O projecto foi apoiado pelo NIH Contrato n º HHSN2662000500021C NIAID. Agradecemos Ming Chen por sua assistência técnica.

1. O programa Amaxa 96 poços Nucleofector shuttle <ol><li> Abra um arquivo novo parâmetro. </li><li> Selecione o número de poços que será utilizado para a transfecção padrão, arrastando o cursor sobre o diagrama de placa de 96 poços. Use um mínimo de três poços para piscina para cada amostra experimental. </li><li> Introduza o código do programa: em part1 selecionar &#39;FF&#39; e em part2 selecionar &#39;168 &#39;do pull down menus </li><li> De &#39;monócitos humanos, &quot;Solução caixa de seleção </li><li> Na Opção de Controle, selecione &#39;standard&#39;. </li><li> Clique em Aplicar. </li><li> Para incluir um controle não-transfecção, selecione poços adicionais a partir do diagrama conforme necessário e, em seguida, escolha a opção &#39;Sem Controle Program&#39; Opção de Controle e clique em Aplicar. </li><li> Selecione qualquer cavidades remanescentes não utilizadas no diagrama de placa e clique em Cancele. </li></ol> 2. Prepare DCs para transfecção <ol><li> Todo o trabalho deve ser feito sob condições estéreis em uma capa de cultura de células, sempre que possível. Preparar a solução adicionando nucleofection de 96 poços suplemento para monócitos humanos de 96 poços Solução Nucleofector na proporção de 450 a 2025. Misture e deixe à temperatura ambiente. Você vai precisar de 20μl de solução nucleofection por poço. Faça um excesso de 10% para permitir a pipetagem erro. </li><li> Coloque o número de módulos nucleocuvette que você precisa na placa nucleocuvette no ie orientação correta de inserir o primeiro em que as linhas 1 e 2. </li><li> Determinar o número de DCs necessários para a sua experiência em cálculo de 500.000 células por poço e pellet por centrifugação a 400g por 10min. Remova cuidadosamente o sobrenadante. </li><li> Ressuspender as células na solução nucleofection cuidado pipetando para cima e para baixo algumas vezes. </li><li> Divida o volume correto de células ressuspendido em eppendorfs etiquetados para o tratamento especial por exemplo GLO e RIG-I. </li><li> Adicionar siRNA 0.25μg por 500.000 células e misture por pipetagem. Use siRNA não-alvo na sua amostra de controlo não-transfecção. </li><li> Pipetar 20μl das misturas acima para os módulos de nucleocuvette, de acordo com seu layout experimental, garantindo que o líquido é enviado para o fundo do poço. </li><li> Cobrir a placa nucleocuvette com a tampa e toque a placa sobre uma superfície dura um par de vezes para ajudar a garantir a remoção de bolhas de ar. </li></ol> 3. Transfectar DCs <ol><li> Inserir placa na bandeja de shuttle Nucleofector de 96 poços, clique em Enviar e, em seguida, começar. </li><li> Acompanhar o progresso do processo de transfecção no visor lap top, uma cruz negra sobre um fundo verde significa uma transfecção bem sucedido em que o bem, ao passo que uma barra preta em um fundo vermelho significa que ele não teve sucesso. </li><li> Após a conclusão do processo de transfecção, retire a placa e adicionar Pipete 80 da DC meio de crescimento a cada poço usando uma pipeta multicanal. </li><li> Incubar placa por 10 min a 37 ° C e 5% CO 2. </li><li> Transferir o volume de 100μl do nucleocuvettes em tubos de matriz contendo 100μl de pré-aquecido meio de crescimento DC, mantendo a orientação correta. </li><li> Retire e deite fora os tubos onde transfecção não ocorreu. </li><li> Incubar a 37 ° C e 5% CO 2 para o intervalo de tempo de 24 horas ou outro desejado. </li></ol> 4. Infectar as células com NDV <ol><li> Remova os tubos da matriz da incubadora para a capa de cultura de células e tubos piscina para cada amostra experimental em eppendorfs </li><li> Agregar as células delicadamente girando em uma centrífuga desk top por 5 minutos e retire o sobrenadante. </li><li> Ressuspender as células no soro livre de meio de crescimento contendo NDV em um MOI de 1 e incubar a 37 ° C e 5% CO 2 por 45 min, com eppendorfs vagamente coberta de uma forma estéril. </li><li> Adicionar 900μl de meio de crescimento DC, e re-incubar por 8-10 h. </li></ol> 5. Células colheita <ol><li> Células de pelotas por fiação eppendorfs em uma centrífuga desk top e remover o sobrenadante. </li><li> Células colheita para RNA ou extração de proteínas de acordo com o protocolo. </li></ol> 6. Resultados representativos: Usando nosso protocolo otimizado que transfectadas DCs com RIGI-targeting siRNA por 24 horas e, em seguida, as células infectadas com o NDV (a paramixovírus detectado pelo RIG-I) para estimular o trajeto resposta interferon. Por qRT-PCR demonstramos knock down do gene em 75% no nível de transcrição. Observamos também uma redução semelhante na expressão de IFNβ, que é uma a jusante efetoras de RIG-I na cascata de sinalização IFN. Além disso, observou-se que a expressão de IFNβ em não-infectados, controle de células transfectadas não foi detectado enquanto o de MxA, um gene de resposta IFNβ a jusante, foi mínima (Figura 1A). A transfecção segunda DCs com RIGI-targeting siRNA foi realizada utilizando células suficientes para incluir a análise de Western blot. Onde oRT-PCR, os resultados foram semelhantes ao que vimos anteriormente (62% e 66% de derrubar RIG-I e expressão IFNβ respectivamente) (Figura 1B), o Western blot sondado para RIG-I revelou que a expressão desse gene foi completamente bloqueada (Figura 1C). <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Figura 1A"/> Figura 1. (A) MoDCs foram transfectadas com siRNA alvo tanto RIG-I ou siRNA GLO inespecíficos eo efeito sobre a expressão de RIG-I IFNβ e MxA determinada por qRT-PCR. O protocolo de transfecção utilizado um buffer de monócitos específico e um programa de nucleoporation FF168 (Lonza Walkersville Inc.) Após a incubação por 24 h, as células transfectadas ou foram infectadas com o NDV (+) ou para a esquerda não infectados (-). Após incubação de mais de 10 h, as células foram colhidas e os níveis de Transcrição RNA extraído representam os resultados de dois experimentos replicar. Tomado de Bowles et al 16. (B) MoDCs obtidos a partir de um revestimento buffy diferentes, usado para a Figura 1A foram novamente transfectadas com um RIG-I-alvo siRNA siRNA ou GLO inespecífica utilizando o protocolo de transfecção mesmo que acima. Um controle adicional usando MoDCs untransfected foi incorporado ao experimento. Após uma 24 horas de incubação todas as células foram infectadas com o NDV e incubados por um h mais 10 antes de ser colhida para ambos RNA e extração de proteínas. Níveis de transcrição de RIG-I IFNβ e MxA conforme determinado pelo qRT-PCR representam os resultados de dois experimentos replicar. Tomado de Bowles et al 16. (C) Lisados ​​de células descrito na Figura 1B acima foram analisados ​​por Western blot. Pistas 1 e 2, lisados ​​de células untransfected; faixas 3 e 4, lisados ​​de células transfectadas siRNA GLO; pistas 5 e 6, lisados ​​de células transfectadas com siRNA RIG-I-alvo. As amostras foram sondados para RIG-I e também GAPDH (como um controle de carga) e foram executados em duplicata. Tomado de Bowles et al 16.

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Transfecção eficiente de células dendríticas naïve primário é importante para a análise de elevada capacidade e engenharia reversa de celulares vias inflamatórias nesta célula-chave mediando a transição inata-imune adaptativo. No entanto, a maioria dos investigadores acham que essas células são difíceis de transfecção tanto de forma eficiente e sem a maturação das células transfecção procedimento de indução ao usar técnicas de transfecção padrão. Nós investigamos se essas limitações poderi...

<table border="1"><tbody><tr><th> Equipamento / Reagente </th><th> Companhia </th><th> Catálogo # </th><th> Comentários </th></tr><tr><td> Amaxa Nucleofector de 96 poços Shuttle </td><td> Lonza </td><td> 108S0109 </td><td> Número de série </td></tr><tr><td> Amaxa Humanos monócitos de 96 poços Kit Nucleofector </td><td> Lonza </td><td> VHPA de 2007 </td><td> Contém os monócitos humanos de 96 poços Solução Nucleofector, o Suplemento de 96 poços e as Nucleocuvettes e placas </td></tr><tr><td> RIG-I siRNA </td><td> Dharmacon </td><td> L-012.511-00 </td><td></td></tr><tr><td> SiRNA GLO </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> Suplementado com 10% SFB, 2 mM L-glutamina, 100 U / ml de penicilina e estreptomicina 100 mg / ml para fazer DC meio de crescimento </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> Soro fetal bovino </td><td> HyClone </td><td> 3.070,03 </td><td></td></tr><tr><td> As células dendríticas </td><td> New York Blood centro </td><td></td><td> DCs são purificados de casacos buffy usando um procedimento padrão </td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

As células dendríticas (DCs) podem ser considerados sentinelas do sistema imune que desempenham um papel fundamental no seu início e resposta à infecção 1. Detecção de antígeno patogênicos por DCs naïve é através de receptores de reconhecimento de padrões (PRRs) que são capazes de reconhecer estruturas específicas conservadas referido como patógeno-padrões moleculares associados (PAMPs). Detecção de PAMPs por DCs desencadeia uma cascata de sinalização intracelular, resultando em sua ativação e transformação para DCs maduras. Este processo é normalmente caracterizada pela produção de interferon tipo 1, juntamente com outras citocinas pró-inflamatórias, upregulation de marcadores da superfície celular como MHCII e CD86 e migração da DC maduro para os linfonodos de drenagem, onde a interação com as células T inicia a resposta imune adaptativa 2, 3. Assim, DCs ligação dos sistemas imune inata e adaptativa. A capacidade de dissecar as redes moleculares subjacentes DC resposta a vários patógenos é fundamental para uma melhor compreensão da regulação destas vias de sinalização e seus genes induzidos. Ele deve também ajudar a facilitar o desenvolvimento de DC baseado em vacinas contra doenças infecciosas e tumores. No entanto, esta linha de pesquisa tem sido severamente impedido pela dificuldade de transfecting DCs primários 4. Métodos de transdução de vírus, como o sistema lentiviral, são normalmente utilizados, mas carregam muitas limitações, como a complexidade e bio-perigosos risco (com os custos associados) 5,6,7,8. Além disso, a entrega de produtos do gene viral aumenta a imunogenicidade dos transduzidas DCs 9,10,11,12. Eletroporação tem sido usado com resultados mistos 13,14,15, mas somos os primeiros a relatar o uso de um protocolo de transfecção de alto rendimento e demonstrar conclusivamente a sua utilidade. Neste relatório, resumem um protocolo otimizado comercial para high-throughput transfecção de DCs humanas primárias, com limitada toxicidade celular e uma ausência de maturação DC 16. Eficiência de transfecção (GFP de plasmídeo) e viabilidade celular foram mais de 50% e 70%, respectivamente. FACS análise estabeleceu a ausência de aumento da expressão do CD86 e marcadores de maturação MHCII em células transfectadas, enquanto qRT-PCR não demonstrou upregulation de IFNβ. Usando este protocolo eletroporação, que fornecem evidências de sucesso da transfecção com siRNA DCs e eficaz knock down do gene alvo RIG-I, um receptor de reconhecimento chave viral 16,17, em ambos os mRNA e os níveis 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

Apresentamos nosso protocolo nucleofection high-throughput otimizado como uma forma eficiente de transfecting humanos primários derivados de monócitos com células dendríticas ou DNA plasmídeo ou siRNA sem causar maturação das células. Nós ainda fornecer provas para silenciar siRNA sucesso do gene alvo RIG-I em ambos os mRNA e os níveis de proteína.

Protocolo otimizado para transfecção eficiente de células dendríticas, sem maturação das 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 visualizações.  Mount Sinai School of Medicine. Apresentamos nosso protocolo nucleofection high-throughput otimizado como uma forma eficiente de transfecting humanos primários derivados de monócitos com células dendríticas ou DNA plasmídeo ou siRNA sem causar maturação das células. Nós ainda fornecer provas para silenciar siRNA sucesso do gene alvo RIG-I em ambos os mRNA e os níveis de proteína.

Vídeo: 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.

Geração e Rotulagem de murino derivadas da medula óssea Células Dendríticas com Nanocristais Qdot de Estudos de rastreamento

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

Imunologia e Infecção

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.

Análise da maturação das células dendríticas e migração pulmonar durante a inflamação alérgica das vias aéreas

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.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

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.

A transfecção altamente eficiente de THP-1 por macrófagos humanos 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.

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

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.

Geração de imatura, madura e tolerogênicas células dendríticas com diferentes fenótipos Metabolic

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.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana Interferon-α

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.

Novo protocolo para a geração de células dendríticas imunogênicas 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 triagem de célula única para a seleção e recuperação de anticorpos com especificidades desejadas de populações de células B de memória humana enriquecida

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.

Transfecção altamente eficiente de macrófagos primários com mRNA 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 ...