Este trabalho foi financiado pelo Prêmio família Smith de Excelência em Pesquisa Biomédica e RO1 AI083426 NIH-01 (FW). Patrick Maschmeyer e Melanie Flach são apoiados por bolsas de doutoramento da Boehringer Ingelheim Foundation.

Camundongos C57BL / 6 deve ser usado em ~ 20 semanas de idade ou mais. O uso de camundongos machos pesando 25-30g é recomendado. O rendimento das células estreladas pode ser aumentado pela alimentação camundongos uma vitamina Uma dieta enriquecida por 2 meses antes do isolamento de células estreladas. Cerca de 2x10 5 células estreladas hepáticas pode ser purificado a partir do fígado de um camundongo C57BL / 6, enquanto o rendimento de células estreladas de camundongos Balb / c é consideravelmente maior. O protocolo que se segue é ajustada para 5 camundongos. Cuidados com os animais e experimentação foram realizados em conformidade com o Animal Care aprovado Institucional e protocolos Comitê Use. 1. Na perfusão in situ de fígado de camundongos com enzimas digestivas <ol><li> Aquecer o tampão SC1 e soluções para perfusão enzima em banho-maria a 37 ° C. </li><li> Montar um set de infusão com asas no tubo de silicone de uma bomba peristáltica. </li><li> Calibrar a bomba com PBS para obter um fluxo laminar de 6.5ml/min, que é a vazão que serão utilizados para a situ na perfusão do fígado. Equilibrar o tubo de silicone com SC1 solução. </li><li> Anestesiar o rato por injeção intraperitoneal de ketamina (90 mg / kg) e xilazina (10 mg / kg) e solução de teste de perda de reflexos para garantir narcotização profundo. </li><li> Corrigir o mouse na posição supina em uma superfície apropriada. </li><li> Use uma tesoura e uma pinça para realizar uma incisão longitudinal na pele abdominal e expor o peritônio. </li><li> Abra cuidadosamente o peritônio e mover os intestinos para fora da cavidade abdominal para o lado esquerdo do animal a fim de expor a veia portal. </li><li> Inserir a cânula do conjunto de perfusão na veia portal. Para esta etapa, o uso de um microscópio estereoscópico é recomendado. </li><li> Iniciar a perfusão do fígado com solução SC1 30ml. Abra a veia cava inferior imediatamente após o início do fluxo. Rubor bem sucedida do fígado é indicada por uma perda de cor do tecido do fígado. </li><li> Perfundir o fígado com a solução de 30ml pronase E. Após a digestão bem-sucedido, os lobos do fígado vai inchar, e os lóbulos aparecerá distintas através da cápsula. </li><li> Perfundir o fígado com a solução de colagenase P 30ml. O fígado neste momento perdeu sua forma e aparência atônica e amorfo. </li><li> Separe cuidadosamente o fígado a partir do diafragma e órgãos circundantes e armazená-lo em 70ml de solução SC2 no gelo. </li><li> Repita o procedimento para ratos adicionais. </li></ol> 2. Digestão in vitro de fígado de camundongos Todas as outras etapas de trabalho deve ser realizado sob condições estéreis em capela de fluxo laminar. <ol><li> Corte o fígado em pedaços de aproximadamente 2x2x2mm 3 usando uma tesoura afiada. </li><li> Combine a suspensão de 70ml SC2 incluindo os pedaços de fígado com 50ml de solução pronase P E-colagenase e adicionar 1 ml de solução de DNase I. </li><li> Digerir os fígados por 20min a 37 ° C, agitando. </li></ol> 3. Centrifugação em gradiente de densidade <ol><li> Filtrar a suspensão de células através de filtros de células 70μm em 6 tubos de 50ml Falcon e adicionar até 50ml com SC2 buffer. Centrifugar por 10 minutos em 600g e 4 ° C. </li><li> Aspirar cuidadosamente 40ml do sobrenadante, em seguida, adicione 150μl DNase solução que eu a cada tubo e ressuspender as células. </li><li> Piscina das suspensões de células em 4 tubos de 50ml Falcon e lave com GBSS-B, centrifugar por 10 minutos em 600g e 4 ° C. </li><li> Aspirar cuidadosamente o máximo de sobrenadante quanto possível, sem perturbar o sedimento e adicionar 150μl DNase solução que eu a cada tubo. Ressuspender o pellets de células em 10ml GBSS-B por tubo. </li><li> Piscina as células em dois tubos de 50ml Falcon e adicione GBSS-B para um volume total de 36ml por tubo. Adicionar 14ml de solução Nycodenz a cada tubo e misture bem. </li><li> Transferência de 10ml da suspensão de células em um tubo de centrifugação 12ml gradiente, resultando em 10 tubos no total. Delicadamente sobrepor a suspensão de células com 1,5 ml GBSS-B por tubo. </li><li> Centrifugar os gradientes de 15 min a 1500g e 4 ° C, sem freio. Posteriormente, hepatócitos será peletizada na parte inferior do tubo enquanto que as células estreladas são encontrados na interfase como um anel branco. </li><li> Cuidadosamente a colheita interfase contendo as células estreladas e lavá-los com GBSS-B; centrifugar por 10 minutos em 600g e 4 ° C. </li><li> Aspirar o sobrenadante e ressuspender as células em DMEM suplementado com 20ml inativado pelo calor de 10% FBS, HEPES 1% Penicilina / Estreptomicina, 2mM L-Glutamina, piruvato de sódio 1 mM e 10 mM. Transferência das células em frascos de cultura de tecidos com uma concentração de 2x10 4 células / cm 2. Incubar as células a 37 ° C e 5% CO 2. </li><li> Troque a mídia assim que as células estreladas hepáticas são aderentes (cerca de 2h após a preparação) para lavar as células mortas e restos celulares. </li><li> No dia seguinte, a célula estreladass deve desenvolver sua morfologia em forma de estrela com característica perinuclear vesículas vitamina A-armazenar lipídios. </li><li> Para os experimentos subseqüentes, as células estreladas hepáticas pode ser facilmente destacado das superfícies não-revestido de plástico leve usando soluções enzimáticas, tais como Accutase, por exemplo, </li></ol> 4. Resultados representativos: Após a preparação de células estreladas hepáticas utilizando o protocolo fornecido, a pureza da população isolada podem ser testadas considerando três principais características deste tipo de célula, como estrelas, como a forma, gotículas lipídicas perinuclear, e expressão da proteína glial fibrilar ácida (GFAP ). Imagens representativas para o aspecto característico de células estreladas hepáticas 2h após o isolamento de células, bem como no dia 1 e 3 de cultura in vitro são apresentadas na Figura 1. A Figura 2 mostra uma coloração representante de imunofluorescência para GFAP em células estreladas hepáticas, que foram cultivadas por 3 dias após o isolamento de células. Células estreladas hepáticas diferenciar em células miofibroblástica durante a cultura in vitro. A marca característica dessas células estreladas ativadas é a expressão de alfa actina de músculo liso (αSMA). A Figura 3 mostra a coloração de imunofluorescência para ASMA e IIA miosina em células estreladas ativadas no dia 7 de cultura in vitro. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Figura 1" /> Figura 1. Morfologia característica de células estreladas hepáticas. Células estreladas foram isoladas de fígado de camundongos usando o protocolo fornecido. As imagens retratam 2h células estreladas após o isolamento da célula (a, b), bem como no dia 1 (c) e dia 3 (d) de cultivo in vitro. Células estreladas hepáticas apresentam grandes quantidades de vesículas lipídicas em locais perinuclear e adquirir sua morfologia astral distintivo como durante os primeiros dias de cultivo in vitro (Ampliação de 200x). <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Figura 2" /> Figura 2. Células estreladas hepáticas especificamente expressar GFAP no fígado. Células estreladas foram isoladas e coradas para immunofluorescently GFAP (vermelho) no dia 3 de cultura in vitro. Núcleos das células são representadas em azul (Hoechst mancha). <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Figura 3" /> Figura 3. Células estreladas hepáticas diferenciar em miofibroblastos. Células estreladas hepáticas isoladas de camundongos C57BL / 6 foram cultivadas por 7 dias. Posteriormente, eles foram transferidos para câmara de slides e corados para ASMA (mostrada em vermelho) e IIA miosina (representado em verde). Os núcleos das células foram contracorados com Hoechst (azul). <table border="1"><tbody><tr><td colspan="2"> SC1 buffer </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> Glicose </td><td> 900mg </td></tr><tr><td> HEPES </td><td> 10 ml de solução 1M de ações </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Vermelho de fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> preencher até 1l </td></tr><tr><td colspan="2"> SC2 buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10ml de solução estoque 1M </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Vermelho de fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> preencher até 1l </td></tr><tr><td colspan="2"> GBSS-A Tampão </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glicose </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Vermelho de fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> preencher até 1l </td></tr><tr><td colspan="2">GBSS-B buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glicose </td><td> 991mg </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Vermelho de fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> preencher até 1l </td></tr><tr><td colspan="2"> Pronase solução de perfusão E </td></tr><tr><td> E pronase </td><td> 100mg (4000 PU / mg min) </td></tr><tr><td> SC2 buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Colagenase solução de perfusão P </td></tr><tr><td> Colagenase P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Pronase E-colagenase Solução P </td></tr><tr><td> E pronase </td><td> 50mg (4000 PU / mg min) </td></tr><tr><td> Colagenase P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 buffer </td><td> 50ml </td></tr><tr><td colspan="2"> Solução DNase I </td></tr><tr><td> DNase I </td><td> 6mg (ca. 2000U/mg) </td></tr><tr><td> GBSS-B </td><td> 3ml </td></tr><tr><td colspan="2"> Nycodenz Solution </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28ml </td></tr></tbody></table> Tabela 1. Buffers e soluções de enzima necessária para o isolamento de células estreladas hepáticas. O pH de todos os buffers deve ser ajustado para 7,3-7,4. Além disso, a filtração estéril de todos os buffers é recomendado. É importante adaptar a quantidade de enzima utilizada de acordo com a determinada atividade enzimática.

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <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+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

Não há conflitos de interesse declarados.

Células estreladas hepáticas regular essenciais processos fisiológicos e fisiopatológicos no fígado. Além disso, as células estreladas possuem propriedades apresentadoras de antígenos, tornando-os um importante componente da resposta imune hepática. Embora as células estreladas hepáticas compreendem 10-15% do número total de células no fígado, o isolamento dessas células é um desafio devido à sua localização no espaço de Disse perisinusoidal. Aqui apresentamos um método ...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Catalog No. </th></tr><tr><td> 70μm Filtro celular </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Colagenase P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNase I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031 cv- </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> Soro fetal bovino </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Glicose </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Tubos de centrifugação de gradiente </td><td> Greiner Bio-One </td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15630 </td></tr><tr><td> KCL </td><td> Sigma-Aldrich </td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> Sigma-Aldrich </td><td> P9791 </td></tr><tr><td> L-Glutamina </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6H 2 O </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 x 7H 2 O </td><td> Sigma-Aldrich </td><td> M2773 </td></tr><tr><td> Na 2 HPO 4 x 2H 2 O </td><td> Fluka </td><td> 71643 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> Fluka </td><td> 71507 </td></tr><tr><td> NaHCO 3 </td><td> Fluka </td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td> Penicilina / estreptomicina </td><td> Gibco </td><td> 15140 </td></tr><tr><td> Bomba peristáltica </td><td> Cole Parmer </td><td> HV-7523-70 </td></tr><tr><td> Vermelho de fenol </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> E pronase </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Tubo de silicone </td><td> Masterflex </td><td> HV-96440-14 </td></tr><tr><td> Piruvato de sódio </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Frasco de cultura de tecidos (25 cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Set de infusão com asas </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Células estreladas hepáticas são fígado residente células de estrelas, como morfologia e estão localizados no espaço de Disse entre fígado sinusoidal células endoteliais e hepatócitos 1,2. Células estreladas são derivados de precursores da medula óssea e armazenar até 80% do total do corpo de vitamina A 1, 2. Após a ativação, as células estreladas diferenciar em miofibroblastos para produzir matriz extracelular, contribuindo para a fibrose hepática 3. Base na sua capacidade de contratar, as células estreladas miofibroblástica pode regular o tom vascular associada à hipertensão portal 4. Recentemente, nós demonstramos que as células estreladas hepáticas são células antígeno apresentando potentes e podem ativar as células NKT, bem como convencionais linfócitos T 5. Aqui apresentamos um método eficiente para a preparação de células estreladas hepáticas do fígado do rato. Devido à sua localização perisinusoidal, o isolamento de células estreladas hepáticas é um processo multi-passo. A fim de tornar as células estreladas acessíveis ao isolamento do espaço de Disse, fígado de camundongos são perfundidos in situ com a enzimas digestivas pronase E e perfusão colagenase P. Em seguida, o tecido hepático é submetido a tratamento enzimático adicionais com pronase E e P colagenase em vitro. Posteriormente, o método tira proveito da enorme quantidade de gotículas de vitamina A de armazenamento de gordura nas células estreladas hepáticas. Este recurso permite a separação de células estreladas de outros tipos de células hepáticas por centrifugação em um gradiente Nycodenz 8%. O protocolo aqui descrito produz uma população altamente pura e homogénea de células estreladas. Pureza das preparações podem ser avaliados através da coloração para a molécula de proteína glial fibrilar ácida marcador (GFAP), antes da análise por microscopia de fluorescência ou citometria de fluxo. Além disso, microscopia de luz revela a aparência única em forma de estrela de células hepáticas estreladas que abrigam grandes quantidades de gotículas lipídicas. Tomados em conjunto, nós apresentamos um protocolo detalhado para o isolamento eficiente de células estreladas hepáticas, incluindo imagens representativas de sua aparência morfológica e expressão GFAP que ajudam a definir a entidade de células estreladas.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Aqui nós descrevemos um método para o isolamento de células estreladas hepáticas do fígado do rato. Para a purificação de células estreladas, fígado de camundongos são digeridos<em&gt; In situ</em&gt; E<em&gt; In vitro</em&gt; Por pronase colagenase-tratamento antes da centrifugação em gradiente de densidade. Esta técnica produz células estreladas hepáticas de alta pureza.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Research

JoVE Journal

Immunology and Infection

33.5K visualizações.  Harvard Medical School. Aqui nós descrevemos um método para o isolamento de células estreladas hepáticas do fígado do rato. Para a purificação de células estreladas, fígado de camundongos são digeridos In situ E In vitro Por pronase colagenase-tratamento antes da centrifugação em gradiente de densidade. Esta técnica produz células estreladas hepáticas de alta pureza.

Vídeo: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Avaliar a resposta estomática ao vivo células bacterianas usando imagens Folha Inteira

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Imunologia e Infecção

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Uma variante do método EPA 1623 que usa fluxo tangencial ultrafiltração de fibra oca e Passos de calor de dissociação para detectar Waterborne<em&gt; Cryptosporidium</em&gt; E<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Transdução de adenovírus Naive CD4 células T para estudar a diferenciação Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Desenvolvimento de uma colite Modelo Antigen-driven para o Estudo de apresentação de antígenos por células apresentadoras de antígenos às células T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

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 ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Um método All-on-chip para análise rápida de quimiotaxis de neutrófilos diretamente de uma gota de sangue

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpressão e purificação do ser humano<em&gt; Cis</em&gt; -preniltransferase em<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Um novo sistema alimentador-livre para produção em massa de murino Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

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

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...