El apoyo financiero para este trabajo fue proporcionado por la Comunidad Europea (ERC-STG INTRACELLTB Grant n° 260901, MM4TB Grant n° 260872), la Agence Nationale de Recherche, el Feder (12001407 (D-AL) Equipex Imaginex BioMed) y la Region Nord Pas de Calais. Agradecemos la asistencia técnica de Gaspard Deloison, Elizabeth Werkmeister, Antonino Bongiovanni y Frank Lafont de la plataforma BICeL.

1. Detección de siRNA en todo el genoma de alto rendimiento
La investigación realizada en un tipo humano de los pneumocytes del tipo II modelo A549 la línea de células sobre la infección con mtb H37Rv que expresa la proteína fluorescente verde (GFP). Este procedimiento se describe en la Figura 1A.
<ol><li>Resuspend la biblioteca de siRNA seca que se almacena en placas madre (placas de 96 pozos) con 1x tampón de siRNA para alcanzar una concentración de 4 μM, luego transfiera 10 μl de la mezcla en una placa hija de 384 pozos (placa hija 1).</li>
	<li>Añadir 10 μl de 1x tampón de siRNA en la placa hija 1 para diluir el siRNA por 2 veces. Después de la resuspensión de siRNA, las placas se sellan con sello de aluminio pelable y se pueden almacenar a -20 ° C al menos 6 meses y hasta 2-3 años, pero el tiempo de almacenamiento puede variar dependiendo de las recomendaciones del fabricante de la biblioteca de siRNA.</li>
	<li>Diluir los siRNAs en la placa hija 1 en la placa hija 2 para alcanzar una concentración de 500 nM. Después de la resuspensión de siRNA, las placas se sellan con sello de aluminio pelable y se pueden almacenar a -20 ° C al menos 6 meses y hasta 2-3 años, pero el tiempo de almacenamiento puede variar dependiendo de las recomendaciones del fabricante de la biblioteca de siRNA.</li>
	<li>Antes de su uso, descongelar la placa hija 2 a temperatura ambiente.</li>
	<li>Tome 2,5 μl de siRNA de la placa hija 2 y colóquelos en una placa de ensayo de 384 pozos.</li>
	<li>En la misma placa de ensayo de 384 pozos en el paso 1.5, agregue 2.5 μl de siRNA de control negativo y positivo a sus respectivos pozos.</li>
	<li>Diluya el reactivo de transfección en 1x D-PBS para producir suficiente solución para proporcionar un reactivo de transfección de 0,1 μl en cada pozo y preincuba la solución de transfección diluida a temperatura ambiente durante 5 min.</li>
	<li>Añadir 7,5 μl de la mezcla de reactivo de transfección/solución de PBS a cada pozo de la placa de ensayo e incubar durante 30 min a temperatura ambiente.</li>
	<li>Añadir 40 μl de células A549 (1.500 células/pozo) suspendidas en medio RPMI 1640 suplementado con suero fetal bovino al 10% (FBS). Mantener las células durante un período de incubación de 3 días a 37 °C en una atmósfera que contenga un 5% deCO2. Estas células se dividen cada 24 horas, por lo que unas 12.000 células están en los pozos tres días después de la transfección.</li>
	<li>Lave un cultivo de Mtb H37Rv que exprese GFP de dos semanas de edad con D-PBS (libre de MgCl2 y CaCl2)por centrifugación a 4,000 x g durante 5 min y deseche los lavados. Repita este paso 3x. (Para las condiciones de cultivoGFP-Mtb H37Rv, consulte el Protocolo 3).</li>
	<li>Suspender el pellet bacteriano en 10 ml de RPMI 1640 medio suplementados con 10% de FBS y decantar durante 1 hora a temperatura ambiente para permitir que los agregados bacterianos se sedimenten.</li>
	<li>Recoger el sobrenadante bacteriano y medir OD600 (OD600 debe estar entre 0.6-0.8) y GFP-fluorescencia (valor de RFU) utilizando un lector de microplacas para determinar la concentración bacteriana. Calcule el título de la suspensión utilizando una línea de regresión de referencia que muestre el valor de RFU = f (valor de CFU) que se había generado antes del experimento en otro cultivo que se había preparado en las mismas condiciones. Preparar suspensión bacteriana que contenga 2,4 x 106 bacterias/ml, que corresponde a una multiplicidad de infección (MOI) de 5.</li>
	<li>Retire el medio en la placa de ensayo de 384 pozos y agregue 25 μl de suspensión bacteriana recién preparada.</li>
	<li>Incubar la placa de ensayo de 384 pozos a 37 °C durante 5 horas en una atmósfera que contenga un 5% de CO2.</li>
	<li>Retire el medio y lave suavemente las células con el medio RPMI complementado con 10% FBS 3x.</li>
	<li>Para matar las bacterias extracelulares restantes, trate las células con 50 μl de medio RPMI-FBS fresco que contenga 50 μg/ml de amikacina a 37 °C durante 1 hora en una atmósfera que contenga 5% de CO2.</li>
	<li>Retire la amikacina que contiene el medio y agregue 50 μl de medio RPMI fresco complementado con 10% de FBS. Incubar la placa de ensayo de 384 pozos a 37 °C durante 5 días en una atmósfera que contenga un 5% deCO2.</li>
	<li>Antes de la adquisición de la imagen, añadir 10 μl de 30 μg/ml de DAPI recién preparados en PBS (concentración final de 5 μg/ml) e incubar durante 10 min a 37 °C.</li>
	<li>Cargue la placa en un microscopio confocal automatizado. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Establezca los parámetros de exposición. Registre la fluorescencia DAPI utilizando láser de excitación de 405 nm con filtro de emisión de 450 nm y fluorescencia GFP utilizando láser de excitación de 488 nm con filtro de emisión de 520 nm. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Seleccione los pozos y los campos en cada pozo que se va a adquirir, que luego se conoce como parámetros de diseño y subdiseño. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Genere el archivo de experimento utilizando los parámetros de los pasos 1.20 y 1.21 y ejecute la adquisición automática. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Transferir imágenes al servidor remoto. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Evalúe las imágenes utilizando el software de análisis de imágenes. Detectar núcleos celulares del canal DAPI utilizando el algoritmo de detección de núcleos y el área bacteriana del canal GFP utilizando el algoritmo de propiedades de intensidad de píxeles (Figura 4A).</li>
</ol>Nota: Este protocolo está optimizado para estudiar el efecto del silenciamiento génico sobre el crecimiento intracelular de Mtb. Mtb es una bacteria de crecimiento lento que se divide cada 20 horas en condiciones óptimas. Después de 5 días después de la infección, la cantidad de Mtb extracelular sigue siendo baja en ausencia de lisis celular y no afectó la calidad del análisis. Este protocolo debe optimizarse en términos de duración del tratamiento con antibióticos y tiempo de incubación para adaptarse a las pantallas de siRNA utilizando bacterias de crecimiento rápido como Mycobacteria smegmatis y Escherichia coli que se liberan ampliamente y pueden infectar nuevas células.
2. Cribado de compuestos de alto rendimiento
La investigación realizada en las células huésped infectadas mtb H37Rv. Este procedimiento se describe en la Figura 1B.
<ol><li>Descongelar las placas madre de 384 pozos que contienen la biblioteca compuesta solubilizada en DMSO al 100%. Transferir 0,5 μl de los compuestos en placas hijas de 384 pozos que contienen 10 μl de medio RPMI 1640 complementado con FBS al 10%.</li>
	<li>Lave un cultivo de Mtb H37Rv que exprese GFP de dos semanas de edad con D-PBS (libre de MgCl2 y CaCl2)por centrifugación a 4,000 x g durante 5 min y deseche los lavados. Repita este paso 3x (para las condiciones de cultivoGFP-Mtb H37Rv, consulte el Protocolo 3).</li>
	<li>Suspender el pellet bacteriano en 10 ml de RPMI 1640 medio suplementados con 10% de FBS y decantar durante 1 hora a temperatura ambiente para permitir que los agregados bacterianos se sedimenten.</li>
	<li>Recoger el sobrenadante bacteriano y medir OD600 (OD600 debe estar entre 0.6-0.8) y GFP-fluorescencia (valor de RFU) utilizando un lector de microplacas. Calcule el título de la suspensión utilizando una línea de regresión de referencia que muestre el valor de RFU = f (valor de CFU) que se había generado antes del experimento. La concentración típica es de 1 x 108 bacterias/ml.</li>
	<li>Cosechar 6 días de edad de macrófagos humanos primarios a 4 x 105 células/ml en rpmi 1640 medio complementado con 10% de suero bovino fetal (FBS) y 50 ng/ml M-CSF humano recombinante (Para la purificación de células de monocitos de sangre periférica humana y la diferenciación de macrófagos ver Protocolo 4).</li>
	<li>Incubar las células primarias diluidas con bacilos a diferentes MOI, variando de 1-5, en suspensión con temblor leve a 90 rpm durante 2 horas a 37 °C.</li>
	<li>Lave las células infectadas por centrifugación a 350 x g para eliminar las bacterias extracelulares. Después de cada paso de centrifugación, resuspend el pellet en rpmi 1640 medio complementado con 10% FBS. Repita este paso 2x.</li>
	<li>Suspender las células infectadas en medio RPMI 1640 suplementado con FBS al 10% y amikacina a 50 μg/ml e incubar la suspensión con temblores leves durante 1 hora a 37 °C.</li>
	<li>Retire el medio de cultivo celular que contiene amikacina por centrifugación a 350 x g y lave las células infectadas con un medio RPMI 1640 completo complementado con 10% de FBS y 50 ng/ml de M-CSF humano recombinante. Repita una vez.</li>
	<li>Añadir 40 μl de la suspensión de macrófagos infectados en la misma placa de ensayo en el paso 2.1, que ya contiene 10 μl de las diluciones compuestas. La concentración final de DMSO en cada pozo ha alcanzado ahora el 1%.</li>
	<li>Incubar las placas de ensayo durante 5 días a 37 °C en una atmósfera que contenga un 5% deCO2.</li>
	<li>Manche las células vivas con el tinte fluorescente lejano-rojo célula-permeante.</li>
	<li>Cargue la placa en un microscopio confocal automatizado. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Establezca los parámetros de exposición. Registre la fluorescencia de color rojo lejano utilizando láser de excitación de 640 nm con filtro de emisión de 690 nm y fluorescencia GFP utilizando láser de excitación de 488 nm con filtro de emisión de 520 nm. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Seleccione los pozos y los campos en cada pozo que se va a adquirir, que luego se conoce como parámetros de diseño y subdiseño. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Genere el archivo de experimento utilizando los parámetros de los pasos 2.14 y 2.15 y ejecute la adquisición automática. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Transferir imágenes al servidor remoto. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a></li>
	<li>Evalúe las imágenes utilizando el software de análisis de imágenes. Detecte el área celular del canal rojo lejano utilizando el algoritmo de propiedades de intensidad de píxeles y el área bacteriana del canal GFP utilizando el algoritmo de propiedades de intensidad de píxeles (Figura 4B).</li>
</ol>Nota: Este protocolo se puede adaptar para la detección de la biblioteca de mutantes Mtb mediante la sustitución de compuestos por mutantes que expresan una proteína fluorescente (un pozo / un mutante) (Figura 1C, véase también Brodin et al. 4). Los mutantes fluorescentes se siembran por primera vez en pozos (20 μl de suspensión bacteriana por pozo). Las bacterias se recuperan entonces por 30 μl de suspensión celular. Después de la centrifugación a 350 x g durante 1 min, la placa se incuba a 37 °C en una atmósfera que contiene un 5% deCO2. El tiempo de incubación y el MOI dependen del ensayo. Como ejemplo, para la visualización de eventos celulares tempranos como la acidificación del fagosoma, las células pueden infectarse durante 2 horas con MOI que van desde 1-20. Los lisosomas se tiñen con tinte Lysotracker a 2 μM durante 1,5 horas a 37 °C en una atmósfera que contiene 5% de CO2 y luego se fijan con formol al 10% o al 4% de paraformadehído (PFA). Las imágenes confocales se adquieren y finalmente se analizan utilizando scripts de análisis de imágenes con algoritmos apropiados para la detección de lisosomas y la localización subcelular4.
3. Proteína fluorescente verde que expresa mycobacterium tuberculosis H37Rv (GFP-H37Rv) Condiciones de cultivo
Para el almacenamiento a largo plazo, GFP-H37Rv se congelaron en D-PBS (alrededor de 1 x 108 micobacterias por vial).
<ol><li>Resuspend un vial de GFP-H37Rv congelado en un matraz Erlenmeyer que contenga 50 ml de medio caldo 7H9 complementado con enriquecimiento de Middlebrook OADC al 10%, glicerol al 0,5%, tween-80 al 0,05% e higromicina B (50 μg/ml).</li>
	<li>Incubar 8 días a 37 °C.</li>
	<li>Mida OD600 de la cultura GFP-H37Rv.</li>
	<li>Diluir el cultivo de GFP-H37Rv para obtener OD600= 0,1 en medio caldo fresco 7H9 suplementado con enriquecimiento de Middlebrook OADC al 10%, Glicerol al 0,5%, Tween-80 al 0,05% e Higromicina B (50 μg/ml).</li>
	<li>Incubar el GFP-H37Rv a 37 °C durante 8 días más antes de usarlo para el ensayo.</li>
</ol>4. Purificación de células de monocitos de sangre periférica humana de sangre entera o preparación de capa de Buffy
<ol><li>Diluir la bolsa de sangre 2x en 1x D-PBS (libre de MgCl2 y CaCl2)que contiene 1% de FBS.</li>
	<li>Aislar los monocitos por Centrifugación de gradiente de densidad de Ficoll a 400 x g durante 20 min.</li>
	<li>Recoger los monocitos aislados.</li>
	<li>Lavar los monocitos 3x con 1x D-PBS (libre de MgCl2 y CaCl2)que contiene 1% de FBS por centrifugación a 400 x g durante 10 min a temperatura ambiente.</li>
	<li>Concentrar las células hasta 1 x 107 células/ml.</li>
	<li>Purificar monocitos usando perlas magnéticas CD14 de acuerdo con el protocolo del fabricante (ver Materiales).</li>
	<li>Después de la purificación de los monocitos CD14, sembraron las células a 1,5 x10 6 células/ml en RPMI 1640 complementadas con 10% de FBS y 40 ng/ml de factor estimulante de colonias de macrófagos humanos (hM-CSF) e incubadas durante 4 días a 37 °C en una atmósfera que contiene 5% de CO2.</li>
	<li>Después de 4 días sustituir el medio por RPMI 1640 fresco complementado con 10% de FBS y 40 ng/ml de hM-CSF e incubado durante 2 días a 37 °C en una atmósfera que contiene un 5% deCO2.</li>
	<li>Después de 6 días, las células se pueden utilizar para el ensayo.</li>
</ol>

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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=Confocal-based+method+for+quantification+of+diffusion+kinetics+in+microwell+plates+and+its+application+for+identifying+a+rapid+mixing+method+for+high-content/throughput+screening.">Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.</a> J. Biomol. Screen. 15, 138-147 (2010).</li></ol>

No se declaran conflictos de intereses.

Se describen aquí los métodos necesarios para un ensayo fenotípico utilizando una cepa de Mtb H37Rv que expresa GFP para infectar las células huésped etiquetadas fluorescentemente, lo que lo hace apropiado para pantallas de alto contenido / alto rendimiento. Este protocolo podría aplicarse a una amplia gama de compuestos, sondas fluorescentes y mutantes mtb. Para cada protocolo descrito anteriormente, se podrían realizar pasos de fijación e inmunoetiquetado antes de la adquisición de imágenes....

Entre los patógenos infecciosos emergentes y reemergentes reportados durante los últimos años, Mycobacterium tuberculosis (Mtb)ocupa un lugar destacado siendo responsable de 1,4 millones de muertes y 8,7 millones de nuevas infecciones en 2011 (Informe Mundial de Tuberculosis 2012, www.who.int/topics/tuberculosis/en/). A pesar de la disponibilidad de terapias multidrogas, el número de personas infectadas sigue en aumento y el Mtb multirresistente (MDR) y ampliamente farmacorresistente (XDR) se está extendiendo rápidamente por todo el mundo1. Por otra parte, si se tiene en cuenta la presencia de antígenos mtb, es evidente que un tercio de la población mundial se considera que está infectada latentemente por mtb. Estadísticamente, en un caso de cada diez, hay evolución hacia la forma activa de la enfermedad con síntomas clínicos posteriores2. Por lo tanto, se necesitan con urgencia nuevos medios para luchar contra el Mtb. En este contexto, desarrollamos un ensayo fenotípico visual in vitro basado en el seguimiento de la invasión de Mtb y la multiplicación en las células huésped mediante microscopía de fluorescencia confocal automatizada3. La adaptación del ensayo en placas de microtíter de 384 pozos en combinación con la adquisición y el análisis automatizados de imágenes, permitió la detección de alto contenido / alto rendimiento (HC / HTS) de bibliotecas de escala media de compuestos, siRNAs y mutantes bacterianos. La investigación de una biblioteca amplia del genoma RNAi en este análisis fenotípico permitió así la identificación de los anfitrión-factores dominantes implicados en tráfico de Mtb y la réplica intracelular pero también la aclaración de los anfitrión-caminos explotados por el bacilo del tubérculo. Otra adaptación de este análisis fenotípico particular estaba para la identificación de los factores bacterianos esenciales a la persistencia intraphagosomal del Mtb. Por ejemplo, la detención de la maduración del fagosoma se considera como uno de los principales mecanismos que facilita la supervivencia y replicación de Mtb en los macrófagos. El monitoreo de la localización subcelular de mutantes knock-out mtb en compartimentos fluorescentemente etiquetados como ácidos permitió la identificación de genes bacterianos involucrados en el proceso de supervivencia4. Finalmente, la imagen de alto contenido de Mtb también ofrece un excelente método para cuantificar la eficiencia de los fármacos para inhibir diversos fenómenos como el crecimiento bacteriano intracelular3. En conjunto, este tipo de ensayo fenotípico de alto rendimiento permite acelerar el descubrimiento de fármacos contra la TB y los datos recogidos por estos diferentes enfoques contribuyen a una mejor comprensión de la manipulación del huésped ejercida por Mtb.

<table border="0" cellpadding="0" cellspacing="0" width="1010">
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		<col />
		<col /> 
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#181;clear-plate black, 384 well</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781091</td>
			<td style="width:388px;">127.8/86/15 MM with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CellCarrier 384 well plate</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;">6007550</td>
			<td>Black, Clear Bottom, with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">V-bottom white , 384 well-plate</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
			<td style="width:160px;">Corning</td>
			<td style="width:175px;">3345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMax</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">13778150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">34943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI 1640 + GlutaMAX-I</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">61870-010</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [-]MgCl2/[-]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-094</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [+]MgCl2/[+]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-091</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Fetal bovine serum</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">2610040-79</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">FICOLL PAQUE PLUS</td>
			<td style="width:160px;">DUTSCHER</td>
			<td style="width:175px;">17-1440-03</td>
			<td>Ficoll for Peripherical Blood Monocyte Cells purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD14 MicroBeads, human</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-050-201</td>
			<td>Purification of CD14+ Monocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Human M-CSF, premium gr. (1000 &#956;g)</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-096-493</td>
			<td>Macrophage Colony Stimulating Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS Columns</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-042-401</td>
			<td>Columns for CD14+ Monocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tween 80</td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">2002-A</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glycerol high purity </td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">50405-EX</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;"> Middlebrook OADC enrichment</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">211886</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7H9</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">W1701P</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Versene 1X</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">15040033</td>
			<td>Non enzymatic cell dissociation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DAPI</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hoechst 33342</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">H3570</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syto60</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">S11342</td>
			<td>Nuclei/cytoplasm dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formalin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">HT5014</td>
			<td>Cell fixation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siRNA targeting Dectin-1</td>
			<td style="width:160px;">Santa-Cruz</td>
			<td style="width:175px;">sc-63276</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">*siGenome* Non targeted siRNA pool </td>
			<td style="width:160px;">Dharmacon</td>
			<td style="width:175px;">D-001206-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Rifampicin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">R3501</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoniazid (INH)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">I3377-50G</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hygromycin B</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">10687-010</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Amikacin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">A1774</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Automated Confocal Microscope OPERA</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Columbus 2.3.1 Server Database</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Data transfert, storage and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acapella 2.6 software</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image-based analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GraphPad Prism5 software</td>
			<td style="width:160px;">GraphPad </td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:287px;">Excel 2010</td>
			<td style="width:160px;">Microsoft</td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening

A pesar de la disponibilidad de terapia y vacuna, la tuberculosis (TB) sigue siendo una de las infecciones bacterianas más mortales y extendidas del mundo. Desde hace varias décadas, la explosión repentina de cepas multirresistentes y ampliamente resistentes a los medicamentos es una grave amenaza para el control de la tuberculosis. Por lo tanto, es esencial identificar nuevas dianas y vías críticas para el agente causal de la tuberculosis, Mycobacterium tuberculosis (Mtb)y buscar nuevos productos químicos que podrían convertirse en fármacos contra la tuberculosis. Un enfoque es establecer métodos adecuados para las pantallas genéticas y químicas de bibliotecas a gran escala que permitan la búsqueda de una aguja en un pajar. Con este fin, desarrollamos un ensayo fenotípico que se basa en la detección de Mtb marcado fluorescentemente dentro de las células huésped etiquetadas fluorescentemente utilizando microscopía confocal automatizada. Este ensayo in vitro permite una cuantificación basada en imágenes del proceso de colonización de Mtb en el huésped y se optimizó para el formato de microplaca de 384 pozos, que es adecuado para pantallas de bibliotecas de mutantes de siRNA, compuestos químicos o Mtb. Las imágenes se procesan entonces para el análisis multiparamétrico, que proporciona la lectura hacia fuera que infiere en la patogenesia del Mtb dentro de las células huesped.

Detección de siRNA en todo el genoma de alto rendimiento
Mtb es capaz de colonizar las células inmunes in vitro, así como varias otras células epiteliales pulmonares. Por ejemplo, Mtbes capaz de infectar y dañar las células epiteliales A549 que se utilizan comúnmente como modelo para los neumocitos humanos tipo II5-7. La dectina-1 fue reportada como un receptor de células huésped implicado en la captación de Mtb, la respuesta proinflamatori...

Watch this Scientific Journal Video about A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening at JoVE.com

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Aquí, se describe un ensayo fenotípico aplicable a las pantallas de alto rendimiento / alto contenido de ARN sintético de interferencia pequeña (siRNA), compuesto químico, y bibliotecas de mutantes de Mycobacterium tuberculosis. Este método se basa en la detección de tuberculosis de la micobacteria fluorescentemente etiquetada dentro de la célula huésped etiquetada fluorescentemente usando microscopia confocal automatizada.

Un Ensayo Fenotípico Microscópico Para La Cuantificación De Micobacterias Intracelulares Adaptadas Para La Detección De Alto Rendimiento / Alto Contenido

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

a-microscopic-phenotypic-assay-for-quantification-intracellular

Research

JoVE Journal

Immunology and Infection

7.6K vistas.  Université de Lille. Aquí, se describe un ensayo fenotípico aplicable a las pantallas de alto rendimiento / alto contenido de ARN sintético de interferencia pequeña (siRNA), compuesto químico, y bibliotecas de mutantes de Mycobacterium tuberculosis. Este método se basa en la detección de tuberculosis de la micobacteria fluorescentemente etiquetada dentro de la célula huésped etiquetada fluorescentemente usando microscopia confocal automatizada.

Video: A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Un ensayo funcional de sangre completa para medir la viabilidad de las micobacterias, usando Reporter-Gene Tagged BCG o M. tuberculosis (BCG Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Investigación

Inmunología e Infección

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

A Parasite Rescate y Transformación de ensayo para la detección antileishmanial contra intracelular<em&gt; Leishmania donovani</em&gt; Amastigotes en THP1 Humanos aguda línea celular de leucemia monocítica

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Un método de screening para la evolución dirigida de enzimas termoestables bacteriolítica

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

La visualización de la resazurina prueba de carbón en agar para semi-cuantitativa, Enumeración de mediano rendimiento de Micobacterias

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Un simple ensayo de fluorescencia para la cuantificación de los neutrófilos extracelular canina Trampa de lanzamiento

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Una de linfocitos ensayo basado en la fluorescencia adecuado para el cribado de alto rendimiento de pequeñas moléculas

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Cuantificación del crecimiento intracelular dentro de los macrófagos es un rápido y confiable método para evaluar la virulencia de los parásitos de Leishmania

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

Un alto rendimiento, alto contenido líquido y basada en C. elegans patosistema

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...