Gostaríamos de agradecer a M. Höxter de assistência técnica para a classificação AECII murino primário de nível de biossegurança 2 amostras. Este trabalho foi financiado por doações da Fundação Alemã de Pesquisa (DFG) para DB (SFB587, TP B12 e BR2221/1-1) e uma bolsa da Escola de Pesquisa Biomédica Hannover (DFG GSC 108) para AA. DB é apoiada por iniciativa do Presidente e do Fundo de Networking da Associação Helmholtz de Centros de Pesquisa Alemão (HGF), sob W2/W3-029 número do contrato.

Os detalhes sobre os reagentes e materiais necessários estão listados na tabela, no final do protocolo abaixo. Antes de iniciar o trabalho, preparar tubos de 15 ml (um por rato) contendo 4 ml de dispase e pré-aquecê-los a 37 ° C num banho de água. Num bloco de aquecimento, logo aquecer pequenas alíquotas de 1% de agarose de baixa fusão (em água) a 95 ° C até liquefeito e, subsequentemente, arrefecer a 45 ° C até à sua utilização. 1. Preparação do pulmão do rato <ol><li> Sacrificar o mouse por asfixia CO 2. Nota: Não realizar o deslocamento cervical como isso irá prejudicar a traqueia, de modo que na sequência de passos do protocolo, tal como a instalação do pulmão com um líquido, não pode ser realizada com sucesso. Assegurar a perda de reflexos nociceptivos. </li><li> Pulverizar o rato com etanol e fazer um corte de comprimento ao longo da linha média ventral do corpo. Puxe a pele ventral e pele e, posteriormente, também cuidadosamente cortar e remover o peritônio. </li><li> Desangrar orato, cortando a veia jugular esquerda e para a direita (Vena jugularis), bem como a artéria renal (renalis Arteria). Remover o sangue fluindo com tecido. </li><li> Punção cuidadosamente e retire o diafragma para expor o coração e pulmão cortando as costelas. Tome especial cuidado para não ferir o pulmão. </li><li> Com uma cânula 26G e uma seringa de 10 ml cheia com solução salina tamponada com fosfato frio (PBS), a punção do ventrículo direito do coração e o pulmão perfundir com PBS até ficar livre de sangue. </li><li> Cortar e remover as glândulas salivares para expor a traquéia. Também corte cuidadosamente o músculo ao redor da traquéia. </li><li> Insira uma cânula 22G na traquéia, retire a agulha e empurrar o cateter plástico para o pulmão. Fixar o cateter, amarrando um pequeno pedaço de fio em torno traquéia e cateter. </li></ol> 2. A digestão enzimática do tecido pulmonar Se desejado, uma amostra de fluido de lavagem broncoalveolar pode serpreparada por lavagem dos pulmões por meio do cateter inserido na traqueia com PBS ou meio antes do passo seguinte. <ol><li> Utilizando uma seringa de 2 ml, cuidadosamente incutir um volume máximo de 2 ml de dispase (do alíquota de 4 ml) para o pulmão através do cateter de modo a que todos os lóbulos estão completamente expandida. Trocar a seringa com uma seringa de 1 ml contendo 0,5 ml de agarose a liquefeito e também instilar para o pulmão. </li><li> Deixar a seringa no cateter para evitar o refluxo da agarose e cobrir imediatamente o pulmão com papel de laboratório tecido e gelo. Deixe o gel de agarose para um par de minutos. </li><li> Remover o papel de tecido, o gelo, seringa e cateter, cortou a traqueia e excisão do coração, pulmão e timo do peito. Lavar o pulmão num prato com PBS e remover o coração timo e traqueia restante. </li><li> Coloque o pulmão para a 2 ml de dispase restante e incubar à temperatura ambiente durante 45 min. </li></ol> 3. Preparação do PulmãoSuspensão de células <ol><li> Remover o pulmão do dispase e transferi-lo para um prato contendo 7 ml de anti-CD16/32 anticorpo (1 ug / ml de concentração final) em meio DMEM e 100 ul de DNase. </li><li> Usando fórceps, completamente desintegrar o tecido pulmonar, puxando-o à parte e incubar durante 10 min à temperatura ambiente enquanto agitando suavemente em um balancim fixado a 200 rpm. Se desejado, a suspensão de células podem então ser armazenadas a 4 ° C, até ao passo seguinte. </li><li> Filtra-se a suspensão de células através de malhas de nylon com 100 um primeiro e, subsequentemente, com 70 uM, 48 uM e 30 uM poros. Ter o cuidado de lavar cada malha, bem como os tubos e pratos cuidadosamente com DMEM para minimizar a perda de células e maximizar o rendimento. Se estiver a juntar amostras, isto pode ser conseguido por aqui posteriormente passando suspensões de células a partir de ratos de um grupo através do mesmo filtro. Substituir o filtro, em caso de entupimento. </li><li> Dependendo do volume e transferir o filtrado para um ou mais tubos de 50 ml ecentrifugar durante 15 min a 160 xg e 4 ° C. Remover o sobrenadante. </li><li> Ressuspender o sedimento de células em 2 ml de tampão de lise de eritrócitos (0,15 M NH 4 Cl, 0,01 M de KHCO3, 0,1 mM de EDTA, pH 7,2) e rapidamente encerrar a lise por adição de 13 ml de meio DMEM. Transferir a solução para um tubo de 15 ml e centrifugar durante 12 min a 160 xg e 4 ° C. </li></ol> 4. A coloração de anticorpo por separação celular por citometria de fluxo- <ol><li> Ressuspender as células em 3 ml de cocktail de anticorpo primário, preparado em meio DMEM em diluições apropriadas para citometria de fluxo. (Os anticorpos foram previamente titulado para óptimos diluições de trabalho através da coloração de 1 x 10 6 esplenócitos num volume de 100 ul. Uso de 3 ml do cocktail de anticorpo primário que contém as concentrações ideais para cada um dos anticorpos usados ​​para as células reunidas a partir de um máximo de cinco ratinhos. Se mais camundongos são reunidas para uma amostra, ajustar o volume). </li><li> Para a coloração, incubar as células durante 10 min no escuroa 4 ° C. </li><li> Lave as células através do enchimento do tubo de 15 ml com DMEM e centrifugação durante 12 min a 160 xg e 4 ° C. </li><li> No caso de anticorpos ou desacoplados biotinilados foram usados ​​em 4.1: Remover o sobrenadante e ressuspender as células em 2-3 ml de cocktail de anticorpo secundário. Mancha durante 10 min a 4 ° C no escuro. </li><li> Lave as células através de enchimento do tubo com DMEM e centrifugação durante 12 min a 160 xg e 4 ° C. </li><li> Ressuspender as células em 1 ml de DMEM e pré-filtro através de um filtro de 50 um para um tubo de triagem de células. Opcional: Contar as células para se obter o número total de células na suspensão de células do pulmão. </li></ol> 5. De separação de células- <ol><li> Misturar as células através de agitação em vórtice antes da triagem. Usar um bocal 100 para uM célula triagem de AECII. Pressão do invólucro de fluido, bem como de emissão laser e comprimentos de onda de detecção dependem do instrumento utilizado para a triagem de citometria de fluxo de células, bem como os fluorocromos utilizados na Antibody procedimento de coloração. </li><li> Portão sobre as células SSC elevados que são negativas para os fluorocromos utilizados para coloração e tipo para um tubo contendo DMEM. Use a frente altura de dispersão (FSC-H) versus área (FSC-A) e para o lado a altura de dispersão (SSC-H) versus área (SSC-A) janelas para excluir quaisquer doublets ou agregados celulares (veja detalhes da estratégia de propagação em Figura 1 a). </li><li> A fim de recolher a ordenada centrífuga células durante 20 min a 280 xg e 4 ° C. </li><li> Ressuspender as células em meio de cultura ou de tampão conforme necessário para a análise subsequente. </li></ol>

<ol>
	<li>Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+type+II+cell:+defender+of+the+alveolus+revisited.">Alveolar epithelial type II cell: defender of the alveolus revisited.</a> Respir. Res. 2, 33-46 (2001).</li><li>Folkerts, G., Nijkamp, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+epithelium:+more+than+just+a+barrier.">Airway epithelium: more than just a barrier.</a> Trends Pharmacol. Sci. 19, 334-341 (1998).</li><li>Gereke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+alterations+in+type+II+alveolar+epithelial+cells+in+CD4++T+cell+mediated+lung+inflammation.">Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation.</a> Respir. Res. 8, 47 (2007).</li><li>Gereke, M., Jung, S., Buer, J., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+type+II+epithelial+cells+present+antigen+to+CD4(+)+T+cells+and+induce+Foxp3(+)+regulatory+T+cells.">Alveolar type II epithelial cells present antigen to CD4(+) T cells and induce Foxp3(+) regulatory T cells.</a> Am. J. Respir. Crit Care Med. 179, 344-355 (2009).</li><li>Gribar, S. C., Richardson, W. M., Sodhi, C. P., Hackam, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+longer+an+innocent+bystander:+epithelial+toll-like+receptor+signaling+in+the+development+of+mucosal+inflammation.">No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.</a> Mol. Med. 14, 645-659 (2008).</li><li>Herold, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+cells+direct+monocyte+transepithelial+migration+upon+influenza+virus+infection:+impact+of+chemokines+and+adhesion+molecules.">Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules.</a> J. Immunol. 177, 1817-1824 (2006).</li><li>Knight, D. A., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+structural+and+functional+properties+in+health+and+disease.">The airway epithelium: structural and functional properties in health and disease.</a> Respirology. 8, 432-446 (2003).</li><li>Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+distribution+in+lung+parenchyma+early+after+pulmonary+infection+with+Pseudomonas+aeruginosa.">Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.</a> Cell Tissue Res. 342, 67-73 (2010).</li><li>Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-+and+Strain-Specific+Host+MicroRNA+Molecular+Signatures+Associated+with+Swine-Origin+H1N1+and+Avian-Origin+H7N7+Influenza+A+Virus+Infection.">Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection.</a> J. Virol. 86, 6109-6122 (2012).</li><li>Marriott, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infect. Immun. 80, 1140-1149 (2012).</li><li>Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetyl-L-cysteine+(NAC)+inhibit+mucin+synthesis+and+pro-inflammatory+mediators+in+alveolar+type+II+epithelial+cells+infected+with+influenza+virus+A+and+B+and+with+respiratory+syncytial+virus+(RSV).">N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).</a> Biochem. Pharmacol. 82, 548-555 (2011).</li><li>Zarbock, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surfactant+protein+C+mutation+A116D+alters+cellular+processing,+stress+tolerance,+surfactant+lipid+composition,+and+immune+cell+activation.">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15 (2012).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+influenza+virus+infections.">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li><li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+alveolar+type+II+cells.">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li><li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+murine+alveolar+type+II+cells.">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li><li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+synthesis,+and+processing+of+surfactant+protein+SP-C+in+rat+lung+analyzed+by+epitope-specific+antipeptide+antibodies.">Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies.</a> J. Biol. Chem. 269, 20318-20328 (1994).</li><li>Phelps, D. S., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+pulmonary+surfactant+proteins+using+immunohistochemistry+and+tissue+in+situ+hybridization.">Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.</a> Exp. Lung Res. 17, 985-995 (1991).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+(IFN-lambda+1)+in+response+to+influenza+A+infection.">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+to+influenza+A+virus+in+differentiated+human+alveolar+type+II+cells.">Innate immune response to influenza A virus in differentiated human alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).</li></ol>

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O protocolo para o isolamento de AECII murina por citometria de fluxo proporciona uma forma rápida de aceder a partir de células primárias do pulmão do rato para uma ampla gama de estudos funcionais e moleculares. O procedimento descrito proporciona populações altamente viáveis ​​e puras dos AECII que são em número suficiente para directos análises subsequentes, tais como o isolamento de ARN (ver figura 2b) e os estudos de transcriptoma. Para aplicações funcionais, também é possível a...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> habitação cânula Introcan 22G </td><td> Braun </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispase, 100 ml (5000 unidades caseinolítica) </td><td> BD Biosciences </td><td> 354235 </td><td> alíquota de 4 ml em tubos de 15 ml, armazenar a -20 ° C </td></tr><tr><td> Placa Biozym Agarose </td><td> Biozym </td><td> 840101 </td><td> 1% w / v de H 2 O </td></tr><tr><td> Desoxirribonuclease I a partir de pâncreas bovino, 2000 unidades Kunitz / frasco </td><td> Sigma-Aldrich </td><td> D4263 </td><td> recém dissolver o conteúdo de 1 frasco em 300 ul de DMEM </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> utilizado como fornecido pelo fabricante (G Lowlucose, piruvato, HEPES) </td></tr><tr><td> Os filtros celulares (100 um, 75 um) </td><td> BD Falcon </td><td> 352360, 352350 </td><td></td></tr><tr><td> malha de nylon (48 um, 30 um) </td><td> Buckmann GmbH </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50 mM filtro </td><td> Partec </td><td> 04-0042-2317 </td><td></td></tr><tr><td> anti-rato CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> clone 93; purificada </td></tr><tr><td> anti-rato F4/80 </td><td> BioLegend </td><td> 123116 </td><td> clone BM8; APC acoplado </td></tr><tr><td> anti-rato CD11b </td><td> BioLegend </td><td> 101208 </td><td> clone M1/70; PE acoplado </td></tr><tr><td> anti-rato CD11c </td><td> BioLegend </td><td> 117310 </td><td> clone N418; APC acoplado </td></tr><tr><td> anti-ratoCD45 </td><td> BioLegend </td><td> 103102 </td><td> clone 30-F11; purificada </td></tr><tr><td> anti-mouse CD19 </td><td> eBioscience </td><td> 12-0193-83 </td><td> eBio 1D3; PE acoplado </td></tr><tr><td> anticorpo policlonal de cabra anti-rato IgG </td><td> BD Pharmingen </td><td> 550767 </td><td> policlonal, PE acoplado </td></tr><tr><td></td><td></td><td></td><td> Anticorpos acoplados a fluorocromos alternativos podem ser utilizados, dependendo do citómetro de fluxo e lasers disponível. </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

Ao longo dos últimos anos, a contribuição das células alveolares tipo II epiteliais (AECII) a vários aspectos da regulação imune no pulmão tem sido cada vez mais reconhecida. AECII têm sido mostrados para participar na produção de citoquina em inflamação das vias aéreas e ao mesmo actuar como células apresentadoras de antigénio, em ambos infecção e de células-T auto-imunidade mediada por 1-8. Por isso, eles são particularmente interessantes também em contextos clínicos, tais como hiper-reactividade ao estrangeiro e auto-antigénios, bem como infecções que, directa ou indirectamente como alvo AECII. No entanto, a nossa compreensão das funções imunológicas detalhadas servidas por células de tipo II alveolares epiteliais no pulmão saudável, bem como na inflamação permanece fragmentária. Muitos estudos sobre a função AECII são realizadas usando o mouse ou humanos alveolares linhas de células epiteliais 9-12. Trabalhando com linhas celulares certamente oferece uma série de vantagens, tais como a disponibilidade de grandes quantidades of células para análise extensiva. No entanto, nós acreditamos que a utilização de AECII murino primário permite um melhor entendimento sobre o papel deste tipo de células em processos complexos como infecção ou inflamação autoimune. AECII murino primário pode ser isolado directamente a partir de animais que sofrem de tais condições respiratórias, o que significa que foram sujeitos a todos os outros factores extrínsecos que desempenham um papel na definição analisados. Como um exemplo, AECII viável pode ser isolado a partir de murganhos intranasalmente infectados com o vírus influenza A, que tem como alvo as células principalmente para a replicação de 13. É importante notar que com a infecção ex vivo de AECII isolado de ratos saudáveis, os estudos das respostas celulares montadas sobre a infecção pode ser ainda mais alargado. O protocolo para o isolamento de AECII murino primário é baseada na digestão enzimática do pulmão de rato seguido de rotulagem da suspensão de células resultante com anticorpos específicos para CD11c, CD11b, F4/80,CD19, CD45 e CD16/CD32. AECII granulares são, em seguida, identificada como a dispersão não marcado e para o lado de alta (SSC alta) da população de células e são separados por classificação de células activadas por fluorescência 3. Em comparação com outros métodos de isolamento de células primárias epiteliais de pulmão de rato, o nosso protocolo para o isolamento de citometria de fluxo de AECII por selecção negativa produz intacta, AECII altamente viável e pura em tempo relativamente curto. Além disso, e em contraste com os métodos convencionais de isolamento por filtração e depleção de linfócitos através de ligação do anticorpo-esferas magnéticas acopladas 14, 15, por citometria de fluxo de células para triagem de discriminação permite que, por meio de tamanho celular e granularidade. Dado que a instrumentação de separação de células por citometria de fluxo está disponível, o processo descrito pode ser aplicado a custos relativamente baixos. Ao lado de anticorpos normais e enzimas para a desintegração do pulmão, não reagentes adicionais, tais como magnéticoGrânulos são necessários. As células isoladas são adequadas para uma ampla gama de estudos funcionais e moleculares, que incluem a cultura in vitro e ensaios de estimulação de células T, bem como transcriptoma, proteoma ou análises secretoma 3, 4.

Ao classificar suspensões de pulmão de células isoladas a partir de ratos saudáveis, o portão AECII normalmente responsáveis ​​por cerca de 42 ± 10% de todos os eventos. Esse percentual pode ser notavelmente menor quando os ratos com doenças respiratórias, como uma infecção viral são usadas, como a suspensão de células inicial vai conter uma proporção consideravelmente maior de linfócitos e outras células imunológicas recrutados para as vias aéreas. Para AECII isolada do pulmão IAV infectados no...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Nós descrevemos o isolamento rápido de murinos primários células tipo II alveolares epiteliais (AECII) por meio de seleção por citometria de fluxo negativo. Estes AECII mostram viabilidade e pureza elevados e são adequadas para uma ampla gama de estudos funcionais e moleculares relativas ao seu papel em condições respiratórias, tais como doenças auto-imunes ou infecciosas.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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Immunology and Infection

26.1K visualizações.  Helmholtz Centre for Infection Research. Nós descrevemos o isolamento rápido de murinos primários células tipo II alveolares epiteliais (AECII) por meio de seleção por citometria de fluxo negativo. Estes AECII mostram viabilidade e pureza elevados e são adequadas para uma ampla gama de estudos funcionais e moleculares relativas ao seu papel em condições respiratórias, tais como doenças auto-imunes ou infecciosas.

Vídeo: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

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 ...

Imunologia e Infecção

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análise do dano epitelial Produzido pela<em&gt; Entamoeba histolytica</em&gt; Infecção

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolamento e Caracterização de citometria de fluxo de linfócitos de murino Intestino Delgado

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestão do fígado murino para um fluxo Cytometric análise de células endoteliais linfáticas

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...