Este trabalho foi financiado por doações CIHR e CF Canadá ao Dr. Jim Hu e por uma bolsa de estudo de doutorado concedida para Rahul Kushwah pela CF Canadá.

1. Sensibilização de camundongos com ovalbumina <ol><li> Preparar uma solução de OVA (grau V; Sigma, MO) em PBS estéril a uma concentração de 1 mg / ml (solução pode ser armazenada a -80 ° C). </li><li> A fim de preparar OVA Alum-mistura, toma Alum num tubo e adicionar solução de OVA em uma forma gota a gota, enquanto o tubo de vórtice em uma proporção de 1:1. Agita-se a mistura durante 30 minutos e utilizar imediatamente após a mistura. </li><li> Utilizando uma seringa de 1 ml, 0,2 ml de injectar a mistura em rato cavidade peritoneal e repetir a injecção de novo depois de 2 semanas. </li></ol> 2. Desafio intranasal de camundongos com ovalbumina Identificada FITC <ol><li> 7 dias após a segunda injecção de OVA-Alum, os ratinhos estão prontos para serem desafiados intranasalmente com OVA-FITC. </li><li> Preparar uma solução de OVA-FITC (Sigma) utilizando PBS estéril a uma concentração de 1 mg / ml e armazenar em alíquotas a -80 ° C. </li><li> Colocar uma gaze estéril, na parte inferior de um tubo Falcon de 50 ml e em um h químicaood, adicionar 5 ml de Aerrane sobre a gaze. A fim de anestesiar ratinhos, dirigir o animal para dentro do tubo para segundo aproximadamente 5-10. </li><li> Segurar o animal anestesiado em posição vertical e utilizando pontas de pipeta estéreis, Pipetar 100 da solução de OVA-FITC para as narinas de ratinhos de uma forma gota a gota,. </li><li> Repita desafio intranasal com OVA-FITC para os próximos dois dias. </li></ol> 3. Preparação de uma única célula Suspensão dos pulmões e os linfonodos de drenagem <ol><li> Sacrificar os ratos usando injeção intraperitoneal de Euthanyl. </li><li> A fim de reduzir a contaminação de macrófagos, que pode complicar análise DC, lavagem pulmonar é realizada. Para a lavagem dos pulmões, traqueias de rato são brevemente exposta e canulada com um cateter e uma seringa; usando PBS gelado com 5 mM de EDTA, o tracto respiratório inferior é enxaguado 3 vezes para remover as células dos espaços alveolares. </li><li> Perfundir os pulmões com 10 ml de PBS contendo 10 U / ml heparem através do ventrículo direito do coração, para remover as células de sangue a partir da vasculatura do pulmão. Executar as perfusões até que os pulmões virar branco, indicativo de remoção da maior parte das células do sangue. Isto é especialmente importante para remover as células contaminantes a partir do sangue periférico. </li><li> Dissecar os pulmões para fora e remover os gânglios linfáticos do mediastino (MLN) e coloque os linfonodos em um prato separado. (Tente minimizar a exposição dos tecidos à luz para evitar extinção de fluorescência FITC). </li><li> Colocar os pulmões em uma placa de Petri e mediu utilizando uma tesoura. Em seguida digerir os pulmões durante 25 minutos a 37 ° C, utilizando 250 U / ml de colagenase D (Roche) e adicionar EDTA (10 mM de concentração final) para os próximos 5 minutos para parar a actividade da colagenase. </li><li> Passar os fragmentos digeridos de pulmões através de um filtro de células de 100 um e realizar a lise hipotónico para remover eritrócitos. A suspensão de células única está pronto para posterior análise de DCs. </li><li> A fim de preparar a suspensão de célula única a partir do MLN, taliviar a MLN utilizando agulhas finas e digerir com colagenase tal como descrito acima, seguido por esforço através de um filtro de célula. </li></ol> 4. A coloração para DC Marcadores para Avaliar a maturação / migração <ol><li> A fim de identificar DCs nos pulmões, a coloração é realizada para CD11c e CD11b. Além disso para avaliar a maturação, a coloração é realizada para CD86 e CD80, que são upregulated como DCS sofrer maturação. CD11c + CD11b + células OVA-FITC + no MLN são identificados como DCs pulmonares migratórias dos pulmões para o MLN em resposta ao desafio das vias aéreas OVA. </li><li> Suspender as células a uma concentração de 10 x 10 6 células / ml em tampão FACS (PBS com 1% de FBS e EDTA 1 mM) e uL aliquota de suspensão de células 150 / tubo para a coloração de FACS. Para a identificação dos DCs de pulmão, manchas seguintes são necessários: controle corado (células pulmonares de camundongos não expostos à OVA-FITC), controle de OVA-FITC, controle de CD11c único, controle de CD11b único, CD11b+ CD11c + duplo controlo, CD11b + OVA-FITC + controle duplo, controle de CD11c + OVA-FITC + dupla, MHC II único controle, CD86 único controle, CD80 único controle e amostras coradas para OVA-FITC + CD11c + CD11b + + MHCII , OVA-FITC + + CD11c CD11b + CD86 + e OVA-FITC + + CD11c CD11b + CD80 +. </li><li> Para avaliar a migração DC, realizar a contagem de células absolutos da suspensão de células única a partir de MLN e, subsequentemente, preparar os tubos seguintes: controlo não corado (células MLN de ratinhos não expostos à OVA-FITC), OVA-FITC de controlo, CD11c + controlo único, CD11b + de duplo controlo, CD11b + OVA-FITC + duplo controle, CD11c + OVA-FITC + duplo controle e CD11c + CD11b + de amostras OVA-FITC A +. </li></ol> 5. Os resultados representativos Os pontos de tempo necessários para a sensibilização intraperitoneal para induzir a inflamação das vias aéreas alérgica é importante e deve ser realizada como descrito na Figura 1. Após sensibilização intraperitoneal e desafio com OVA das vias aéreas, para confirmar a indução da inflamação das vias aéreas alérgica, alguns ratinhos pode ser sacrificado e as análises histológicas pode ser efectuada nas secções de pulmão como mostrado na Figura 2. Presença de células inflamatórias pode ser confirmada por hematoxilina e eosina (Figura 2A) e presença de produção de muco pode ser avaliada por ácido periódico de Schiff-mancha (Figura 2B). No conjunto, este irá confirmar a indução de inflamação das vias aéreas alérgica após o desafio com OVA de OVA-sensibilizados ratos 8. Em contraste, as secções de pulmão a partir de ratinhos tratados com solução salina são esperados para estar livre de qualquer inflamação, juntamente com a ausência de qualquer produção de muco. Além disso, o desafio com OVA seguinte, pulmonar DCs unde<html> RGO maturação e posterior migração para os linfonodos de drenagem. Análise de CD11c + células dos nodos linfáticos mediastinal (MLN) de ratinhos sensibilizados e desafiados com OVA é esperado para mostrar uma maior proporção de células CD11c + em comparação com ratinhos tratados com solução salina (Figura 3A). Além disso, a análise da contagem de células absoluta de CD11c + CD11b + de OVA-FITC + de células no MLN de ratinhos OVA-sensibilizadas e desafiados, espera-se que mostram uma significativamente maior (isto é, várias vezes superior) de contagem que os homólogos de salinas ratinhos tratados ( A Figura 3B). <img alt="A Figura 1" src="/files/ftp_upload/4014/4014fig1.jpg" /> Figura 1. Protocolo experimental para a indução de ovalbumina (OVA) a inflamação das vias aéreas induzida em ratinhos alérgica, juntamente com desafio das vias aéreas com FITC ovalbumina rotulada (OVA-FITC). fig2.jpg &quot;/&gt; Figura 2. Sensibilização OVA seguido por desafio das vias aéreas OVA conduz a indução de inflamação das vias aéreas alérgica como identificado por hematoxilina e eosina coloração de secções de pulmão mostrados em (A) e conduz também à produção de muco nas vias aéreas tal como identificado por coloração pelo ácido periódico de Schiff de pulmão secções como mostrado em (B). <img alt="A Figura 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> Figura 3. OVA-FITC desafio induz DC migração dos pulmões para os gânglios linfáticos do mediastino (MLN). (A) parcelas de citometria de fluxo que descreve proporções de CD11c + células no MLN de controlo ou de OVA-sensibilizadas ratinhos. (B) As contagens absolutas de CD11c + CD11b + células OVA-FITC + no MLN de ratos salina ou OVA-sensibilizados. * P &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

<ol>
	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

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O método aqui apresentado oferece uma abordagem baseada em citometria de fluxo para análise de DCs pulmonares, com base na entrega de OVA-FITC para o desafio da via aérea. Isto permite a monitorização selectiva de DCs pulmonares, que ocupam OVA-FITC e, portanto, as populações de CC, que são acompanhados são efectivamente as que participam na resposta das vias aéreas imune durante o curso de OVA induzida por inflamação das vias aéreas alérgica. Em ratinhos de controlo, na ausência de inflamação das vias ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Ovalbumina </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Alúmen </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Colagenase D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Antibodies </td><td> e-Bioscience </td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

As células dendríticas (DCs) são os principais agentes envolvidos na iniciação da resposta imune adaptativa por ativação de células T antígeno-específicas. DCs estão presentes nos tecidos periféricos em estado estacionário, no entanto, em resposta à estimulação de antigénio, DCs assumir o antigénio e rapidamente migram para os nódulos linfáticos drenantes onde eles iniciam resposta de células T contra o antigénio 1,2. Além disso, DCs também desempenham um papel-chave na iniciação auto-imune, bem como alérgica resposta imunitária 3. DCs desempenham um papel essencial em ambos iniciação da resposta imune e indução de tolerância na configuração do ambiente de pulmão 4. Ambiente de pulmão é em grande parte tolerogénico, devido à exposição a vasta gama de antigénios ambientais 5. No entanto, em alguns indivíduos, há um intervalo de tolerância, o que leva a indução de alergia e asma. Neste estudo, descrevemos uma estratégia, o que pode ser utilizado para monitorizar das vias aéreas maturação DC e MIGração em resposta ao antigénio utilizado para a sensibilização. A medição da via aérea maturação DC e migração permite a avaliação da cinética da resposta imune durante inflamação das vias aéreas alérgica e também auxilia na compreensão da magnitude da resposta imune subsequente, juntamente com os mecanismos subjacentes. A nossa estratégia é baseada no uso de ovalbumina como um agente sensibilizante. Ovalbumina induzida asma alérgica é um modelo largamente utilizado para reproduzir a eosinofilia das vias aéreas, inflamação pulmonar e níveis elevados de IgE encontrados durante a asma 6,7. Após a sensibilização, os ratos são desafiados por administração intranasal de FITC ovalbumina rotulado, que permite a marcação específica de DCs das vias aéreas que ovalbumina absorção. Em seguida, usando vários marcadores específicos DC, podemos avaliar a maturação desses DCs e também pode avaliar a sua migração para os linfonodos de drenagem através do emprego de citometria de fluxo.

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

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

Nós descrevemos uma estratégia para monitorar a maturação e migração de células dendríticas pulmonares em resposta à ovalbumina na configuração de ovalbumina inflamação das vias aéreas induzida alérgica. Esta estratégia pode ser modificado para avaliar a migração de células dendríticas pulmonares nas configurações de infecção.

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 ...

analysis-pulmonary-dendritic-cell-maturation-migration-during

Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  McMaster University, Hamilton. Nós descrevemos uma estratégia para monitorar a maturação e migração de células dendríticas pulmonares em resposta à ovalbumina na configuração de ovalbumina inflamação das vias aéreas induzida alérgica. Esta estratégia pode ser modificado para avaliar a migração de células dendríticas pulmonares nas configurações de infecção.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

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 ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análise por Citometria de fluxo das células imunológicas Dentro aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

No método de imagem in vivo Distinguir inflamação aguda e crônica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caracterização e exploração funcional da célula de rede gengival Imune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Administração intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; No mouse modelos para o Estudo de inflamação das vias aéreas

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Um ensaio baseado em CFSE para estudar a migração de Murino pele as células dendríticas em drenagem gânglios linfáticos durante a infecção com<em&gt; Mycobacterium bovis</em&gt; Bacilo de Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Rato pulmão Slices para Visualize células vivas pulmonar dendríticas precisão de corte

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Análise de cisalhamento induzida pelo fluxo de migração de murino Marginal Zone B células In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...