transfectaron de nuevo con cualquiera de RIG-I de metas de siRNA o siRNA GLO no específica utilizando el protocolo de transfección igual que el anterior. Un control adicional con MoDCs untransfected fue incorporado en el experimento. Tras una incubación de 24 horas todas las células fueron infectadas con el NDV y se incubaron durante 10 h antes de ser cosechada, tanto para el ARN y extracción de proteínas. Los niveles de transcripción de RIG-I, IFNβ y MxA según lo determinado por QRT-PCR representan los resultados de dos experimentos de replicar. Tomado de Bowles et al 16. (C) lisados ​​de las células se describe en la Figura 1B anteriores fueron analizados por Western blot. Calles 1 y 2, lisados ​​de células no transfectadas, carriles 3 y 4, lisados ​​de células transfectadas siRNA GLO, carriles 5 y 6, lisados ​​de células transfectadas con RIG-I-targeting siRNA. Las muestras se probaron para la RIG-I y GAPDH (como control de carga) y se realizaron por duplicado. Tomado de Bowles et al 16.

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Transfección eficiente de ingenuo células dendríticas primaria es importante para el análisis de alto rendimiento e ingeniería inversa de celulares vías inflamatorias en esta celda mediador clave de la transición inmune innato-adaptación. Sin embargo, la mayoría de los investigadores encuentran que estas células son difíciles de transfectar de forma eficiente y sin la maduración procedimiento de transfección de células inducen al uso de técnicas estándar de transfección. Nosotros investigamos si estas l...

<table border="1"><tbody><tr><th> Equipo / Reactivo </th><th> Empresa </th><th> Catálogo # </th><th> Comentarios </th></tr><tr><td> Amaxa Nucleofector 96 y Traslados </td><td> Lonza </td><td> 108S0109 </td><td> Número de serie </td></tr><tr><td> Amaxa monocitos humanos de 96 pozos Kit Nucleofector </td><td> Lonza </td><td> VHPA-2007 </td><td> Contiene los monocitos humanos de 96 pozos de solución Nucleofector, el suplemento de 96 pozos y la Nucleocuvettes y placas </td></tr><tr><td> RIG-I siRNA </td><td> Dharmacon </td><td> L-012511-00 </td><td></td></tr><tr><td> GLO siRNA </td><td> Dharmacon </td><td> D-001600-01-20 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td><td> Complementado con un 10% de FCS, 2 mM L-glutamina, 100 U / ml penicilina y 100 ug / ml de estreptomicina para el crecimiento de mediano DC </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> L-glutamina </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> Suero fetal de ternero </td><td> HyClone </td><td> 3.070,03 </td><td></td></tr><tr><td> Las células dendríticas </td><td> New York Blood Center </td><td></td><td> Los países en desarrollo se purificó a partir de capas de leucocitos mediante un procedimiento estándar </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

Las células dendríticas (DC) se puede considerar centinelas del sistema inmunitario que juegan un papel crítico en la iniciación y respuesta a la infección 1. La detección de antígenos patogénicos por los países en desarrollo ingenuo es a través de los receptores de reconocimiento de patrones (RRP), que son capaces de reconocer estructuras específicas de conservación conocido como patógeno de patrones moleculares asociados (PAMP). La detección de PAMP por los países en desarrollo desencadena una cascada de señalización intracelular que resulta en su activación y transformación de los países en desarrollo maduro. Este proceso se caracteriza típicamente por la producción de interferón tipo 1, junto con otras citocinas proinflamatorias, la regulación positiva de los marcadores de superficie celular, como MHCII y CD86 y la migración de las DC maduras a los ganglios linfáticos de drenaje, donde la interacción con las células T se inicia la respuesta inmune adaptativa 2, 3. Por lo tanto, los países en desarrollo enlace del sistema inmune innato y adaptativo. La capacidad de diseccionar las redes moleculares que subyacen a la respuesta de DC a varios patógenos es fundamental para una mejor comprensión de la regulación de estas vías de señalización y de sus genes inducidos. También debe ayudar a facilitar el desarrollo de la DC, las vacunas contra las enfermedades infecciosas y tumores. Sin embargo, esta línea de investigación se ha visto gravemente obstaculizada por la dificultad de la transfección de primaria DC 4. Métodos de transducción de virus, como el sistema de lentiviral, se utilizan normalmente, pero tienen muchas limitaciones, como la complejidad y la bio-peligrosos de riesgo (con los costos asociados) 5,6,7,8. Además, la entrega de los productos de los genes virales aumenta la inmunogenicidad de los países en desarrollo transducidas 9,10,11,12. La electroporación se ha utilizado con resultados mixtos 13,14,15, pero nosotros somos los primeros en reportar el uso de un protocolo de transfección de alto rendimiento y demuestran de forma concluyente su utilidad. En este informe se resume un protocolo optimizado comerciales de alto rendimiento para la transfección de los países en desarrollo humano primario, con una toxicidad celular limitado y la falta de maduración de las DC 16. Eficiencia de transfección (del plásmido GFP) y la viabilidad celular fueron más del 50% y 70% respectivamente. FACS análisis estableció la ausencia de aumento en la expresión de la maduración de los marcadores CD86 y MHCII en las células transfectadas, mientras que QRT-PCR demostró que no había regulación al alza de IFNβ. El uso de este protocolo de electroporación, aportar pruebas para la transfección exitosa de países en desarrollo con siRNA y efectiva de derribar gen diana RIG-I, un receptor clave reconocimiento viral 16,17, tanto en el mRNA y los niveles de proteína.

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Presentamos nuestra optimizado de alto rendimiento protocolo nucleofection como una forma eficiente de la transfección de primaria monocitos humanos derivados de células dendríticas, ya sea con el ADN de plásmido o siRNA sin causar la maduración celular. Nos proporcionan evidencia adicional para silenciar siRNA éxito de gen diana RIG-I tanto en el mRNA y los niveles de proteína.

Protocolo optimizado para la transfección eficiente de células dendríticas, sin maduración de las células

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

optimized-protocol-for-efficient-transfection-dendritic-cells-without

Research

JoVE Journal

Immunology and Infection

20.2K vistas.  Mount Sinai School of Medicine. Presentamos nuestra optimizado de alto rendimiento protocolo nucleofection como una forma eficiente de la transfección de primaria monocitos humanos derivados de células dendríticas, ya sea con el ADN de plásmido o siRNA sin causar la maduración celular. Nos proporcionan evidencia adicional para silenciar siRNA éxito de gen diana RIG-I tanto en el mRNA y los niveles de proteína.

Video: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Generación y Etiquetado de murino médula ósea procedentes de células dendríticas con nanocristales Qdot de Estudios de seguimiento

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigación

Inmunología e Infección

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

El análisis de la maduración pulmonar de células dendríticas y migración durante la inflamación de las vías alérgica

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Artificial células presentadoras de antígenos (KaAPC) para la utilización eficiente<em&gt; In Vitro</em&gt; El agotamiento de células T humanas específicas de antígeno

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfección altamente eficiente de humanos THP-1 macrófagos por Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Un método de rendimiento eficiente y de alta para el aislamiento de ratón subconjuntos de células dendríticas

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Generación de inmaduras, maduras y tolerogénico células dendríticas con diferentes fenotipos metabólico

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desarrollo y validación de una sola molécula ultrasensible matriz Digital análisis de enzima-ligado del inmunosorbente para interferón-α humano

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Nuevo protocolo para la generación de células dendríticas inmunogénicas fisiológicas

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

Método de cribado de una sola célula para la selección y recuperación de anticuerpos con especificidades deseadas de poblaciones de células B de memoria humana enriquecidas

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Transfección altamente eficiente de macrófagos primarios con ARNm transcrito in Vitro

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...