Agradecemos Aaron Rae (Emory University Departamento de Pediatria e Saúde Infantil de Atlanta Núcleo de fluxo) para separação de células. Este trabalho foi financiado pelo NIH concessão AA01787001, um prêmio de Desenvolvimento de Carreira da Fundação de Crohn e Colite da América, e um Crianças Emory-Egleston de Pesquisa semente Centro de concessão de TLD

1. Dissecção e dissociação das células epiteliais intestinais Preparação de reagentes e equipamento: <ul><li> Quente Ca 2 + / Mg2 + livre de PBS (CMF PBS) à temperatura ambiente. </li><li> Ca quente 2 + / Mg + 2-free HBSS com FBS a 5% (CMF HBSS / FBS) e EDTA 2 mM à temperatura ambiente. </li><li> Quente agitador orbital a 37 ° C. </li></ul> Nota: Os passos 1,1 a 1,7 deve ser realizado tão rapidamente quanto possível para minimizar o grau de morte celular e para atingir o rendimento máximo de célula. <ol><li> Euthanize ratos em uma câmara de CO 2 e etanol spray de 70% sobre o abdômen e tórax. </li><li> Fazer uma pequena incisão horizontal no meio do abdómen com uma tesoura e retirar a pele para expor o peritoneu. </li><li> Continuar a separar o estômago do intestino delgado superior, cortando no esfíncter pilórico. Tease longe do mesentério com uma pinça e cortar novamentea válvula íleo-cecal para libertar todo o intestino pequeno a partir do intestino grosso. Faça um corte na borda anal e novamente provocar uma separação do mesentério até o intestino grosso é gratuito. </li><li> Corte abrir o cólon longitudinalmente com tesoura e lave conteúdo fecal e muco do lúmen intestinal em CMF PBS à temperatura ambiente. </li><li> Use tesouras e pinças para dissecar macroscopicamente placas de Peyer ao longo da superfície anti-mesentérica do intestino delgado e cortado e aberto no intestino delgado longitudinalmente. </li><li> Lave as lumen do intestino delgado de conteúdos fecais e muco em CMF PBS à temperatura ambiente. </li><li> Separadamente cortar o intestino delgado / grande em aproximadamente 1,5 cm pedaços e colocá-las separadas tubos de 50 ml cónico contendo 30 ml de pré-aquecida CMF HBSS / FBS e 2 mM de EDTA. Não adicione mais de 1 intestino por 50 tubo ml. </li><li> Horizontalmente colocar cada tubo cónico de 50 ml em um agitador orbital e agitar durante a 250 rpm durante 20 min a 37 ° C. &lt;/ Li&gt; <li> Coloque um coador de malha de arame única ao longo de um balde de resíduos e despejar o conteúdo de cada tubo cónico de 50 ml através de recuperar os pedaços de 1,5 cm de intestino e colocá-los em separadas de 50 ml contendo tubos cónicos de 30 ml de pré-aquecidos CHBSS / FBS com 2 mM de EDTA. </li><li> Repita 1,8. </li></ol> 2. A digestão do tecido e isolamento de células intestinais Preparações de reagentes e equipamento: <ul><li> Solução de colagenase: 1,5 mg / ml Tipo VIII colagenase dissolvido em pré-aquecida CMF HBSS / FBS com 40 ug / ml de DNase I. </li><li> Pré-aquecida CMF HBSS / FBS e 2 mM de EDTA. </li><li> Pré-aquecida agitador orbital a 37 ° C. </li><li> Ice-frio CMF HBSS / FBS. </li></ul><ol><li> Após a segunda rodada de agitação, despejar o conteúdo de cada tubo cónico de 50 ml através do filtro e transferir 1,5 cm pedaços de intestino de um plástico pequeno barco pesar depois esfregando afastado o excesso de mídia, utilizando uma toalha de papel. </li><li> Rapidamente mediu a 1.5 Peças cm de intestino usando tesouras directamente no barco peso e adicionar intestino picada a 20 ml de solução de colagenase. Horizontalmente colocar cada tubo cónico de 50 ml em um agitador orbital e digerir a 200 rpm durante 10-20 min a 37 ° C. Favor ver discussão abaixo em relação optimização. </li><li> Resumidamente vórtice para assegurar a dissociação completa de qualquer tecido restante intestinal e filtrar através de um coador de célula 100 uM directamente num tubo cónico de 50 ml. </li><li> Top fora de cada tubo cónico de 50 ml com CMF HBSS / FBS e centrifugar a 1500 rpm durante 5 min a 4 ° C. Se um sedimento sólido não é observado para as amostras de cólon depois da centrifugação, as amostras devem ser novamente centrifugado durante 3,5 min. Repetir este passo de lavagem mais uma vez. </li><li> Decantar o sobrenadante e ressuspender o pellet celular em HBSS CMF gelada / FBS e amostras de colocar no gelo. </li><li> Vá para a Seção 3 para aquisição FACS / análise ou na Seção 4 para magnética talão de enriquecimento para alta velocidade célula Sórting. </li></ol> 3. Coloração do anticorpo para Multi-Color citometria de fluxo e DCs e macrófagos Preparações de reagentes e equipamento: <ul><li> Ice-frio CMF PBS. </li><li> Tampão gelado de coloração (CMF PBS + 5% de FBS). </li><li> Prepare célula morta mancha na gelada CMF PBS a diluição 1:1000 utilizando VIVO / MORTO fixável do Aqua Kit Dead Cell Stain. </li><li> Preparar o cocktail de coloração de anticorpo, adicionando os seguintes fluorescência-rotulados anticorpos monoclonais (mAbs) para o tampão gelado de coloração: CD45-PerCP, CD103-PE, CD11c-APC, MHC-II (IA b)-Alexa Fluor 700, CD11b -eFluor 450, F4/80-PE-Cy7. </li></ul><ol><li> Transferir as células em um ml de poliestireno 5 de fundo redondo de tubo (FACS). </li><li> Lave as células duas vezes em gelada CMF PBS. </li><li> Incubar as amostras com célula morta mancha durante 15 min em gelo, no escuro. </li><li> Lave as células duas vezes em gelada CMF PBS. </li><li> Bloco com células anti-FcγRIII/II 2.4G2 na gelada coloraçãotampão durante 10 min em gelo. </li><li> Lave as células em tampão gelado de coloração. </li><li> Incubar as amostras com cocktail de anticorpos de coloração durante 20 min em gelo, no escuro. </li><li> Lave as células com tampão gelado de coloração duas vezes e ressuspender as amostras em 400 uL de tampão gelado de coloração e passar através de 40 uM tampa de filtro em tubos de FACS. </li><li> Adquirir amostras em LSR II citómetro (BD), definidas pela estratégia de propagação na Seção 5 e Figura 1. </li></ol> 4. Enriquecimento de DCs e macrófagos do Intestino Preparações de reagentes e equipamento: <ul><li> Tampão gelado de coloração (CMF PBS + 5% de FBS). </li></ul><ol><li> Incubar única suspensão celular obtida a partir do passo 2.5 com CD11b e grânulos CD11c MACS de acordo com as instruções do fabricante. </li><li> Lave as células com tampão gelado de coloração seguido de centrifugação. </li><li> Descartar o sobrenadante e ressuspender o pellet celular em 1 mL de tampão de coloração geladae passar através de um filtro de células de 100 um seguido de um filtro de célula 40 uM. </li><li> Enriqueça para magnéticos talão inscritos células por seleção positiva usando MACS coluna LS magnético. </li><li> Repita o passo 4,2 e elimine o sobrenadante. </li><li> Incubar as células com mAbs de superfície do marcador, como descrito no passo 3.7. </li><li> Lavar magnéticos grânulo enriquecidas células duas vezes com tampão gelado de coloração. Ressuspender sedimentos celulares em 500 uL de tampão de coloração gelada sem azida de sódio, e passar através do filtro 40 uM de células para um tubo de FACS. </li><li> Vá para a FACS-ordenação no separador de células BD ARIA II para classificar DC intestinal e / ou subconjuntos de macrófagos de interesse. </li></ol> 5. Estratégia para Gating LP APCs Nota: Note-se que as células intestinais não coradas pode ser utilizado como um controlo negativo para ajudar na colocação adequada das portas para separar as populações positivas e negativas. <ol><li> Como mostrado na Figura1, criar um gráfico de pontos e não incluem células que são positivas para a célula morta mancha (Fig. 1A), seguido pela exclusão de eventos dupleto (Fig. 1B e C). Então, a porta sobre as células de interesse de acordo com a encaminhar e dispersão lateral certificando-se de excluir os detritos (Fig. 1D). </li><li> Criar um outro lote de ponto e portão mais adiante CD45 + e + b IA células, o que caracteriza fenotipicamente APCs (Fig. 1E). </li><li> Em um gráfico de pontos separados, para analisar CD11b e expressão CD11c para distinguir DC específico e subconjuntos de macrófagos (R1, R2, e R3;. Fig. 1F). Células de R1 são CD11c + CD11b maçantes / - células. A região, R2, é delineada de tal modo que estas células têm níveis semelhantes de expressão de superfície para CD11c como em células de R1 mas as células de R2 também expressa CD11b. Depois disso, a região R3 é designada para as células que são CD11b + e CD11c opaca / -. </li><li> Umalyze regiões R1, R2, e de expressão adicional para F4/80 e CD103 R3 para diferenciar macrófagos e as populações de CC, respectivamente. O CD11c + CD11b opaca / - células de níveis elevados de R1 expressas a integrina αE, CD103, e baixos níveis de F4/80 (Fig. 1G). CD11b + + CD11c células de R2 são compostos de ambos os DCs e macrófagos com base na sua expressão dicotômica de CD103 e F4/80 (Fig. 1H). Por último, CD11b + CD11c baços / - células de R3 constituem macrófagos com base no perfil fenotípica de F4/80 + e CD103 - (Fig. 1I) 16. </li></ol> 6. Os resultados representativos <img alt="A Figura 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> Figura 1. Estratégia gating para DCs intestinais e macrófagos. Células mortas (A) e dupletos (B e C) foram primeiro excluídos da análise e, em seguida intesti pequenaAs células foram nal gated em conformidade para transmitir e do lado de dispersão (D), e APCs foram definidos como CD45 + IA b + (E). Os macrófagos e DCs foram identificados pela expressão de CD11b e CD11c (F). CD103 e F4/80 expressão para células pré-gated em R1 (G), R2 (H) e R3 (I) populações foi analisada. <img alt="A Figura 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> Figura 2. Rendimento celular e de qualidade de coloração de anticorpo depende do tempo de digestão. CD11b e padrão de coloração CD11c e rendimento celular total de sob-(A, D), optimamente-(B, E) ou sobre-digerido (C, F) de tecido intestinal. As células intestinais foram isoladas a partir de um intestino C57BL / 6 pequeno rato e DCs e macrófagos foram analisadas por FACS sobre o LSR BD II. Tensão e compensação foram criados usando imaculado e único fluorocromo manchadas de esplenócitos. As células mortas (Fig. 1A) e dupletos (Fig. 1B e C) foram excluídos da primeiraa análise. Células de interesse foram então analisados ​​de acordo com a transmitir e do lado de dispersão (Fig. 1D), seguido por gating em CD45 + e IA b + células (Fig. 1E). Posteriormente, CD11b e expressão CD11c foi avaliada entre o CD45 + b + células IA para delinear três regiões (R1, R2, e R3;. Fig. 1F). CD103 e F4/80 expressão nas três regiões foi avaliada para distinguir entre DCs e macrófagos, respectivamente. O CD11c + CD11b / aborrecido - células de R1 expressa níveis elevados da integrina αE, CD103, e baixos níveis de F4/80 (Fig. 1G). CD11b + + CD11c células de R2 foram compostas de ambos os DCs e macrófagos com base na sua expressão dicotômica de CD103 e F4/80 (Fig. 1H), enquanto CD11b + de CD11c baços / - células de R3 constituem macrófagos com base no perfil fenotípica de F4 / 80 + e CD103 - ( <strong&gt; fig. 1I) 16. Macrófagos dentro do portão R2 e macrófagos no portão R3 têm a frente semelhante e propriedades de dispersão laterais e são distinguíveis por expressão CD11c. A dicotomia funcional desses subconjuntos permanece incompletamente compreendida. A relação entre a duração da digestão do tecido sobre o rendimento de total de células e expressão de CD11b e CD11c é ilustrado na Figura 2. Tecido intestinal que foi digerido durante 3 min (sob a digestão-) rendeu o número de células total baixo (Fig. 2D) e DCS assim poucos e macrófagos disponíveis para a caracterização (Fig. 2A). Digestão de tecido para 11 min produziu um rendimento robusto de células vivas (fig. 2E) com populações de DCs e macrófagos que expressavam níveis elevados de CD11b e CD11c e foram fenotipicamente distinta (Fig. 2C). Em contraste, a digestão durante 50 min (mais de digestão-) resultou num rendimento celular semelhante, quando compared à digestão optimizado (2E Fig. e F), no entanto, o delineamento da populações de células diferentes, utilizando CD11b e CD11c tornou-se mais obscura como a expressão de diminuída CD11c (Fig. 2C) e do número de células mortas aumentada (dados não mostrados).

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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="A Figura 3"/> Figura 3. Factores importantes para a optimização do rendimento celular e de expressão antigénio de superfície. Rendimento celular e de expressão antigénio de superfície estão directamente afectada pela duração da digestão do tecido, as características específicas da colagenase, o grau de tecido picagem, ea presença ou ausência de inflamação, o que pode afetar a integridade dos tecidos e celularidade. Digestão prol...

<table border="1"><tbody><tr><td> Nome do Reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> 1X PBS, Ca 2 + - + e Mg 2-livre </td><td></td><td></td><td></td></tr><tr><td> Solução salina equilibrada de Hank (HBSS) com vermelho de fenol </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> O bicarbonato de sódio </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> HEPES 1M em NaCl 0,85% </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Soro fetal de bovino (FBS) </td><td> Atlanta biológicos </td><td> S11150H </td><td> Inativado pelo calor </td></tr><tr><td> EDTA 0,5 M (pH 8,0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> Collagenase tipo VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> 14785000 </td><td> Solução stock: 100mg/ml </td></tr><tr><td> AO VIVO / MORTO fixável do Aqua Kit Dead Cell Stain para 405 nm de excitação </td><td> Invitrogen </td><td> L34957 </td><td> Use a 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Use a 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Use a 1:100 </td></tr><tr><td> FcγRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Use a 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Use a 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Use a 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Use a 1:200 </td></tr><tr><td> F4/80-PE-Cy7 mAb (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b micro </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c micro </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> Tubos de 50 ml cónicos </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Filtro único fio de malha </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Pequeno barco pesar </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 filtro célula mM </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 filtro de célula mM </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 mL de poliestireno de fundo redondo de tubos </td><td> BD Falcon </td><td> 352235 </td><td> Use a 1:100 </td></tr><tr><td> MaxQ 4450 bancada agitador orbital </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS coluna MACS </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

Dentro do intestino residem populações únicas de inata e adaptativa células do sistema imunológico que estão envolvidas na promoção da tolerância para a flora comensal e antígenos alimentares enquanto concomitantemente pronta restante para montar respostas inflamatórias para 1,2 patógenos invasivos. Células apresentadoras de antígenos, particularmente DCs e macrófagos, desempenham papéis críticos na manutenção da homeostase imune intestinal através de sua capacidade de sentir e responder adequadamente à microbiota 3-14. Isolamento eficaz de DCs intestinais e macrófagos é um passo crítico para a caracterização do fenótipo e função destas células. Enquanto muitos métodos eficazes de isolar as células imunes intestinais, incluindo DCs e macrófagos, têm sido descritos 6,10,15-24, muitos dependem de longos tempos de digestões que podem influenciar negativamente célula expressão antigénio de superfície, a viabilidade das células, e / ou o rendimento celular. Aqui, nós detalhe uma metodologia para o isolamento rápido de grandes números de concretiza-seE, DCs e macrófagos intestinais. Caracterização fenotípica de DCs intestinais e macrófagos é realizada por coloração directamente isoladas células intestinais com específicos de fluorescência-rotulados anticorpos monoclonais para multi-cor análise de citometria de fluxo. Além disso, DC altamente puro e populações de macrófagos são isolados para estudos funcionais utilizando CD11c e CD11b esferas magnéticas activadas classificação celular seguido de separação de células.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Aqui, nós detalhe uma metodologia para o isolamento rápido de células de ratinho intestinais dendríticas (DC) e macrófagos. Caracterização fenotípica de DCs intestinais e macrófagos é realizada utilizando várias cores análise de fluxo citométrico enquanto enriquecimento grânulo magnético seguido por separação de células é usado para produzir populações altamente puros para estudos funcionais.

Isolamento e caracterização de células dendríticas e macrófagos a partir do intestino do rato

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K visualizações.  Emory University. Aqui, nós detalhe uma metodologia para o isolamento rápido de células de ratinho intestinais dendríticas (DC) e macrófagos. Caracterização fenotípica de DCs intestinais e macrófagos é realizada utilizando várias cores análise de fluxo citométrico enquanto enriquecimento grânulo magnético seguido por separação de células é usado para produzir populações altamente puros para estudos funcionais.

Vídeo: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento de células dendríticas pulmonares do rato

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Imunologia e Infecção

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

As células T Capturar Bactérias por Transinfection das dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e análise de citometria de fluxo de células imunes do cérebro isquêmico Rato

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caracterização de derivados de monócitos células dendríticas humanas por imagem Citometria de Fluxo: Uma Comparação entre dois protocolos de isolamento de monócitos

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.