Este trabalho apoiado em parte pelo NIH Grant sob CA137499 R15-01 (FB) e um fundo de arranque da Universidade de Ohio (FB).

1. Dissecção dos fêmures e tíbias de rato e cultura de células da medula óssea <ol><li> Sacrifício 2 camundongos por asfixia CO 2 e dissecar cuidadosamente tíbias e fêmures sem cortar as extremidades dos ossos. </li><li> Limpa os ossos de todos os tecidos anexado usando lenço de papel. Tenha cuidado para não quebrar os ossos. </li><li> Esterilizar os ossos por imersão em etanol 70% por 10 min em uma placa de Petri 35 mm. A partir deste momento, o trabalho dentro de uma capa de biossegurança para evitar a contaminação das culturas de células. </li><li> Recuperar os ossos a partir do etanol e deixe-os secar por 5 min em uma placa de Petri dentro do armário de biossegurança. </li><li> Cortar os fêmures no meio, e da tíbia por sua ponta mais fina. Infundir o interior do osso com 1 ml de meio RPMI (sem soro, mas com antibióticos), utilizando uma seringa estéril sobre uma placa de Petri estéril. </li><li> A suspensão de células são coletadas e lavadas em meio RPMI 2X por 10 min de centrifugação em um tubo de centrífuga de 15 ml a 1.100 RPM em uma centrífuga refrigerada (4 ° C) com um rotor de caçamba móvel. </li><li> Após a última lavagem, ressuspender as células em 2 ml de tampão de lise ACK e incubar por 5 min à temperatura ambiente, a fim de eliminar as células vermelhas do sangue. </li><li> Adicionar 13 ml de RPMI com 10% de FBS, ressuspender e lavar 2X neste meio com as mesmas configurações, conforme descrito em 1.6. </li><li> Contagem de células, ajustar a 2 x 10 5 células / ml com RPMI 10% FBS, e adicionar rm-GM-CSF (20 concentração ng / ml final) 5. </li><li> Adicionar 10 ml dessa suspensão para uma qualidade microbiológica estéril, 10 cm placa de Petri, e cultura em uma incubadora de CO 2 (37 ° C CO, 5% 2). </li><li> Três dias depois, adicionar outra de 10 ml de RPMI 10% FBS com 20 ng / l de rmGM-CSF a cada um dos pratos preparados. </li><li> Três dias depois, 10 ml de suspensão de células são recuperados a partir de cada placa de Petri, centrifugados como em 1.6, ressuspendido em igual volume de RPMI com 10% FBS rmGM-CSF e voltou para a placa de Petri. As células são cultivadas por 2 dias adicionais no 2 CO incubadora. </li></ol> 2. Coleta e rotulagem das DCs <ol><li> Depois de 8 dias em cultura de células frouxamente aderente são recuperados pela lavagem das placas de Petri com meio fresco. Este protocolo torna em torno de 2-4 x10 8 DCs em nossas mãos. As células podem ser analisados ​​para DC fenótipo por citometria de fluxo. </li><li> Células coletadas são então rotulados com o Qtracker Labeling Kit 655 celular seguindo estritamente as instruções do fabricante. </li><li> Temos excelentes resultados rotulagem derivadas da medula óssea de camundongos DCs com Qdots em uma concentração 10 nM. Para conseguir isso, alíquotas de Qtracker Celular componentes Kit Labeling A e B são misturados em volumes iguais, incubadas por 5 min à temperatura ambiente, e imediatamente diluído 1 / 100 em meio fresco com vórtex por 30 s. </li><li> Para manchar 5 x 10 6 DCs, mistura de 5 mL de cada componentes do kit A e B em um tubo eppendorf e incubar por 5 min à temperatura ambiente. Em seguida, adicionar 1 ml de RPMI incubar FBS 10%, e vortex por 30 segundos. </li><li> Adicionar 0,5 ml de suspensão de células contendo 5 6 x10 DCs, e incubar a 37 ° C por 60 min. </li><li> Então, lave as células em RPMI 2X 10% FBS (1.100 RPM). As células podem ser ressuspendido em RPMI para cultura, nem em PBS para injeção em camundongos experimental. </li></ol> 3. Avaliação do DC rotulados <ol><li> Rotulagem célula pode ser avaliada por meio de microscopia de fluorescência. Para conseguir isso, uma gota de suspensão de células é adicionado a uma lâmina de microscópio, coberto com uma lamela de vidro, e avaliada com um microscópio fluorescente tendo em conta que estes têm Qdots de emissão e excitação espectros de 565NM e 405-525nm, respectivamente (Fig. 3 ) </li><li> Neste ponto, as células rotuladas podem ser usadas para injetar animais, ou de maturação pode ser induzida pela incubação destas células por 48 h (37 ° C, 5 CO 2) na presença de LPS (100 ng / ml) e TNF-( 20 ng / ml). Fluorescentes de rotulagem não é afetado por DC maturação (Fig. 4). </li></ol> 4. Resultados representativos: Com isto, descrito como preparar derivadas da medula óssea de camundongos DCs e como rotulou-os com Qdots fluorescente. Fig. 1 resume o procedimento para obter as células, enquanto a fig. 2 mostra o procedimento para classificá-los com Qdots. Como mostrado na figura. 3, quase todas as células são rotulados por este procedimento, e este não é afetada pela DC maturação com fatores inflamatórios (Fig. 4). Fig. 5 mostra que Qdot manchada de DCs (Fig.5a) comportam-se semelhante a células não-manchada quando tratados com um cocktail inflamatórias. Ambas as preparações de células de forma semelhante upregulate expressão de moléculas co-estimulatórias (Fig. 5b) e produzir quantidades semelhantes de IL-6 (Fig. 5c) e óxido nítrico (Fig. 5d) sobre os estímulos de maturação. Isto concorda com a PREVIous dados publicados, indicando que a coloração Qdot não afeta a capacidade de DC para se transformar em células maduras capazes de induzir respostas imunes [6]. Estas células podem ser usadas para injetar animais experimentais. Como mostrado na figura. 6, dois dias após a injeção intravenosa de DCs rotulados em camundongos, que foram capazes de detectar Qdot manchada de células do baço. Este também é de acordo com dados publicados previamente mostrando o uso de nanocristais Qdot para avaliar a migração de DC in vivo [6], não-invasivo de imagens e citometria de fluxo. <img src="/files/ftp_upload/2785/2785fig1.jpg" alt="Figura 1"/> Figura Fluxograma 1. DC de geração a partir de medula óssea. Com isto, descrevem o processo para obter derivadas da medula óssea DC. Ambas as tíbias e fêmures são dissecados e limpa do tecido circundante. Dicas óssea são preservadas e do interior dos ossos são lavados com meio. Depois de eliminar os glóbulos vermelhos, células da medula óssea são cultivados por 8 dias na presença de GM-CSF para diferenciá-los em DCs. <img src="/files/ftp_upload/2785/2785fig2.jpg" alt="Figura 2"/> Figura Fluxograma 2. Do procedimento de rotulagem. Alíquotas iguais de Qtracker Celular componentes Kit Labeling A e B são misturados em um tubo eppendorf. As células dendríticas são recolhidos a partir de 8 dias culturas de medula óssea com a GM-CSF e misturado com o corante de rotulagem para os 60 minutos a 37C. Em seguida, as células são lavadas na mídia para eliminar as partículas rotulagem excesso. <img src="/files/ftp_upload/2785/2785fig3.jpg" alt="Figura 3"/> Figura 3. Microfotografia fluorescente de DCs imediatamente após rotulagem. Uma gota de células marcadas foi depositado em uma lâmina de vidro e coberto por uma lamínula. Então, as amostras foram avaliadas em um microscópio de fluorescência e as imagens foram adquiridas através de uma câmera digital 5,0 Micropublisher Cor CCD (Qimaging, Surrey, BC Canada). <img src="/files/ftp_upload/2785/2785fig4.jpg" alt="Figura 4"/> Figura 4. Microfotografia fluorescente de DCs após 48 h de maturação in vitro. DCs rotulados foram cultivadas por 48 h com LPS (100 ng / ml) e TNF-α (20 ng / ml) em lâminas de vidro em uma incubadora de CO 2 (37 ° C CO, 5% 2). Então, as amostras foram lavadas com PBS (5 min, 2X), fixado com acetona (15 min, 4 ° C) e contrastado com DAPI. As células foram avaliadas em um microscópio de fluorescência como acima. <img src="/files/ftp_upload/2785/2785fig5.jpg" alt="Figura 5"/> Figura 5. A atividade biológica de Qdot manchada DCs maduras. DCs rotulados maturados in vitro como acima foram analisados ​​por citometria de fluxo (A) e (B). (A) Qdot manchada de células dão um sinal forte no canal FL3 (PerCP). Sombreada histograma: controle imaculada. (B) DCs Qdot manchadas e sem manchas foram adicionalmente corados com anticorpos específicos contra maturação marcadores CD86 e CD40; e controles isotipo. As células foram então analisados ​​em um citômetro de fluxo FACSort (Becton Dickinson, San Jose, CA). Além disso, IL-6 (C) e óxido nítrico (NO) foram determinados em sobrenadantes de madura Qdot manchada de DCs e controles. Níveis de IL-6 e óxido nítrico foi determinada por meio de ELISA análise e ensaio de Griess como temos descrito anteriormente 7, 8. Todos os dados mostraram nesta figura é representativa de dois ou três experimentos independentes mostrando resultados semelhantes. Os dados foram analisados ​​por ANOVA, utilizando o software GraphPad. <img src="/files/ftp_upload/2785/2785fig6.jpg" alt="Figura 6"/> Figura 6. Detecção de DCs rotuladas de tecidos dissecados. Rotulados DCs maduras (1x10 6 células) foram injetadas em ratos por via intravenosa C57BL / 6. (A) Dois dias depois, os ratos foram sacrificados e os baços coletados, snap congelados, embutidos em outubro e 8 seções mM preparada com a criostato. Em seguida, amostras foram fixadas com acetona (15 min, 4 ° C) e contrastado com DAPI. As células foram avaliadas em um microscópio de fluorescência como acima Fig. 6a:. Ampliação de 40x, Fig. 6b: aumento de 100X, fig. Ampliação, 6C 400X.

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Murino mielóide) DCs têm sido amplamente utilizados, a fim de determinar a eficácia e melhoria da DC baseado em vacinas; investigar DC: interações de células T ou desenvolvimento DC, e determinar seu papel em várias doenças 9-11. Com isto mostramos como gerar DCs a partir de precursores recuperado de medulas ósseas de tíbias e fêmures. Nós recuperar os ossos sem cortar as pontas, permitindo-nos para esterilizá-los por submersão em etanol 70%, reduzindo assim a probabilidade de contaminação. Para diferenciar CDs a partir de células de medula óssea só usamos GM-CSF como 5 descritos anteriormente. Apesar de alguns protocolos também usar IL-4, tem sido relatado que essa citocina não é necessário quando se trabalha com altos níveis de GM-CSF 12. Na verdade, nós já demonstraram que essas DCs são capazes de induzir respostas imunes 13. Além disso, o cuidado tem que ser tomadas para recuperar as células aderentes apenas vagamente de 8 dias-culturas, lavando os pratos de Petri com meio desde células anexados mostram um fenótipo mais monócitos-like. Aqui nós mostramos a rotulagem de DCs com partículas Qdot fluorescente. Esta rotulagem tem algumas vantagens relativamente a outros métodos. Primeiro, as partículas Qdots são facilmente incorporados nas células. Segundo, o sinal fluorescente é muito alto e não é alterada pela maturação DC. Terceiro, a fluorescência não é perdido quando as células ou tecidos são fixados com solventes, como acetona, ao contrário do que acontece se GFP é utilizada para tag DCs 14, dando mais flexibilidade no momento de escolher protocolos de coloração. Finalmente, o sinal de alta fluorescentes dada por essas partículas permite a visualização das células, apesar de tecidos auto-fluorescência. Como descrito anteriormente 6, coloração Qdot não afetou a capacidade de maturação dessas células. Com isto mostramos que Qdot manchada de DCs se comportam de maneira semelhante como não-manchada DCs, upregulating moléculas co-estimulatórias e produção de IL-6 e óxido nítrico em resposta a estímulos inflamatórios. Embora DCs são células especializadas no desencadear respostas imunes, eles foram mostrados para participar em condições patológicas, como câncer e aterosclerose 4, 15, 16. Eles foram também alegou a participar de processo angiogênico 17, 18, ​​chegou a sugerir como estruturalmente participantes no desenvolvimento de novos vasos 19, 20. Assim, os métodos que permitem o rastreamento de DC in vivo, e determinar a sua localização geográfica em diferentes tecidos 4, 21, 22 são muito valiosos.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Camundongos C57BL / 6 </td><td> Jackson laboratórios </td><td></td><td> Fêmeas, 6-8 semanas de idade </td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 11875-119 </td><td></td></tr><tr><td> Soro fetal bovino, qualificado </td><td> Invitrogen </td><td> 10437-028 </td><td></td></tr><tr><td> Antibiótico antimicótico </td><td> Invitrogen </td><td> 15240-096 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 10010-049 </td><td></td></tr><tr><td> Tampão de Lise ACK </td><td> Lonza Walkersville, Inc </td><td> 10-548E </td><td></td></tr><tr><td> Recombinante murino GM-CSF </td><td> PeproTech Inc </td><td> 315-03 </td><td></td></tr><tr><td> Qtracker 655 Kit Labeling celular </td><td> Invitrogen </td><td> Q25021MP </td><td></td></tr><tr><td> Lipopolissacarídeo </td><td> Invivogen </td><td> tlrl-eblps </td><td></td></tr><tr><td> Recombinante murino TNF alpha </td><td> Peprotech </td><td> 315-01A </td><td></td></tr><tr><td> CD86 anticorpo </td><td> BD Biosciences </td><td> 553691 </td><td></td></tr><tr><td> CD40 anticorpo </td><td> BD Biosciences </td><td> 553791 </td><td></td></tr><tr><td> Sistema reagente de Griess </td><td> Promega </td><td> G2930 </td><td></td></tr><tr><td> IL-6 anticorpo de captura </td><td> eBioscience </td><td> 13-7061-81 </td><td></td></tr><tr><td> Anticorpo de detecção de IL-6 </td><td> eBioscience </td><td> 13-7062-81 </td><td></td></tr></tbody></table>

generation and labeling of murine bone marrow-derived dendritic cells with qdot nanocrystals for tracking studies

As células dendríticas (DCs) são células antígeno apresentando profissionais (APCs) encontrados em tecidos periféricos e em órgãos imunológicos, como timo, medula óssea, baço, linfonodos e placas de Peyer 1-3. DCs presentes na amostra de tecidos periféricos do organismo para a presença de antígenos, que eles ocupam, processar e apresentar em sua superfície, no contexto de moléculas de histocompatibilidade (MHC). Então, o antígeno-carregado DCs migrar para órgãos imunológicos onde apresentam o antígeno processado aos linfócitos T desencadear respostas imunes específicas. Uma forma de avaliar a capacidade migratória de DCs é classificá-los com corantes fluorescentes 4. Com isto, demonstrar o uso de Qdot nanocristais fluorescentes para rotular derivadas da medula óssea de camundongos DC. A vantagem desta é que a rotulagem Qdot nanocristais possuem estável e de longo fluorescência duradoura que os tornam ideais para a detecção de células marcadas em tecidos recuperados. Para conseguir isso, as primeiras células será recuperado a partir marrows óssea de camundongos e cultivados por 8 dias na presença do fator macrófago-colônia de granulócitos estimulante, a fim de induzir a diferenciação DC. Estas células serão então marcadas com Qdots fluorescentes por curto incubação in vitro. Células coradas podem ser visualizados com um microscópio fluorescente. As células podem ser injetadas em animais experimentais, neste ponto, ou pode ser em células maduras sobre incubação in vitro com estímulos inflamatórios. Em nossas mãos, a maturação DC não determinam a perda do sinal fluorescente nem coloração Qdot afetam as propriedades biológicas dos DCs. Após a injeção, essas células podem ser identificadas em órgãos imune por microscopia fluorescente após dissecção típicos e os processos de fixação.

Watch this Scientific Journal Video about Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies at JoVE.com

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

Dendríticas células antígenos captação e migrar para órgãos imunes a antígenos presentes processados ​​às células T. Qdot nanocristais rotulagem fornece um sinal duradouro e estável fluorescente. Este permite o rastreamento de células dendríticas a diferentes órgãos, por microscopia fluorescente.

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 ...

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Research

JoVE Journal

Immunology and Infection

24.7K visualizações.  Ohio University. Dendríticas células antígenos captação e migrar para órgãos imunes a antígenos presentes processados ​​às células T. Qdot nanocristais rotulagem fornece um sinal duradouro e estável fluorescente. Este permite o rastreamento de células dendríticas a diferentes órgãos, por microscopia fluorescente.

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Investigação de polarização de macrófagos Usando medula macrófagos derivados

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Imunologia e Infecção

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Medula Óssea derivadas de macrófagos Produção

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolamento e injecção intravenosa de Murino de Medula Óssea Derivado monócitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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. ...

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.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

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. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Geração de um grande número de progenitores mieloides e células dendríticas precursores de murino medula óssea usando um celular romance classificação estratégia

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Deteção de ativação de Inflammasome e morte celular de Pyroptotic nos macrófagos da medula óssea-derivado murino

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Um modelo de rato In Vivo para ativação de células T Naïve CD4 medida, proliferação e diferenciação de células Th1 induzida pelas células dendríticas derivadas de medula óssea

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Protocolo Econômico e Eficiente para Isolar e Culturing Células Dendríticas Derivadas da Medula Óssea de camundongos

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...