Este trabajo fue financiado por el Premio de la familia Smith a la Excelencia en la Investigación Biomédica y el NIH RO1 AI083426-01 (FW). Patrick Maschmeyer Flach y Melanie son compatibles con becas de doctorado de la Fundación Boehringer Ingelheim.

Ratones C57BL / 6 se debe utilizar en aproximadamente 20 semanas de edad o más. El uso de ratones macho de 25 a 30 g se recomienda. El rendimiento de las células estrelladas se puede aumentar por la alimentación de los ratones de la vitamina A dieta enriquecida por 2 meses antes de que el aislamiento de células estrelladas. Aproximadamente 2x10 5 células estrelladas hepáticas pueden purificar a partir del hígado de un C57BL / 6 del ratón, mientras que el rendimiento de las células estrelladas de ratones Balb / c es mucho mayor. El siguiente protocolo se ajusta a 5 ratones. Cuidado de los animales y la experimentación se realizaron de acuerdo con el cuidado aprobado Animales institucional y protocolos de uso Comité. 1. En la perfusión in situ de los hígados de los ratones con las enzimas digestivas <ol><li> Caliente el buffer SC1 y soluciones enzimáticas de perfusión en un baño de agua a 37 ° C. </li><li> Montar una infusión alado conjunto en el tubo de silicona de una bomba peristáltica. </li><li> Calibrar la bomba con PBS para obtener un flujo laminar de 6.5ml/min, que es el caudal que se utilizará para el de la perfusión in situ del hígado. Equilibre el tubo de silicona con SC1 solución. </li><li> Anestesiar el ratón por inyección intraperitoneal de ketamina (90 mg / kg) y xilazina (10 mg / kg) y la solución de prueba de la pérdida de los reflejos para asegurar narcotización de profundidad. </li><li> Fijar el ratón en la posición en decúbito supino sobre una base adecuada. </li><li> Use las tijeras y pinzas para realizar una incisión longitudinal en la piel del abdomen y exponer el peritoneo. </li><li> Abra cuidadosamente el peritoneo y mover los intestinos fuera de la cavidad abdominal en el lado izquierdo del animal con el fin de exponer la vena portal. </li><li> Inserte la cánula de la infusión en la vena porta. Para este paso, el uso de un microscopio estereoscópico se recomienda. </li><li> Iniciar la perfusión del hígado con una solución de 30 ml SC1. Abrir la vena cava inferior inmediatamente después de iniciar el flujo. Lavado éxito del hígado está indicado por una pérdida de color de los tejidos del hígado. </li><li> Perfundir el hígado con una solución de 30 ml E Pronasa. Durante la digestión con éxito, los lóbulos del hígado se hinchan, y los lóbulos se parecen distintos a través de la cápsula. </li><li> Perfundir el hígado con una solución de 30 ml de colagenasa P. El hígado en este momento ha perdido su forma y aspecto atónico y amorfo. </li><li> Separar cuidadosamente el hígado desde el diafragma y los órganos circundantes y lo almacenan en 70 ml SC2 solución en el hielo. </li><li> Repita el procedimiento con otros ratones. </li></ol> 2. En la digestión in vitro de hígados de los ratones Todas las nuevas medidas de trabajo debe llevarse a cabo en condiciones estériles en una campana de flujo laminar. <ol><li> Cortar el hígado en trozos de aproximadamente 3 2x2x2mm usando unas tijeras afiladas. </li><li> Combine la suspensión de 70 ml SC2 incluyendo las piezas de hígado con 50 ml de pronasa E-colagenasa solución P y añadir 1 ml de solución de DNasa I. </li><li> Recopilación de los hígados de 20 minutos a 37 ° C mientras se agita. </li></ol> 3. Centrifugación en gradiente de densidad <ol><li> Filtrar la suspensión celular a través de filtros de 70μm de células en tubos Falcon de 50 ml 6 y añadir hasta 50 ml con buffer de SC2. Centrifugar durante 10 minutos a 600g y 4 ° C. </li><li> Con cuidado aspirar 40 ml del sobrenadante, a continuación, añadir 150μl DNasa solución que a cada tubo y resuspender las células. </li><li> Piscina las suspensiones de células en tubos Falcon de 50 ml 4 y lavar con GBSS-B, centrifugar durante 10 minutos a 600g y 4 ° C. </li><li> Con cuidado aspirar la mayor cantidad de sobrenadante como sea posible sin perturbar el precipitado y añadir 150μl DNasa solución que a cada tubo. Resuspender los pellets de células en 10 ml GBSS-B por tubo. </li><li> Piscina de las células en tubos Falcon de 50 ml 2 y añadir GBSS-B con un volumen total de 36ml por tubo. Añadir 14 ml solución Nycodenz a cada tubo y mezcle bien. </li><li> Transferencia de 10 ml de la suspensión celular en un tubo de centrifugación en gradiente de 12 ml, lo que resulta en 10 tubos en total. Suavemente superposición de la suspensión celular con 1,5 ml GBSS-B por tubo. </li><li> Centrifugar los gradientes de 15 min a 1500 g y 4 ° C sin freno. Posteriormente, los hepatocitos se sedimentan en el fondo del tubo, mientras que las células estrelladas se encuentran en la interfase como un anillo blanco. </li><li> Con cuidado, la cosecha de la interfase que contiene las células estrelladas y los lavarás con GBSS-B, centrifugar durante 10 minutos a 600g y 4 ° C. </li><li> Aspirar el sobrenadante y resuspender las células en 20 ml de DMEM suplementado con 10% de SFB inactivado por calor, HEPES 1% de penicilina / estreptomicina, 2 mM L-glutamina, piruvato de sodio 1 mM y 10 mM. Transferencia de las células en frascos de cultivo de tejido con una concentración de 2x10 4 células / cm 2. Se incuban las células a 37 ° C y 5% CO 2. </li><li> Cambiar los medios de comunicación tan pronto como las células estrelladas hepáticas son adherentes (aproximadamente 2 horas después de la preparación) de lavar las células muertas y restos celulares. </li><li> Al día siguiente, las células estrelladass debe desarrollar su característica morfología en forma de estrella con perinuclear vesículas de lípidos de almacenamiento de la vitamina A. </li><li> Para los experimentos posteriores, las células estrelladas hepáticas pueden separarse fácilmente de las superficies de plástico sin revestimiento con leves soluciones enzimáticas como Accutase, por ejemplo, </li></ol> 4. Los resultados representativos: Después de la preparación de las células estrelladas hepáticas mediante el protocolo previsto, la pureza de la población aislada se puede probar en cuenta tres características principales de este tipo de células, tales como estrellas, como la forma, las gotas de lípidos perinuclear, y la expresión de proteína ácida glial fibrilar (GFAP ). Imágenes representativas de la apariencia característica de las células estrelladas hepáticas 2h después del aislamiento de células, así como en el día 1 y 3 de cultivo in vitro se muestran en la Figura 1. La figura 2 muestra una tinción representativa de inmunofluorescencia para GFAP en las células estrelladas hepáticas, que han sido cultivadas durante 3 días después del aislamiento celular. Las células estrelladas hepáticas se diferencian en células miofibroblástico durante el cultivo in vitro. Un sello característico de las células estrelladas activadas es la expresión de alfa-actina del músculo liso (αSMA). La Figura 3 muestra la tinción de inmunofluorescencia para el Asma y la miosina II en las células estrelladas activadas en el día 7 de cultivo in vitro. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Figura 1" /> Figura 1. Morfología característica de las células estrelladas hepáticas. Las células estrelladas se aislaron de hígados de los ratones con el protocolo previsto. Las imágenes muestran 2h células estrelladas tras el aislamiento de células (a, b), así como el día 1 (c) y el día 3 (d) de cultivo in vitro. Las células estrelladas hepáticas presentan altas cantidades de vesículas de lípidos en los sitios perinuclear y adquieren su distintiva astral-como la morfología durante los primeros días de cultivo in vitro (Aumento 200x). <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Figura 2" /> Figura 2. Células estrelladas hepáticas específicamente expresar GFAP en el hígado. Las células estrelladas se aislaron y immunofluorescently teñidos para GFAP (rojo) en el día 3 de cultivo in vitro. Los núcleos celulares se muestran en azul (Hoechst mancha). <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Figura 3" /> Figura 3. Células estrelladas hepáticas se diferencian en miofibroblastos. Las células estrelladas hepáticas aisladas de ratones C57BL / 6 fueron cultivadas durante 7 días. Posteriormente, fueron trasladados a la cámara de diapositivas y teñidas de Asma (en rojo) y la miosina IIA (representado en verde). Los núcleos celulares fueron contrastados con Hoechst (azul). <table border="1"><tbody><tr><td colspan="2"> SC1 Buffer </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> Glucosa </td><td> 900 mg </td></tr><tr><td> HEPES </td><td> 10 ml de solución madre 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> NaHCO3 </td><td> 350mg </td></tr><tr><td> Rojo fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> llenar hasta 1 litro </td></tr><tr><td colspan="2"> SC2 Buffer </td></tr><tr><td> De CaCl 2 x 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10 ml de solución madre 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> NaHCO3 </td><td> 350mg </td></tr><tr><td> Rojo fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> llenar hasta 1 litro </td></tr><tr><td colspan="2"> GBSS-A Buffer </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> De CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucosa </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> De 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> NaHCO3 </td><td> 227mg </td></tr><tr><td> Rojo fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> llenar hasta 1 litro </td></tr><tr><td colspan="2">GBSS B-Buffer </td></tr><tr><td> De CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucosa </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> De 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> NaHCO3 </td><td> 227mg </td></tr><tr><td> Rojo fenol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> llenar hasta 1 litro </td></tr><tr><td colspan="2"> E pronasa perfusión Solución </td></tr><tr><td> Pronasa E </td><td> 100 mg (4000 PU / min mg) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Colagenasa P solución de perfusión </td></tr><tr><td> Colagenasa P </td><td> 85 mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Pronasa E-colagenasa P Solución </td></tr><tr><td> Pronasa E </td><td> 50 mg (4000 PU / min mg) </td></tr><tr><td> Colagenasa P </td><td> 85 mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 50ml </td></tr><tr><td colspan="2"> DNasa I Solución </td></tr><tr><td> DNasa I </td><td> 6 mg (2000U/mg CA) </td></tr><tr><td> GBSS-B </td><td> 3 ml </td></tr><tr><td colspan="2"> Nycodenz Solución </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28 ml </td></tr></tbody></table> Tabla 1. Tampones y soluciones de enzima requerida para el aislamiento de las células estrelladas hepáticas. El pH de todos los búferes se debe ajustar a 7,3-7,4. Además, la filtración estéril de todos los búferes se recomienda. Es importante adaptar la cantidad de enzima que se utiliza de acuerdo con la misma actividad 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>

Las células estrelladas hepáticas regular esenciales los procesos fisiológicos y fisiopatológicos en el hígado. Por otra parte, las células estrelladas poseen propiedades presentadoras de antígeno, lo que hace que un componente importante de la respuesta inmune hepática. Aunque las células estrelladas hepáticas constituyen el 10-15% del número total de células en el hígado, el aislamiento de estas células es un reto debido a su localización en el espacio perisinusoidal de Disse. <p class="jove_content...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> N º de catálogo </th></tr><tr><td> 70μm de células colador </td><td> BD </td><td> 352350 </td></tr><tr><td> CACL 2 x 2 H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Colagenasa P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNasa I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21 a 031-cv </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> De suero fetal bovino </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Glucosa </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Los tubos de centrifugación 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> MgCl2 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 2 H 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> NaHCO3 </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> Rojo fenol </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> Pronasa E </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Tubo de silicona </td><td> Masterflex </td><td> HV-96.440-14 </td></tr><tr><td> El piruvato de sodio </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Frasco de cultivo de tejidos (25 cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Equipo de infusión con alas </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Las células estrelladas hepáticas son residentes del hígado de las células de la estrella-como la morfología y se encuentran en el espacio de Disse entre el hígado y las células endoteliales sinusoidales 1,2 hepatocitos. Las células estrelladas se derivan de los precursores de la médula ósea y almacenar hasta 80% de la vitamina A del cuerpo total de 1, 2. Tras la activación, las células estrelladas se diferencian en miofibroblastos para producir la matriz extracelular, contribuyendo así a la fibrosis del hígado 3. Con base en su capacidad para contratar, las células estrelladas miofibroblástico puede regular el tono vascular asociado a la hipertensión portal 4. Recientemente, hemos demostrado que las células estrelladas hepáticas son potentes células presentadoras de antígeno y puede activar las células NKT, así como los linfocitos T convencionales 5. Aquí presentamos un método para la preparación eficaz de las células estrelladas hepáticas del hígado de ratón. Debido a su localización perisinusoidal, el aislamiento de las células estrelladas hepáticas es un proceso de múltiples pasos. A fin de que las células estrelladas accesible para el aislamiento del espacio de Disse, hígados de los ratones son perfundidos in situ con el Pronasa digestivo E enzimas colagenasa y la perfusión P. Después, el tejido hepático se somete a tratamiento enzimático adicional con pronasa E y P en la colagenasa vitro. Posteriormente, el método se aprovecha de la enorme cantidad de gotas de vitamina A de lípidos que almacena en las células estrelladas hepáticas. Esta característica permite la separación de las células estrelladas hepáticas de otros tipos de células por centrifugación en un 8% de pendiente Nycodenz. El protocolo descrito aquí produce una población muy pura y homogénea de las células estrelladas. Pureza de las preparaciones pueden ser evaluados mediante la tinción de la molécula de proteína glial fibrilar marcador ácida (GFAP), previo al análisis por microscopía de fluorescencia o citometría de flujo. Además, la microscopía de luz revela el aspecto único de la forma de estrella, las células estrelladas hepáticas que albergan grandes cantidades de gotitas de lípidos. En conjunto, se presenta un protocolo detallado para el aislamiento eficiente de las células estrelladas hepáticas, incluyendo imágenes representativas de su aspecto morfológico y la expresión de GFAP que ayudan a definir la entidad de células estrelladas.

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

Seven Steps to Stellate Cells

Aquí se describe un método para el aislamiento de las células estrelladas hepáticas del hígado de ratón. Para la purificación de células estrelladas, hígados de los ratones se digieren<em&gt; In situ</em&gt; Y<em&gt; In vitro</em&gt; Por el tratamiento pronasa colagenasa antes de la centrifugación en gradiente de densidad. Esta técnica produce células hepáticas estrelladas de alta pureza.

Siete pasos para células estrelladas

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 vistas.  Harvard Medical School. Aquí se describe un método para el aislamiento de las células estrelladas hepáticas del hígado de ratón. Para la purificación de células estrelladas, hígados de los ratones se digieren In situ Y In vitro Por el tratamiento pronasa colagenasa antes de la centrifugación en gradiente de densidad. Esta técnica produce células hepáticas estrelladas de alta pureza.

Video: 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.

Evaluación de la respuesta estomática de las células bacterianas en vivo utilizando imágenes de Hoja Entera

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

Investigación

Inmunología e Infección

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.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos 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.

Una modificación de la EPA Método 1623 que utiliza el flujo tangencial de fibra hueca de ultrafiltración y la Plaza de calor de disociación para detectar a base de agua<em&gt; Cryptosporidium</em&gt; Y<em&gt; Esp.</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.

La transducción adenoviral de células T CD4 + naïve a estudiar la diferenciación 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).

Desarrollo de un modelo de colitis impulsado por el antígeno para el Estudio de la presentación de antígenos por células presentadoras de antígenos a las 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.

Precisión de corte pulmón de ratón Rebanadas para visualizar las células en vivo pulmonar dendríticas

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.

Un método todo-en-chip para el análisis rápido de la quimiotaxis de neutrófilos directamente de una gota de sangre

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.

Sobreexpresión y Purificación de Humanos<em&gt; Cis</em&gt; -preniltransferasa en<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.

Un novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos 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.

Desarrollo y validación de una sola molécula ultrasensible matriz Digital análisis de enzima-ligado del inmunosorbente para interferón-α humano

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