This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).

1. Establecimiento y mantenimiento de E. Culturas histolytica <ol><li> Crecer axénicamente (totalmente libre de todos los demás organismos contaminantes) 1 x 10 5 trofozoítos de Entamoeba HML cepa histolytica: IMSS clon A 18 en 16 x 125 mm tubos de cultivo con teflón tapones de rosca de línea regular (o 1 x 10 6 trofozoítos en una desechable T- matraz de 25) y 15 ml (o 50 ml en matraz T-25) de TYI-S-33 medio (caldo de TYI suplementado con 3% de mezcla de diamante vitamina, suero inactivado bovino adulto calor al 10%, 0,5 UI / ml de penicilina y 35 mg / ml de estreptomicina) 19 en una incubadora a 37 ° C. </li><li> Trofozoítos de la cosecha durante la fase de crecimiento logarítmica por lo general a intervalos de 48-96 hr (Figura 1) mediante el enfriamiento de los tubos de cultivo durante 5-10 min en un baño de hielo-agua para liberar los trofozoítos unidos al tubo de cultivo de vidrio. </li><li> Transfiera la cultura en un tubo cónico e invertir varias veces para dispersar el cells. Determinar el número de células utilizando un hemocitómetro (cámara de Neubauer), y la transferencia de un inóculo en un tubo de cultivo que contiene medio TYI-S-33 fresco. <ol><li> Utilice un bajo número de amebas por períodos de incubación más largo (~ 3 x 10 5 células de ~ 5 días) y un mayor número de períodos más cortos (~ 1 × 10 6 células para ~ 1 día). Mientras inóculos contados son deseables, culturas establecidas se vuelven predecibles por lo que los volúmenes estimados de inóculos son factibles. </li><li> Valorar la cantidad de trofozoítos para optimizar el número de células para cada experimento. </li><li> Mantener un cultivo duplicado en paralelo para tener una copia de seguridad en caso de contaminación accidental o rotura del tubo. </li></ol></li><li> Tubos de tapa y los incuban a 37 ° C en el 5 ° inclinado ángulo horizontal. </li><li> Compruebe culturas por inspección visual con regularidad, ya que un crecimiento excesivo de la cultura puede contener muchas células lisadas. </li></ol> 2. Establecimiento y mantenimiento de MDCK Cultura &lt;/ P&gt; <ol><li> Crecer células MDCK (Madin Darby de riñón canino) tipo I (de alta resistencia) en matraces desechables T-75 con 10 ml de medio DMEM suplementado con penicilina (100 UI / ml), estreptomicina (100 mg / ml), insulina (0,08 U / ml) y 10% de suero de ternero recién nacido en una atmósfera humidificada de 5% de CO 2 y 95% de aire a 37 ° C. </li><li> Para dividir las células, comprobarlos en un microscopio invertido. Dividir las células cuando son ~ 85-100% confluentes. </li><li> Utilice un aspirador de vacío en una campana estéril y una pipeta Pasteur de vidrio estéril para eliminar los medios de comunicación de la cultura. Para evitar raspar las células, gire el frasco boca abajo y los medios de comunicación aspirado desde la esquina inferior. </li><li> Dispensar 10 ml de PBS precalentado (140 mM de NaCl, 2,7 mM de KCl, 10 mM de Na 2 HPO 4, 1,8 mM KH 2 PO 4, pH 7,4) en un matraz T-75 (5 ml en el caso de un T-25 matraz ) y mueva suavemente el matraz para lavar las células. Retire PBS por aspiración al vacío. Se requiere un prolongado lavado desde el suero inhibe la tripsina. </li><li> Dispensar 1,5 ml de 0,05% de tripsina en un matraz T-75 (T-25 para utilizar un matraz 0,5 ml) y el matraz suavemente roca para cubrir la capa de células con tripsina. </li><li> Incubar durante 5-10 minutos en la incubadora (tiempo exacto depende de la actividad de la tripsina). </li><li> Compruebe si hay desprendimiento de las células mediante la celebración de frasco contra la luz y verificar si hay células que fluye. Desprendimiento celular (células redondeadas), también se puede comprobar con un microscopio. A menudo, las células liberan más rápido con una rápida palmada suave en el lado del matraz. </li><li> Añadir 15 ml de DMEM suplementado a un matraz T-75 (5 ml para un matraz T-25). Resuspender las células pipeteando arriba y abajo de 4 o 5 veces. </li><li> Para propagar células, diluir 1:05 suspensión de células con DMEM fresco suplementado. Número de células también se puede determinar en este punto usando un hemocitómetro pero esto es por lo general sólo es necesario si la división de células en formatos especiales para determinados ensayos. </li></ol> 3. Preparación de Trofozoíto total lisados <ol><li> Separe trofozoítos de cutubos lture enfriando el tubo durante 5 a 10 min en un baño de agua helada. Transferir la cultura asépticamente en un tubo cónico y se centrifuga a 360 xg durante 10 min a 4 ° C. Desechar con cuidado el sobrenadante por decantación, añadir helado de PBS al sedimento, invertir el tubo varias veces para dispersar las células y centrifugar de nuevo a 360 xg durante 10 min. Repita este paso dos veces para eliminar todos los restos de medio TYI-S-33. </li><li> Determinar el número de trofozoítos utilizando un hemocitómetro. </li><li> Lyse trofozoítos por los ciclos de congelación-descongelación. Snap-congelar un inóculo medida de trofozoítos (600.000 trofozoítos / ml) diluido en PBS helado, en nitrógeno líquido durante 1 min. Descongelar la muestra a 4 ° C y agitar vigorosamente. Repita de congelación y descongelación 3 veces para completar la lisis celular. </li><li> Activar las proteasas presentes en los lisados ​​con 0,02% β-mercaptoetanol. </li></ol> 4. Preparación de trophozoite secretadas Productos <ol><li> Realice los pasos 3.1 a 3.2. </li><li> Determinarnúmero de células y la viabilidad en este punto utilizando un hemocitómetro y la aplicación de un test de exclusión con azul de tripano 20: <ol><li> Diluir 9 x 10 6 trofozoítos en 3 ml de helado de PBS y las células de transferencia en un tubo cónico de 50 ml. Tomar 10 l de esta suspensión y añadir 1 l de tripano al 0,4% solución de azul stock pH 7,2. </li><li> Utilice una cámara de Neubauer para contar las células a bajo aumento. </li><li> Contar todas las células y separar el número de células de color azul, ya que las células azules han tomado el colorante y deben ser considerados muertos. </li><li> Calcular el porcentaje de células viables mediante la división del número de células viables por el número total de células y multiplicando por 100. </li></ol></li><li> Transferir el tubo cónico que contiene la suspensión de trofozoitos en la incubadora a 37 ° C durante 2 horas en un 5 ° inclinadas ángulo horizontal. </li><li> Para recoger los productos secretados, tubos de centrífuga a 360 xg durante 10 min. Utilice una jeringa desechable y estéril y cuidadosamente recoger el dosobrenadante, evitando la contaminación de muestras con trofozoítos de la pastilla. Eliminar las células transferidas al pasar el sobrenadante a través de una membrana de acetato de celulosa de 0,22 micras. Activar proteasas con 0,02% β-mercaptoetanol. </li><li> Para descartar la contaminación no deseada putativa con moléculas liberadas por las células muertas, vuelva a aplicar la prueba de exclusión con azul de tripano para la viabilidad celular. Viable y número total de células debería ser la misma que la obtenida en la etapa 4.2. Si este no es el caso, el sobrenadante recogido puede no sólo contener las proteínas secretadas y debe desecharse. </li></ol> 5. La interacción de las células MDCK con trofozoítos, Trofozoíto lisados ​​o secretadas Productos <ol><li> Incubar confluente monocapas de células MDCK con trofozoítos vivos (una relación de-MDCK a amebas de 1:01), trophozoite lisados ​​(una relación-MDCK a la ameba de 1:2) o productos secretados (una relación de-MDCK a la ameba de 1 : 10) a 37 ° C durante 2 min y 30 (Figura 2A). </li><li> Lavar las células epiteliales cinco veces con helado de PBS para eliminar las moléculas no unidas o trofozoítos. </li></ol> 6. Preparación de muestras para inmunofluorescencia <ol><li> Células MDCK Cultura sobre cubreobjetos de vidrio estériles colocados dentro de una placa de cultivo celular de 24 pocillos múltiples. Inspeccionar las células en el microscopio invertido hasta que alcanzan el 100% de confluencia. A continuación, sustituir medio con 1 ml de medio DMEM suplementado fresco y transferir la placa a una incubadora a 37 ° C durante 24 h más. </li><li> Retire el medio utilizando un aspirador de vacío y una pipeta Pasteur de vidrio estéril y añadir 1 ml de PBS caliente a cada pocillo. Retire PBS y repetir el lavado con PBS fresco. Tener cuidado de no raspar las células de la parte inferior del pocillo con la pipeta de vidrio. </li><li> Añadir diferentes condiciones de trofozoítos diluido en 1 ml de PBS caliente: trofozoítos vivos (T), lisados ​​totales de trofozoitos (TL) y productos secretados (SP). <ol><li> Coloque la placa de cultivo en la incubadora durante 2 o 30 min. </li><li> Prepare dos condiciones de control: uno llamado tiempo 0 min, donde las células MDCK no deben ser incubadas con E. histolytica pero sólo con 1 ml de PBS caliente; y otra condición de control como anticuerpos secundarios, en este caso incubar MDCK con T, TL o SP para 2 o 30 min y se omite la incubación con anticuerpos primarios. </li></ol></li><li> Lavar las células epiteliales cinco veces con PBS frío para eliminar moléculas no unidas o trofozoítos. </li><li> Fijar y permeabilizar las células con 1 ml de etanol al 96% durante 30 min a -20 ° C. </li><li> Para eliminar el etanol, realizar tres lavados rápidos con 1 ml de PBS a temperatura ambiente. </li><li> Realice los siguientes pasos en una cámara húmeda para evitar el secado de las muestras: <ol><li> Utilice cualquier caja plana disponible que puede ser cerrada y llena de una toalla de papel húmeda en el que las muestras se pueden colocar de forma segura. Por ejemplo, una caja de puntas de pipeta vacía sirve para este propósito. </li><li> Dentro de la cámara, coloque uniformemente una capa Parafilm y pipeta de 25 l de bloqueing solución (0,5% de BSA) para cada cubreobjetos. </li><li> Tome cubreobjetos de los pozos con unas pinzas finas, retire con cuidado el exceso de líquido de la punta con un papel de filtro y poner cubreobjetos en la gota que contiene la solución de bloqueo (BSA al 0,5%) con las células frente a la solución de bloqueo. Se incuban las muestras durante 1 hora a temperatura ambiente. </li></ol></li><li> Preparar una mezcla de anticuerpos primarios en PBS: IgM de ratón anti-EhCPADH112 (mα-EhCPADH112; dilución 1:10) y la IgG de conejo anti-ZO-1 (pα-ZO-1; dilución 1:100). </li><li> Coloque una hoja de Parafilm en la caja húmeda y de pipeta 25 l de solución de anticuerpos para cada cubreobjetos. </li><li> Levante los cubreobjetos de la solución de bloqueo, secar el exceso de líquido en los bordes con un papel de filtro, y colocarlos en las gotas con las células frente a la solución de anticuerpos. Se incuban las muestras durante la noche a 4 ° C. </li><li> Ponga cada cubreobjetos en un pozo de un 24 pocillos múltiples (lado de células en la parte superior) y añadir 1 ml de PBS. <ol><li&gt; Lavar cinco veces con 1 ml de PBS para eliminar las moléculas no unidas. </li></ol></li><li> Preparar una mezcla de anticuerpos secundarios fluorescentes en PBS: ratón anti-IgM de cabra acoplado a FITC (dilución 1:100) y anticuerpo de cabra anti-IgG de conejo acoplado a TRITC (1:50 dilución). </li><li> Coloque una hoja de Parafilm en la caja húmeda y de pipeta 25 l de la solución de anticuerpos secundaria para cada cubreobjetos. </li><li> Transferir las muestras como en 6,10 y se incuba durante 1 hora a temperatura ambiente en la oscuridad para evitar la decoloración de los colorantes fluorescentes. Repita el paso 6.11.1. </li><li> Para la tinción nuclear, incubar durante 3 min con 200 l de solución 0,05 mM DAPI a temperatura ambiente y protegido de la luz. Repita el paso 6.11.1. </li><li> Para conservar los tintes fluorescentes, colocar el cubreobjetos (células hacia abajo) en 5 l Vecta Shield Antifade solución de montaje en portaobjetos de vidrio para microscopio. Selle los cubreobjetos utilizando el esmalte de uñas para evitar la desecación de la muestra. </li><li> Para el almacenamiento a largo plazo, mantener las diapositivas en unacaja de portaobjetos a -20 ° C. </li><li> Analizar los preparativos de la microscopía confocal a través de secciones Z-stack y xz planos (Figura 2A). </li></ol> 7. La incubación de trofozoítos con inhibidores de la proteasa o de anticuerpos específicos <ol><li> Repita los pasos 3.1 a 3.2. Para cada condición experimental, se incuban 1 x 10 5 trofozoítos (resuspendidas en 50 l de PBS) con inhibidores de proteasa (1 mM completos y 40 mg / ml E-64) o 30 mg anticuerpo monoclonal contra EhCPADH112 (mαEhCPADH112) 21 durante 20 min a 4 ° C (Figura 2B). Utilice estos preparativos para los ensayos de co-incubación, como se describe en el paso 8.5. </li></ol> 8. Medición de la resistencia eléctrica transepitelial (TER) <ol><li> Cultura células MDCK en soportes permeables Transwell estériles (0,4 micras de tamaño de poro). En el compartimiento superior, colocar 100 l de suspensión celular y en el compartimiento inferiorcolocar 600 l de medio DMEM suplementado. <ol><li> Compruebe periódicamente el nivel medio. Medio fresco se puede añadir según sea necesario. </li><li> Inspeccionar las células en un microscopio invertido hasta que alcanzan el 100% de confluencia. Cambie medio cada 2-3 días. </li><li> Para mejorar la unión celular (si es necesario), equilibrar los filtros Transwell durante la noche a 37 ° C en DMEM antes de añadir la suspensión celular. </li></ol></li><li> Esterilizar electrodos Stx2 en la campana por inmersión en etanol y luego en PBS estéril durante 15 minutos cada uno, bajo luz UV. Conecte los electrodos a un epitelio voltohmmeter EVOM y seleccione el modo de resistencia. </li><li> Recoger medio del compartimiento superior de cada transwell y agruparlas. Tomar 50 l de esta mezcla de medios y combinarlo con 50 l de trofozoítos suspensión (1 x 10 5 trofozoítos en PBS) o sólo con PBS (control). Use una mezcla similar para cada transwell. </li><li> Añadir las mezclas a la cámara superior de cada containin transwellg las células MDCK. Las condiciones experimentales incluyen células MDCK sin trofozoítos (control), un co-cultivo con trofozoítos o con trofozoítos pre-incubadas con inhibidores de la proteasa o mαEhCPADH112. Para eliminar la resistencia proporcionada por los filtros, utilizar cultivos polarizados se incubaron con medio solo. Para cada condición de uso experimental por lo menos tres cultivos polarizados. </li><li> Inmediatamente, medir TER sumergiendo los electrodos en cada Stx2 transwell. <ol><li> Asegúrese de que el electrodo ya toca la parte inferior de la cámara inferior y que el electrodo más corto está cubierto por medio en el compartimiento superior (véase la figura 2B). </li><li> Asegúrese de sujetar los electrodos perpendicularmente cada vez. Esto mejorará la reproducibilidad, de manera significativa. Evite mover o inclinar el electrodo durante las mediciones, ya que esto dará lugar a la variación de los datos. </li></ol></li><li> Esta medición se debe considerar el valor inicial TER. Entonces, supervisar la TER durante el siguiente30 min. </li><li> Para calcular el valor final TER, restar el valor TER de sólo filtros. Para comparar los resultados de diferentes experimentos normalizar cada punto de datos a los valores iniciales, tomando éstos como 100% (Figura 2B). </li></ol>

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Appendix 3, (2001).</li><li>Arroyo, R., Orozco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+identification+of+an+Entamoeba+histolytica+adhesin.">Localization and identification of an Entamoeba histolytica adhesin.</a> Mol Biochem Parasitol. 23, 151-158 (1987).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenic+cultivation+of+Entamoeba+hitolytica.">Axenic cultivation of Entamoeba hitolytica.</a> Science. 134, 336-337 (1961).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+of+axenic+cultivation+of+Entamoeba+histolytica+Schaudinn,+1903+and+E.+histolytica-like+amebae.">Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae.</a> J Parasitol. 54, 1047-1056 (1968).</li><li>Dukes, J. D., Whitley, P., Chalmers, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDCK+variety+pack:+choosing+the+right+strain.">The MDCK variety pack: choosing the right strain.</a> BMC Cell Biol. 12, (2011).</li><li>Espinosa-Cantellano, M., Martinez-Palomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+intestinal+amebiasis:+from+molecules+to+disease.">Pathogenesis of intestinal amebiasis: from molecules to disease.</a> Clin Microbiol Rev. 13, 318-331 (2000).</li><li>Martinez-Palomo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bases+of+the+cytolytic+mechanisms+of+Entamoeba+histolytica.">Structural bases of the cytolytic mechanisms of Entamoeba histolytica.</a> J Protozool. 32, 166-175 (1985).</li><li>Martinez-Lopez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EhADH112+recombinant+polypeptide+inhibits+cell+destruction+and+liver+abscess+formation+by+Entamoeba+histolytica+trophozoites.">The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.</a> Cell Microbiol. 6, 367-376 (2004).</li><li>Ocadiz-Ruiz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+silencing+of+the+Ehcp112+gene+on+the+in+vitro+virulence+of+Entamoeba+histolytica.">Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.</a> Parasit Vectors. 6, 248 (2013).</li><li>Johnson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+imaging+techniques+to+studies+of+epithelial+tight+junctions.">Applications of imaging techniques to studies of epithelial tight junctions.</a> Adv Drug Deliv Rev. 57, 111-121 (2005).</li></ol>

The authors have nothing to disclose.

Con el fin de estudiar in vitro las interacciones huésped-patógeno durante la infección epitelial por E. histolytica, es fundamental trabajar con culturas bien establecidos de ambas células epiteliales y trofozoítos. Por ejemplo, anteriormente, E. culturas histolytica usualmente se habían establecido en asociación con ciertas especies de bacterias o tripanosomátidos 22,23. Sin embargo, el co-cultivo de E. culturas histolytica es contraproducente pa...

Entamoeba histolytica es un protozoo único responsable de la amibiasis humana, una infección intestinal de células causando inflamación y diarrea. E. histolytica infecta a 50 millones de personas al año, pero sólo alrededor del 10% de las personas infectadas desarrollan los síntomas asociados a la amibiasis 1. La infección se produce por ingestión de alimentos contaminados o agua que contiene E. quistes histolytica. En el intestino, quistes producen trofozoítos en vivo que se adhieren a la mucina de colon y proliferan 2. Los trofozoítos suelen formar quistes que se excretan a través de las heces. En otros casos y por razones aún desconocidas, trofozoítos rompen la capa del epitelio intestinal e invaden los tejidos subyacentes. En el peor de los casos, entran en el torrente sanguíneo y afectan a otros órganos como el hígado 3. Rompiendo la barrera epitelial requiere la interrupción de las estructuras epiteliales transmembrana que mantienen las células unidas. Célula epitelialcontactos se forman por el complejo de unión apical que consiste de apretado (TJ) y uniones adherentes (AJ), y desmosomas 4. Las uniones más apical son TJ, y por lo tanto, que son la primera barrera afrentado por E. histolytica y algunos otros patógenos durante la invasión del huésped. TJ se componen de los receptores de adhesión transmembrana como claudins, occludin y moléculas de adhesión de unión (JAM) que se dedican a la homo-o interacciones heterófilos con receptores de la célula vecina. Están intracelularmente obligados por moléculas de andamio de los zonula occludens (ZO) de la familia que se conectan los receptores de adhesión a citoesqueleto de actina para proporcionar más resistencia mecánica al epitelio. TJ son responsables para el sellado de tejido intestinal desde el lumen intestinal, evitando que el agua excesivo y fugas de soluto. Así, después de TJ se interrumpen por el parásito, los tejidos son invadidos. E. histolytica segrega varias moléculas tales como: (i) los implicados en la adhesión de las amebas a Target células 5; (Ii) factores activos de membrana que participan en la destrucción de las células huésped por exocitosis, por ejemplo, los péptidos que forman canales iónicos denominados amoebapores 6,7; y (iii) las proteinasas que degradan las proteínas de la matriz extracelular y median tejido desintegración 5,8,9. La proteasa cisteína EhCP112 y la molécula de adhesión EhADH112 que en conjunto forman el complejo EhCPADH112 son dos E. histolytica virulencia proteínas que desempeñan un papel importante en el desmontaje de TJ 10. Trofozoítos vivo, sus lisados ​​totales y productos secretados inducen cambios moleculares en la perturbación compleja y funcional TJ de la barrera epitelial. En este estudio, se muestra que EhCP112 y EhADH112 interactúan con ocludina y claudina-1 proteínas que conducen a la internalización y la degradación de proteínas de la célula, facilitando de este modo E. entrada histolytica a través de la ruta paracelular. Nuestros datos y los Of otros grupos 11-17 sugieren fuertemente la necesidad de las interacciones huésped-patógeno específicos que permiten la invasión del parásito. Revelación de la base molecular de estas interacciones es de suma importancia para una mejor comprensión de la patogénesis de la amebiasis. Perturbación selectiva de TJ por trofozoitos, caracterizado por aumento de la permeabilidad paracelular, se puede medir por una disminución en la resistencia eléctrica transepitelial (TER). La transferencia de las proteínas parasitarias hacia epitelios huésped puede ser determinado por tinción de inmunofluorescencia y microscopía láser confocal, un método que también puede revelar co-localización de los factores de virulencia de ameba con marcadores epiteliales de unión que indican posibles interacciones directas. En este artículo, describimos en detalle cómo se cultivan, cosechan y se manipulan para examinar las interacciones huésped-patógeno y sus consecuencias células epiteliales y trofozoítos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Entamoeba histolytica HM1:IMSS, Clone A&#160;</td>
			<td>IMSS Hospital, Mexico</td>
			<td>Without/number</td>
			<td>Virulent trophozoites18&#160;</td>
		</tr>
		<tr>
			<td rowspan="8">TYI broth</td>
			<td rowspan="8">Becton, Dickinson and Company 
			Merck 
			Merck 
			Merck 
			J.T. Baker 
			Reproquifin 
			SIGMA-Aldrich 
			SIGMA-Aldrich</td>
			<td rowspan="8">211862 
			K35625437 626 
			21578 
			4873 
			3252-01 
			CAS 50-81-7 
			C7880 
			F-5879</td>
			<td rowspan="8">3.45% BBL Biosate peptone 
			58 mM glucose 
			39 mM NaCl 
			5 mM KH2PO4 
			6.5 mM K2HPO4 
			16.3 mM ascorbic acid 
			8.1 mM L-cysteine 
			&#160;0.1 mM ferric ammonium citrate, adjust pH 6.819</td>
		</tr>
		<tr>
			<td>Bovine serum adult</td>
			<td>Microlab , Labs., Mex.</td>
			<td>SU146</td>
			<td>Use at 10% and inactivated to 56&#176; C for 30 min</td>
		</tr>
		<tr>
			<td>Diamond&#160; vitamin&#160; mixture- Tween 80</td>
			<td>In vitro</td>
			<td>SR-07</td>
			<td>Use at 3%</td>
		</tr>
		<tr>
			<td>Penicillin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>34564SSA IV</td>
			<td>0.5 IU/mL</td>
		</tr>
		<tr>
			<td>Streptomycin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>75757SSA IV</td>
			<td>35 &#181;g/ mL</td>
		</tr>
		<tr>
			<td>Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps</td>
			<td>Corning-Pyrex</td>
			<td>9826-16X</td>
			<td>16x125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 Well</td>
			<td>Corning-Costar</td>
			<td>3516</td>
			<td>Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2.&#160; Rings on lid prevent cross-contamination</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430168</td>
			<td>Canted neck flasks</td>
		</tr>
		<tr>
			<td>MDCK (Madin Darby canine kidney) type I</td>
			<td>American Type Culture Collection</td>
			<td>CCL34</td>
			<td>Kidney epithelial cells grown between the 60th and 90th passage</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>12800-017</td>
			<td>Dulbecco&#39;s Modified Eagle Medium with high glucose.</td>
		</tr>
		<tr>
			<td>Neonate Calf Serum</td>
			<td>In vitro</td>
			<td>S-02</td>
			<td>Use at 10%.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin mixture&#160;</td>
			<td>In vitro&#160;</td>
			<td>A-01</td>
			<td>Stock solution 10,000 U/&#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;&#160;</td>
			<td>AMSA</td>
			<td>398MJ94SSA IV</td>
			<td>Stock solution 100 IU/mL</td>
		</tr>
		<tr>
			<td>Trypsin solution&#160;</td>
			<td>In vitro</td>
			<td>EN-005</td>
			<td>0.05% enzyme solution without calcium and magnesium</td>
		</tr>
		<tr>
			<td>75cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430720</td>
			<td>Canted neck flasks for trophozoite culture in TYI-S-33 medium</td>
		</tr>
		<tr>
			<td>Transwell permeable supports</td>
			<td>Corning-Costar</td>
			<td>3470</td>
			<td>0.4. &#181;m polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2</td>
		</tr>
		<tr>
			<td>24 well cell culture dish&#160;&#160;</td>
			<td>Corning-Costar</td>
			<td>3524</td>
			<td>Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche</td>
			<td>11836 153 001</td>
			<td>Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM&#160;</td>
		</tr>
		<tr>
			<td>Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)</td>
			<td>SIGMA-Aldrich</td>
			<td>E3132</td>
			<td>Cystein protease inhibitor, final concentration 40 &#181;g/mL</td>
		</tr>
		<tr>
			<td>p&#945;ZO-1&#160;</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td>IgG rabbit policlonal&#160; antibody&#160; against&#160; a synthetic peptide in the middle region of the ZO-1 human protein</td>
		</tr>
		<tr>
			<td>m&#945;EhCPADH112</td>
			<td>Homemade antibody</td>
			<td>Without/ Number</td>
			<td>IgM mouse monoclonal antibody&#160; against&#160; 444-601 epitope located at C-terminal of EhCPADH11221,27</td>
		</tr>
		<tr>
			<td>FITC-goat anti-mouse IgM</td>
			<td>Zymed</td>
			<td>62-6811</td>
			<td>Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary&#160; antibody</td>
		</tr>
		<tr>
			<td>TRITC- goat anti-rabbit IgG (H+L)</td>
			<td>Zymed</td>
			<td>816114</td>
			<td>Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled&#160; goat anti-rabbit IgG&#160; secondary antibody.</td>
		</tr>
		<tr>
			<td>STX2 Electrode</td>
			<td>World Precision Instrument&#160;</td>
			<td>102711</td>
			<td>Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM</td>
		</tr>
		<tr>
			<td>EVOM epithelial voltohmmeter</td>
			<td>World Precision Instrument&#160;</td>
			<td>12111</td>
			<td>Use in resistance mode and maintain unplugged during TER measurements</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>MEARIENFELD</td>
			<td>610610</td>
			<td>Hemocytometer&#160;</td>
		</tr>
		<tr>
			<td>Leica TCS_SP5_MO</td>
			<td>Leica</td>
			<td>Without/number</td>
			<td>Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td>Mounting medium for fluorescence</td>
		</tr>
		<tr>
			<td>4&#180;, 6-diamino-2-phenylindole (Dapi)</td>
			<td>SIGMA</td>
			<td>D-9542</td>
			<td>0.05 mM final concentration</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A-1310</td>
			<td>0.5%&#160; final concentration for blocking solution</td>
		</tr>
	</tbody>
</table>

analysis of the epithelial damage produced by entamoeba histolytica infection

Entamoeba histolytica es el agente causante de la amebiasis humano, una causa importante de diarrea y absceso hepático en los países tropicales. La infección se inicia por la interacción del patógeno con las células epiteliales intestinales. Esta interacción da lugar a una alteración de las estructuras intracelulares tales como uniones estrechas (TJ). TJ asegurar el cierre de la capa epitelial para separar el tejido del huésped de luz intestinal. Estudios recientes demuestran que la interrupción de TJ por la EhCPADH112 proteína parasitaria es un requisito previo para E. invasión histolytica que se acompaña de disfunción de la barrera epitelial. Por lo tanto, el análisis de los mecanismos moleculares implicados en TJ desmontaje durante E. invasión histolytica es de suma importancia para mejorar nuestra comprensión de la patogénesis de la amibiasis. En este artículo se presenta un modelo sencillo que permite la evaluación de las interacciones huésped-patógeno iniciales y el potencial de la invasión del parásito. Parámetros que deben analizarse incluyen tresistencia eléctrica ransepithelial, la interacción de EhCPADH112 con receptores de la superficie epiteliales, cambios en la expresión y localización de marcadores epiteliales de unión y localización de moléculas de parásitos dentro de las células epiteliales.

Para una E. éxito cultura histolytica, dos importantes condiciones debe cumplirse: el crecimiento en condiciones axénicas y cosecha en fase logarítmica. Anteriormente, cultivos de E. histolytica se establecieron fácilmente en asociación con ciertas especies de bacterias o tripanosomátidos 22. Sin embargo, hoy en día es común tener cultivos axénicos de este parásito es decir, un subcultivo indefinida de amebas en un ambiente libre de bacterias que metabolizan, hongos, proto...

Watch this Scientific Journal Video about Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection at JoVE.com

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

La infección humana por Entamoeba histolytica conduce a la amebiasis, una causa importante de diarrea en los países tropicales. La infección se inicia por la interacción de agentes patógenos con células epiteliales intestinales, provocando la apertura de los contactos célula-célula y en consecuencia la diarrea, a veces seguido por infección del hígado. Este artículo proporciona un modelo para evaluar las primeras interacciones huésped-patógeno para mejorar nuestra comprensión de la patogénesis de la amibiasis.

Análisis del daño epitelial Producido por<em&gt; Entamoeba histolytica</em&gt; Infección

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

analysis-epithelial-damage-produced-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

18.6K vistas.  Center for Research and Advanced Studies of the National Polytechnic Institute.  La infección humana por Entamoeba histolytica conduce a la amebiasis, una causa importante de diarrea en los países tropicales. La infección se inicia por la interacción de agentes patógenos con células epiteliales intestinales, provocando la apertura de los contactos célula-célula y en consecuencia la diarrea, a veces seguido por infección del hígado. Este artículo proporciona un modelo para evaluar las primeras interacciones huésped-patógeno para ...

Video: Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Ensayo in vitro de metilación del ADN tRNA con Entamoeba histolytica y Dnmt2 tRNA metiltransferasa (Ehmeth) Enzima

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Investigación

Inmunología e Infección

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desarrollo de un marcador negativo seleccionable para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

La aplicación de un ratón ligado Ensayo placas de Peyer asa intestinal para evaluar la absorción por las células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

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

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

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

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

El análisis de la accesibilidad al disolvente de los residuos de cisteína en<em&gt; Maize rayado fino virus</em&gt; Partículas similares al virus producidas en<em&gt; Nicotiana benthamiana</em&gt; Plantas y reticulación de péptidos a VLPs

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular Serotipificación de<em&gt; Streptococcus pneumoniae</em&gt; Por partículas de látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Análisis rápido de aberraciones cromosómicas en linfocitos B de ratón por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

El uso de la técnica de superposición Soft-agar para la detección de compuestos inhibidores en bacterias producidas

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Imágenes de Ca2 + respuestas durante la infección de Shigella de células epiteliales

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...