La cría de animales peritajes a través de Tina Martin y David Albright es gratamente apreciados. Esta investigación fue apoyada por subvenciones T32-AI-07285 (HN), NIH R25 2GM064133 (TCL), 1R03-HD061671-01, R24-AI-059830-06 de los NIH.

1. Los animales X. laevis x X. gilli híbridos LG-6 y el LG-15 clones isogénicas 1 son de nuestra colonia de cría de la Universidad de Rochester (http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). LG-6 y compartir LG-15 el mismo haplotipo MHC heterocigotos (a / c), pero difieren en menores de histocompatibilidad (H) loci. La progenie de estos clones son producidos por ginogénesis, en el que los huevos diploides producidos por la hembra son activados por irradiados con UV de espermatozoides (sin contribución del ADN a la progenie). El uso de guantes es facultativa. Algunas personas prefieren no usarlos porque es más difícil de manejar a las ranas (resbaladizo) y, de hecho, parece que las ranas incómodo. 2. La purificación de gp96 de 15 / 0 del tumor (expresa tanto del tumor y menores H-Ags) Gp96 purificación se ha descrito anteriormente 2, 3. En pocas palabras, la gp96, se purifica mediante el fraccionamiento de amonio 50-70% de sulfato, seguido por ConA-sefarosa y cromatografía de DEAE. Acerca de 20-50 g de proteína se puede obtener por 1 ml de tejido tumoral. Pureza de la preparación está determinado por SDS-PAGE y tinción de plata. 3. Estimulación y la cosecha de leucocitos peritoneales (LP) de menor H-Ag-dispares LG-6 ranas <ol><li> Crecer un 25 mL noche a la mañana E. Coli cultura en un tubo cónico de 50 ml a 37 ° C con agitación. </li><li> El día siguiente el calor mata las bacterias hirviéndola durante 1 hora. </li><li> Centrifugar el calor mató a E. Coli durante 15 minutos a 2.000 rpm (1.500 g) a 4 ° C. </li><li> Eliminar el sobrenadante y resuspender el sedimento bacteriano en 1 / 10 de volumen de cultivo original (2,5 ml) en APBS. En este punto, el cultivo bacteriano está listo para su uso y debe ser utilizado dentro de las 24 hrs. </li><li> Se inyecta por vía intraperitoneal (ip) 200 (para una rana 2 pulgadas) o 300 L (para una rana de 3 pulgadas) de calor mató a la preparación de bacterias por la rana con un calibre de 25 5 / 8 de aguja. </li><li> Tres días después de la inyección PLs son cosechadas por lavado intraperitoneal. </li><li> Antes de la cosecha PL, ranas adultas son anestesiados por inmersión en una solución acuosa al 0,1% de metano sulfonato tricaína (TMS, MS-222) tamponado con bicarbonato de sodio para un máximo de 5 minutos hasta que todo movimiento cesa (duración depende del tamaño y edad). Los animales despiertan a los 10-20 minutos después del tratamiento. </li><li> Desinfectar el abdomen de la rana con una pequeña cantidad de etanol al 70%. </li><li> Inyectar 5 ml (una rana 2 pulgadas) o 10 ml (una rana de 3 pulgadas) de APBS estéril pre-calentado a temperatura ambiente en la cavidad peritoneal con un calibre de 18 1 ½ de la aguja. Retire la aguja y masajee suavemente la rana por un minuto para asegurar que el buffer se equilibra con la inyección de líquido en la cavidad del cuerpo. </li><li> Use un nuevo calibre 18 de 1 ½ aguja sin una jeringa para recoger el líquido peritoneal que el goteo de la parte posterior de la aguja en un recipiente limpio Tubo de 50 ml cónicos. Asegúrese de obtener la mayor cantidad de volumen inicial inyectada como sea posible. Tenga cuidado de evitar los vasos sanguíneos en la zona central de la región abdominal. </li><li> Una vez que se cosechan PLs poner la rana en un recipiente con agua poco profunda hasta que esté despierto y en ese momento se puede colocar de nuevo en su jaula. </li></ol> 4. Transferencia de pulso y Adoptivos de Lg-6 Dentro de Pls Naive Lg-6 destinatarios <ol><li> Lavar una vez con los LP APBS frío y centrifugar a 1.000 rpm en (750 g) durante 10 min a 4 ° C. </li><li> Eliminar el sobrenadante y resuspender el PLs en APBS. </li><li> Pulso de los LP con gp96 en una concentración de 1 mg por gp96 5 x 10 5 LP. </li><li> Una vez que la cantidad apropiada de gp96 se añade a los LP, mezclarlas pipeta e incubar en hielo durante 1 hora. </li><li> Centrifugar las células ya sea a 1.000 rpm durante 10 min a 4 ° C o menos 14.000 rpm durante 1 minuto para eliminar cualquier consolidados gp96. </li><li> Lavar los LP 3X con APBS frío para asegurar que no hay residual gp96 izquierda. </li><li> Resuspender las PLs pulsado a una concentración de 5 x 10 5 PLs por cada 300 mL. </li><li> Transferencia adoptiva PLs por inyección ip utilizando calibre 25 8.5 aguja. Inyectar 300 L de PLs pulsada (5 x 10 5 células) por animal. </li><li> Las ranas deben ser preparados por lo menos 3 días antes de continuar con injerto de piel o de los experimentos tumor desafío. </li></ol> 5. Ensayo de la piel del injerto El rechazo de injertos de piel es un ensayo bien establecido en Xenopus 4, 5, 6. En Xenopus, un receptor rechaza la piel de los donantes mostrando 1 o 2 desajustes haplotipo MHC dentro de 18-22 días a 21-22 ° C. Por el contrario, los injertos de piel entre LG y LG-6-15 clones que sólo se diferencian por un menor H-Ags son rechazados más lentamente (más de 30 días). Sin embargo, este rechazo se acelera en los receptores de That han sido preparados en contra de los donantes menores H-Ags, ya sea por un injerto de piel anterior o mediante la inmunización con gp96 purificada a partir de los donantes. El no rechazo en todas se produce cuando el donante y el receptor son genéticamente idénticos (por ejemplo, los animales clonados o totalmente puras). Por lo tanto, esta sencilla técnica es muy poderosa para la caracterización in vivo la respuesta inmune provocada por gp96. <ol><li> Anestesiar a la rana de los donantes como se describe en 3.7. Nota: Sólo un mínimo de precauciones deben ser tomadas desde Xenopus producen potentes péptidos antimicrobianos (por ejemplo, Magainin) en la piel que evita la necesidad de total de los procedimientos asépticos. De hecho, el tratamiento de la rana con cualquier desinfectante sería perjudicial para la piel. Además, Xenopus no tienen los agentes patógenos que pueden ser transferidas a los seres humanos. Por lo tanto, el uso de guantes es opcional. Sin embargo, todos los instrumentos de disección utilizados tienen que ser esterilizados en autoclave y todas las soluciones deben ser estériles. </li><li> Cortar y retirar un pequeño (20 mm x 5 mm) el pedazo de piel ventral (la piel del abdomen que parece de plata debido a la presencia de irridophore células pigmentadas) que de una rana donantes LG-15 con unas tijeras. </li><li> Lugar de la piel en un APBS Petridish contener y mantener en hielo. Manipular el tejido de la piel con mucha suavidad, evitar la celebración que en medio de unas pinzas. </li><li> Utilizando una navaja o tijeras cortan injertos individuales en 5 mm x 5 mm piezas. Mantener los fragmentos en APBS en el hielo. </li><li> En este punto, la rana de los donantes se coloca en un recipiente con agua poco profunda hasta que esté despierto y luego se coloca en agua que contiene antibióticos (Penstrep a una concentración de 5 mg / L). La rana se mantiene en el agua con antibióticos durante dos días, momento en el que se devuelve en el agua normal. La pequeña herida en el lugar de donde se extirpó la piel no tiene que ser cosida y se cura en una semana. </li><li> Anestesiar el receptor LG-6 rana. </li><li> Haga una pequeña incisión en el dorso (parte posterior) de la piel del receptor e insertar los 5 mm x 5 mm por debajo del injerto de piel con el lado plateado hacia arriba. Tenga cuidado de no introducir burbujas de aire bajo la piel, ya que puede provocar el desplazamiento o incluso la pérdida del injerto. </li><li> Asegúrese de tomar nota de las marcas de tijeras y pinzas en los injertos, ya los que no se cuentan como el rechazo del injerto una vez que el marcador se inicia. </li><li> 24 horas más tarde una parte de la piel de acogida superposición debe ser eliminado del injerto. </li><li> Anestesiar el receptor LG-6 rana. Manejar los animales como se describe en 5.1. </li><li> Con la tijera autoclave cortar una ventana alrededor del injerto para que el injerto se puede visualizar libremente. Tenga cuidado de no tocar la piel de los donantes o para inducir el sangrado. Vamos a recuperar la rana como se describe en 5.5. </li><li> Inicio anotar el injerto de inmediato mediante la adopción de un esquema. El rechazo de injertos de piel está determinado por el porcentaje de destrucción de la irridophores en la piel injertada. </li><li> Los injertos deben ser controlados cada 2-3 días, pero los animales no deben ser anestesiados para esto. Para visualizar los injertos, las ranas necesitan ser colocados en un petridish bajo un microscopio de disección. </li></ol> 6. Todo el montaje inmunohistología de piel trasplantada Rana de todo el montaje inmunohistología se ha descrito previamente 6. En pocas palabras, la rana se anestesia y se coloca bajo el microscopio sobre una toalla de papel estéril pre-mojado con agua rana (el área de trabajo también es preparado asépticamente). La piel trasplantada es entonces recolectado con una pequeña cantidad de la piel circundante de acogida y se tiñeron con anticuerpos de manera similar a la tinción de células para el análisis de citometría de flujo 6. Instrumentos en autoclave y tampones estériles deben ser usados. Después de la tinción de la piel se coloca sobre un portaobjetos de microscopio y se presiona suavemente con una hoja de cubierta. En este punto, la piel se pueden visualizar mediante un microscopio de fluorescencia para diferentes poblaciones de células que se han infiltrado en el injerto. Después del procedimiento, la rana se mantiene en el agua con antibióticos como se describe en la sección 5.5. y regresó en el agua normal. La pequeña herida a la izquierda donde la piel fue removido cura en una semana. 7. Caracterización de los leucocitos de la sangre <ol><li> Antes de extraer la sangre de la rana, hay que preparar &quot;agujas de cristal&quot; tirando de pipetas Pasteur estériles sobre la llama. El extremo fino de la pipeta se agudiza bajo el microscopio estereoscópico. La pipeta se conecta a un tubo de plástico por aspiración. Todos los instrumentos como las pinzas y tijeras deben ser esterilizados en autoclave antes de su uso. </li><li> También preparan el hielo frío 10 ml de solución de heparina APBS y añadiendo 50 unidades de heparina por 1 mL de APBS. Mantener la solución en hielo. Todas las soluciones que se utilizan deben ser estériles. </li><li> Anestesiar a la rana como se describe en 3.7, y seguir los procedimientos de manejo de los animales mismos como se describe en 5.1. . </li><li> Cortar la piel por encima del pie posterior para exponerla vena dorsal del tarso. </li><li> Llenar la pipeta Pasteur con 2.1 mL de solución APBS + heparina. </li><li> Inserte la &quot;aguja de vidrio&quot; en la vena y se empezará a recoger la sangre lentamente, aspirando con la boca. Es importante tener solución APBS + heparina en la &quot;aguja de cristal&quot;, ya que esto evitará que la coagulación de la sangre y la obstrucción de la punta de la aguja. </li><li> 1 a 2 ml de sangre se puede obtener de una rana de tamaño medio. </li><li> La pequeña incisión en la pierna de la rana no tiene que ser cosida y la herida se cura en una semana. </li><li> Se centrifuga la sangre a 1.000 rpm durante 10 min a 4 ° C. </li><li> Eliminar el sobrenadante y lavar las células 1X con 10 ml de APBS frío. </li><li> Poner 2 ml de sangre (1-5 x 10 6 células) en 1 ml de Ficoll Histopaque 1.077 (Sigma) previamente calentada a temperatura ambiente con el fin de separar los leucocitos de la sangre de las células rojas de la sangre. </li><li> Centrifugar 20 minutos a 1.000 rpm a temperatura ambiente sin frenos, y luego recoger la banda de leucocitos. </li><li> Lavar las células 2 veces con APBS por centrifugación a 1.400 rpm durante 10 min a 4 ° C para eliminar el Ficoll residual. </li><li> En este punto, las células están listas para ser teñidas con anticuerpos para el análisis de citometría de flujo, o que se utilizan en los ensayos de cultivo in vitro. </li></ol> 8. Ensayo de Tumor Trasplante <ol><li> Aproximadamente una semana antes del deshielo experimentar un nuevo lote de células 15 / 0 del tumor y asegurarse de que las células empiezan a crecer bien. Medio de cultivo se describe sucintamente en el siguiente apartado y en más detalle en el 3. </li><li> Expandir las células 15 / 0 del tumor. </li><li> En el día del experimento, el recuento de las células y determinar la muerte celular por exclusión del azul tripano. La muerte celular debe ser inferior al 5%. Lavar las células 1X en APBS frío y centrifugar a 1000 rpm a 4 ° C durante 10 min. </li><li> Resuspender las células en medio de cultivo del tumor, con una densidad de 5 x 10 5 / 15 0 300 células por mL. </li><li> Trasplante de 5 x 10 5 células en 300 l de volumen por la rana mediante una inyección subcutánea con un calibre de 25 5 / 8 en la aguja de un lado de la dorsal (espalda) de la animal. </li><li> El crecimiento del tumor se iniciará a las 2-3 semanas después de la exposición del tumor. La aparición del tumor inicial debe tenerse en cuenta el volumen del tumor y debe ser registrada cada 2-3 días. El volumen del tumor (alto x largo x ancho) se mide con un calibrador. </li><li> Una vez que el tumor crece hasta 3.500 mm 3 o la rana comienza a buscar letárgico, tiene que ser sacrificados para evitar el malestar. </li></ol> 9. Los reactivos necesarios: <ul><li> Anfibios Tampón fosfato salino (APBS): 6,6 g / L NaCl, 1,15 g / L de Na 2 HPO 4, 0,2 g / L KH 2 PO. el pH a 7,5 con NaOH 10N y esterilizar por filtración a través de filtro de 0,2 micras. </li><li> Tricaína metano sulfonato (TMS, MS-222) (Media Luna Roja de Investigación Química CAS # 886-86-2). </li><li> Bicarbonato de sodio (Fisher Scientific-S 233-500). </li><li> Histopaque-1077 (Sigma-Aldrich 10771-100 mL) </li><li> Heparina sal sódica (Sigma-Aldrich H3149-50KU) </li><li> Medio de cultivo para Xenopus 15 / tumores 0 [véase el punto 3 para más detalles]: 1 litro de Iscove medio DMEM basal (Gibco-Invitrogen 11965) con la insulina de 10 ml, 10 ml no aminoácidos esenciales, 10 ml de penicilina-estreptomicina, 10 mg / ml de kanamicina, 3 ml de primatone (Sheffield División de Productos), 1 ml de β2-mercaptoetanol, y 3,02 g de NaHCO3 en agua (pH 7,0). Este medio se diluye a la osmolaridad de anfibios mediante la adición de un 30% de agua bidestilada, y se complementa con suero fetal bovino al 5%, 20% superantant de un A6 riñón Xenopus línea celular, y el 0,25% de suero normal de Xenopus. </li></ul> 10. Resultados representante <img src="/files/ftp_upload/2026/2026fig1.jpg" alt="Figura 1" /> Figura 1. Análisis estereoscópico de rechazo del injerto de piel 12 días después del trasplante. LG-6 clonado ranas recibieron un injerto de piel por parte de (A) un MHC-idénticos LG-6 (no muestra rechazo) o (B) de un donante outbred MHC-dispares (80% de rechazo). Las flechas plateadas muestra células pigmentadas iridophore marca sanos (no rechazo) injertado tejidos. (*) Marca de fórceps no debido al rechazo. <img src="/files/ftp_upload/2026/2026fig2.jpg" alt="Figura 2" /> Figura 2. Gp96, facilita la presentación cruzada de antígenos tumorales en Xenopus. LG-15 PLs (5 x 10 5) fueron pulsadas durante 1 hora en hielo, ya sea con APBS (control negativo), 1 mg de gp96 recombinante purificada a partir de una E. G cultura coli, o 1 ó 0,5 de la gp96 purificada a partir de 15 / 0 tejido tumoral. Después de tres lavados, las células fueron transferidas adoptiva en receptores adultos LG-15 (1 x 10 6 / individual). Tres días más tarde, en vivo 15 / 0 células tumorales (5 x 10 5) setrasplantadas mediante inyección subcutánea. Cada curva representa la cinética del crecimiento tumoral en una rana. Reto días después, cuando los tumores aparecieron por primera vez fueron controlados, y el tamaño del tumor se determina periódicamente con un (largo x ancho x grosor) de la pinza.

<ol>
	<li>Kobel, H. R., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdiploid+species+hybrids+for+gene+mapping+in+Xenopus.">Hyperdiploid species hybrids for gene mapping in Xenopus.</a> Nature. 279, 157-158 (1979).</li><li>Robert, J., Menoret, A., Basu, S., Cohen, N., Srivastava, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+the+molecular+and+immunological+properties+of+the+chaperones+gp96+and+hsp70.">Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70.</a> Eur J Immunol. 31, 186-195 (2001).</li><li>Robert, J., Gantress, J., Cohen, N., Maniero, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+an+experimental+model+for+studying+evolution+of+hsp--immune+system+interactions.">Xenopus as an experimental model for studying evolution of hsp--immune system interactions.</a> Methods. 32, 42-53 (2004).</li><li>Chardonnens, X., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+skin+allograft+tolerance+during+metamorphosis+of+the+toad+Xenopus+laevis:+a+possible+model+for+studying+generation+of+self+tolerance+to+histocompatibility+antigens.">Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens.</a> Eur J Immunol. 3, 569-573 (1973).</li><li>Du Pasquier, L., Bernard, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+suppression+of+the+allogeneic+histocompatibility+reactions+during+the+metamorphosis+of+the+clawed+toad+Xenopus.">Active suppression of the allogeneic histocompatibility reactions during the metamorphosis of the clawed toad Xenopus.</a> Differentiation. 16, 1-7 (1980).</li><li>Ramanayake, T., Simon, D. A., Frelinger, J. G., Lord, E. M., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+T-cell+responses+to+skin+alloantigens+in+Xenopus+using+a+novel+whole-mount+immunohistology+method.">In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method.</a> Transplantation. 83, 159-166 (2007).</li><li>Robert, J., Ramanayake, T., Maniero, G. D., Morales, H., Chida, A. S., S, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+glycoprotein+96+ability+to+interact+with+CD91+and+facilitate+antigen+cross-presentation.">Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation.</a> J Immunol. 180, 3176-3182 (2008).</li><li>Robert, J., Gantress, J., Rau, L., Bell, A., Cohen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+histocompatibility+antigen-specific+MHC-restricted+CD8+T+cell+responses+elicited+by+heat+shock+proteins.">Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins.</a> J Immunol. 168, 1697-1703 (2002).</li><li>Morales, H., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+techniques+for+comparative+study+of+antiviral+T-cell+responses+in+the+amphibian+Xenopus.">In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.</a> Biol Proced Online. 10, 1-8 (2008).</li><li>Maniero, G. D., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+gp96-mediated+antigen-specific+cellular+immunity:+new+evidence+from+adoptive+cell+transfer+in+xenopus.">Phylogenetic conservation of gp96-mediated antigen-specific cellular immunity: new evidence from adoptive cell transfer in xenopus.</a> Transplantation. 78, 1415-1421 (2004).</li></ol>

El anfibio Xenopus es una singular versatilidad de animales no mamíferos modelo para estudiar la inmunidad. Su amplio uso en la investigación biomédica e inmunológica ha dado en diversas herramientas de investigación importantes, como los clones MHC define LG LG-6 y 15, así como diferentes líneas celulares y de anticuerpos monoclonales. El uso de estas herramientas se han establecido diferentes in vitro y en ensayos in vivo para estudiar la capacidad de las proteínas de choque...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents needed:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Amphibian Phosphate Buffered Saline (APBS): </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl, 1.15 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4, 0.2 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10N NaOH </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine Methane Sulfonate (TMS, MS-222) </td>
<td>&nbsp;</td>
<td>Crescent Research Chemicals </td>
<td>CAS#886-86-2</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S-233-500</td>
<td>&nbsp;</td>
<tr>
<td>Histopaque-1077</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>10771</td>
<td>100ml</td>
</tr>
<tr>
<td>Heparin Sodium Salt </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>H3149-50KU</td>
<td>&nbsp;</td>
<tr>
<td>Culture medium for Xenopus 15/0 tumors [see 3 for more details]:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Iscove DMEM basal medium</td>
<td>&nbsp;</td>
<td>Gibco-Invitrogen </td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primatone</td>
<td>&nbsp;</td>
<td>Sheffield Products Division</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&beta;2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>30% double distilled water </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5% featal bovine serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20% superantant from a Xenopus kidney cell line A6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.25% of normal Xenopus serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Materials and Equipment:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 and 15 ml conical centrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 gauge 5/8 Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>18 gauge 1&frac12; Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 ml Tuberculin Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml MicroCentrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 X 15 mm Polystyrene Petri Dishes sterile</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 X 75 mm X 1 mm Premium Microscope Slides </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 cm glass petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9" Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tygon tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Two # 5 Swiss Jeweler&#x2019;s Forceps</td>
<td>&nbsp;</td>
<td>Miltex Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Helios calipers </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High intensity illuminator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Un-bunsen burner</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C shaking incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comparative in vivo study of gp96 adjuvanticity in the frog xenopus laevis

Hemos desarrollado en el Xenopus laevis un único anfibio de animales no mamíferos modelo para estudiar la capacidad de ciertas proteínas de choque térmico (HSP), tales como la gp96, para facilitar la presentación cruzada de antígenos carabina y obtener respuestas T innata y adaptativa de la célula. Xenopus rechazo del injerto de piel proporciona una plataforma excelente para estudiar la capacidad de gp96 para obtener clásico MHC de clase Ia (clase Ia) limita las respuestas de células T. Además, el sistema modelo Xenopus también ofrece una alternativa atractiva a los ratones para explorar la capacidad de gp96 para generar respuestas frente a los tumores que se han reducido regulado su clase Ia moléculas para eludir la vigilancia inmune. Recientemente, hemos desarrollado un ensayo de células de la transferencia adoptiva de clones Xenopus utilizando leucocitos peritoneales como células presentadoras de antígeno (APC), y ha demostrado que puede gp96 primer T CD8 respuestas de las células in vivo contra los antígenos de histocompatibilidad menores piel, así como contra el tumor del timo Xenopus 15 / 0 que no expresan moléculas de clase I bis. Se describe aquí la metodología consistió en llevar a cabo estos ensayos incluida la transferencia de la obtención, pulso y adoptivos de leucocitos peritoneales, así como el injerto de piel y los ensayos de trasplante del tumor. Además también estamos describiendo la recolección y separación de los leucocitos de sangre periférica usado para citometría de flujo y ensayos de proliferación que permiten la caracterización de las poblaciones efectoras implicadas en el rechazo de piel y de las respuestas anti-tumorales.

Watch this Scientific Journal Video about Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis at JoVE.com

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

La rana<em&gt; Xenopus laevis</em&gt; Ofrece una atractiva alternativa de animales no mamíferos modelo para explorar la capacidad de la proteína de choque térmico, tales como la gp96, para promover el antígeno específico de células T CD8. Se presentan métodos para el estudio<em&gt; In vivo</em&gt; Facilitación de presentación cruzada de antígenos de la piel y los tumores de gp96.

Comparativo In vivo Estudio de la gp96 adyuvante de la rana Xenopus laevis</em

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

comparative-vivo-study-gp96-adjuvanticity-frog-xenopus

Research

JoVE Journal

Immunology and Infection

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

10.5K vistas.  University of Rochester. La rana Xenopus laevis Ofrece una atractiva alternativa de animales no mamíferos modelo para explorar la capacidad de la proteína de choque térmico, tales como la gp96, para promover el antígeno específico de células T CD8. Se presentan métodos para el estudio In vivo Facilitación de presentación cruzada de antígenos de la piel y los tumores de gp96.

Video: Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imágenes in vivo de los cultivos transgénicos parásitos Leishmania en un host en vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Investigación

Inmunología e Infección

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

De imagen no invasivas de Homing leucocitos y Migración In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

La leishmaniasis cutánea en la piel dorsal de los hámsteres: un modelo útil para la selección de fármacos antileishmania

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modelo para estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Los cultivos humanos de la placenta y Decidual de órganos para el Estudio de Infecciones en la interfase materno-fetal

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral transducción de células T colaboradoras como un enfoque genético para estudiar mecanismos que controlan su diferenciación y función

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Cuantificación de la Quimiotaxis de monocitos humanos In Vitro y linfocitos murinos, tráfico en Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Monocapas de Colonoid humano para estudiar las interacciones entre patógenos, comensales y epitelio Intestinal Host

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Speed

Subtitles

Methods Article

Comparativo In vivo Estudio de la gp96 adyuvante de la rana Xenopus laevis