Estamos agradecidos a Kaz Nagaosa y Akiko Shiratsuchi por su consejo.

1. Preparación <ol><li> Preparación de placas de agar de jugo de uva fresca <ol><li> Añadir 100 ml de agua a 4,4 g de agar y calentar la mezcla en un horno de microondas para disolver el agar. </li><li> Añadir 80 ml de jugo de uva fresca, 5 ml de ácido acético y 5 ml de etanol en solución de agar. </li><li> Verter aproximadamente 1,5 ml de la solución con una pipeta en cada portaobjetos de vidrio y dejar que se solidifique. </li></ol></li><li> Preparación de placas de 6 cm de placa de agarosa <ol><li> Añadir 50 ml de agua a 0,5 g de agarosa y calentar la mezcla en un horno de microondas para disolver la agarosa. </li><li> Verter aproximadamente 2,5 ml de solución de agarosa en un plato de 6 cm con una pipeta. </li></ol></li></ol> 2. Etapa 16 Colección de embriones <ol><li> Recoger las moscas adultas, y agregar 200 hembras y 200 machos en un tubo cónico de 50 ml. </li><li> Ponga un plato de agar de jugo de uva fresco en la levadura en el tubo de 50 ml y cierre con una esponja capag. </li><li> Incubar las moscas a 16 ° C en la luz durante 2 - 3 días. Cambiar la placa una vez al día. </li><li> Mover las moscas a la oscuridad a 25 ° C durante 1 h. </li><li> Cambiar el plato viejo a uno nuevo sin levadura. Permitir que las moscas pongan huevos durante 2 h. </li><li> Recoger la placa e incubar a 16 ° C durante 26 h. </li><li> Recoger los embriones de la placa con un pincel en 1 mL de PBS que contiene 0,2% (v / v) de Triton X-100. </li><li> Lavar los embriones dos veces con 1 mL de PBS que contiene 0,2% (v / v) de Triton X-100. </li><li> Con el fin de eliminar el corion, agregar 1,2 ml de solución de hipoclorito de sodio (2,2 - 3,4% Cl) a los embriones durante 3 min. </li><li> Lavar los embriones 4 veces con 1 mL de PBS que contiene 0,2% (v / v) de Triton X-100. </li><li> Colocar los embriones en placas de 6 cm de placa de agarosa. Recoge los embriones de la etapa 16 bajo un microscopio como describe Roberts 20 . Recoja aproximadamente 50 embriones en un tubo de prueba micro de Treff de 1,5 ml con una micropipeta. </li></ol> 3. PrepaRación de células embrionarias <ol><li> Lavar los embriones dos veces con 150 μL de PBS. </li><li> Homogeneizar los embriones 30 veces en un tubo de micro test de 1,5 ml y mezclador de pellets en presencia de 200 μl de colagenasa (0,25% (p / v)) en PBS. </li><li> Disperse las células embrionarias pipeteando 10 veces, y mover la suspensión celular a un tubo de 1,5 ml. Incubar las células a 37 ° C durante 1 min en un baño de agua. Repita este paso dos veces. </li><li> Añadir 800 μl de PBS a la suspensión celular y centrifugar a 1400 x g a 4 ° C durante 5 min. </li><li> Eliminar el sobrenadante y añadir 200 μ l de tripsina (0,25% (p / v)) en PBS a las células. Disperse las células embrionarias pipeteando 50 veces. </li><li> Filtrar la suspensión celular con un filtro de células de 70 μm. </li><li> Con el fin de detener la actividad tripsina, añadir 40 μ l de suero fetal bovino inactivado por calor a la suspensión de células filtradas. Añadir 800 μl de PBS y centrifugar a 1.400 x g a 4 ° C durante 5 min. </li><li> Eliminar los supernatosNt, suspender las células precipitadas en 200 μl de PBS y centrifugar a 1400 x g a 4 ° C durante 5 min. </li><li> Se retira el sobrenadante y se suspenden las células precipitadas en 30 μl de PBS. </li><li> Montar la suspensión de células preparada en diapositivas de cristal revestidas con aminopropil trietoxisilano, y esperar 10-15 minutos hasta que las células se adhieran a las diapositivas de vidrio. </li><li> Retire la solución restante en los portaobjetos de vidrio con una pipeta. Montar 60 - 70 μL de PBS que contenga PFA al 4% (p / v) (paraformaldehído) sobre las células durante 10 - 15 min para la fijación. </li><li> Retire la solución de fijación y coloque los portaobjetos de vidrio en PBS durante más de 1 min. </li></ol> 4. Tinción de los hemocitos <ol><li> La inmunotinción con un anticuerpo anti-Croquemort <ol><li> Se empapan en serie los portaobjetos de vidrio en metanol durante 10 min, en PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min y luego en PBS durante 10 min. </li><li> Montar 20 μl de suero de cerdo entero al 5% (v / v) en PBSNg 0,2% (v / v) de Triton X-100 en las células para el bloqueo. Incubar los portaobjetos a temperatura ambiente durante 20 min. </li><li> Eliminar la solución de las diapositivas de vidrio y montar 20 μl de antisuero anti-Croquemort 19 , 21 (0,1% (v / v)) en PBS que contiene 0,2% (v / v) de Triton X-100 en las células. Incubar los portaobjetos a 4 ° C durante la noche. </li><li> Se empapan los portaobjetos de vidrio en PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min 5 veces. </li><li> Remoje los portaobjetos de vidrio en PBS durante 10 min. </li><li> Colocar 20 μl de IgG anti-rata marcada con fosfatasa alcalina (0,5% (v / v)) en PBS que contiene 0,3% (v / v) de suero de cerdo entero Triton X-100 y 5% (v / v) Temperatura durante 1 h. </li><li> Se empapan los portaobjetos de vidrio en PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min 5 veces. </li><li> Remojar portaobjetos de vidrio en tampón que contenía 100 mM Tris-HCl, pH 9,5, NaCl 100 mM, y MgCl 50 mM 2 durante 10 min. </li><li> La solución de sustrato de fosfatasa de montaje que contiene 0,23 mg / ml de 5-bromo-4cloro-3-indolil-fosfato (BCIP), 0,35 mg / ml nitro azul tetrazolio (NBT), 100 mM Tris-HCl, pH 9,5, NaCl 100 mM, y MgCl 50 mM 2 en las células. </li><li> Observar las células bajo un microscopio para una tinción adecuada. Cuando aparecen fuertes señales púrpuras en los gránulos de los hemocitos, se retira la solución del sustrato y se empapa las diapositivas de vidrio en un tampón que consiste en Tris-HCl 10 mM, pH 8,5 y EDTA 1 mM. Se recomienda una tinción de aproximadamente 5 a 10 minutos. </li></ol></li><li> La inmunotinción con un anticuerpo anti-GFP (otra opción para la tinción de hemocitos) <ol><li> Se empapan en serie las láminas de vidrio en metanol durante 10 min, PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min y PBS durante 10 min. </li><li> Monte 20 μl de suero entero de cerdo al 5% (v / v) en PBS que contenga Triton X-100 al 0,2% (v / v) en las células para bloqueo. Incubar los portaobjetos a temperatura ambiente durante 20 min. </li><li> Eliminar la solución en portaobjetos de vidrio y montar 20 μl del anticuerpo anti-GFP de ratón (1% (v / v)) / PBS cObteniendo 0,2% (v / v) de Triton X-100 en las células. Incubar los portaobjetos a temperatura ambiente durante la noche. </li><li> Se empapan los portaobjetos de vidrio en PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min 5 veces. </li><li> Remoje los portaobjetos de vidrio en PBS durante 10 min. </li><li> Colocar 20 μl de IgG anti-ratón marcado con fosfatasa alcalina (0,5% (v / v)) en PBS que contiene 0,3% (v / v) de suero de cerdo entero de Triton X-100 y 5% (v / v) Temperatura durante 1 h. </li><li> Se empapan los portaobjetos de vidrio en PBS que contiene 0,2% (v / v) de Triton X-100 durante 10 min 5 veces. </li><li> Remojar portaobjetos de vidrio en tampón constituido por 100 mM Tris-HCl, pH 9,5, NaCl 100 mM, y MgCl 50 mM 2 durante 10 min. </li><li> Montar la solución de sustrato de fosfatasa que contiene 0,23 mg / ml de BCIP, 0,35 mg / ml de NBT, 100 mM Tris-HCl, pH 9,5, NaCl 100 mM, y MgCl 50 mM 2 en las células. </li><li> Observar las células bajo un microscopio para una tinción adecuada. Cuando las señales de color púrpura aparecen en hemocitos enteros, retire la solución de sustrato y pase las diapositivas en un tampón que consiste enDe Tris-HCl 10 mM, pH 8,5 y EDTA 1 mM. Se recomienda una tinción de aproximadamente 10 - 15 min. </li></ol></li></ol> 5. Stain Apoptotic Cells de TUNEL <ol><li> Remoje los portaobjetos de vidrio en PBS durante 5 min. </li><li> Repita con el nuevo PBS. </li><li> Monte el tampón de equilibrio (véase la Tabla de Materiales) sobre las células durante 10 min. </li><li> Retire la solución de los portaobjetos de vidrio y monte 20 μL de solución de TdT que contenga 5 μL de desoxinucleotidil transferasa terminal y 15 μL de tampón de reacción en las células a 37 ° C durante 1 h. </li><li> Retirar la solución de los portaobjetos de vidrio y empapar las diapositivas en un tampón que consiste en 0,5 mL de STOP / tampón de lavado y 17 mL de agua. </li><li> Remoje las diapositivas en PBS durante 5 min 3 veces. </li><li> Montar 20 μL de Anti-Digoxigenina-Peroxidasa en las células a temperatura ambiente durante 30 min. </li><li> Remoje las diapositivas de vidrio en PBS durante 5 min 4 veces. </li><li> Sumerja los portaobjetos de vidrio en solución de sustrato de peroxidasa que consta de 30 ml de Tris-HCl 50 mM, pH 7,5 conteniendoga completo espátula micro de 3,3&#39;-diaminobencidina tetrahydrichloride (DAB), y 0,002% (v / v) H 2 O 2 durante 30 s. </li><li> Repita el paso 5.9 hasta que las células apoptóticas estén manchadas de color marrón. Sumerja los portaobjetos de vidrio en agua para detener la reacción de la peroxidasa. </li><li> Encierre las células con agua para la observación. </li></ol> 6. Medir el nivel de fagocitosis de las células apoptóticas <ol><li> Observe las muestras bajo un microscopio óptico para evaluar el nivel de fagocitosis. Contar células positivas a Croquemort o células GFP positivas como hemocitos, células positivas a TUNEL como células apoptóticas y células positivas dobles como hemocitos fagocitantes. </li><li> El índice fagocítico se define como el número de células dobles positivas al número total de células positivas a Croquemort o células positivas para GFP. Se recomienda observar más de 300 hemocitos. </li></ol>

<ol>
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<html> En este documento se describió un ensayo de fagocitosis utilizando embriones de Drosophila . Mediante el uso de células embrionarias dispersas para medir la fagocitosis cuantitativamente, los hemocitos, los fagocitos profesionales de Drosophila , son inmunocorados para el marcador de hemocitos Croquemort o srpHemo- dirigido GFP, y las células apoptóticas son detectadas por TUNEL en este protocolo. El nivel de fagocitosis se expresa como un índice fagocítico contando el número tota...

En animales metazoarios, por ejemplo el nematodo Caenorhabditis elegans , la mosca de la fruta Drosophila melanogaster , y ratones y humanos, un gran número de células sufren apoptosis durante el desarrollo para modelar sus cuerpos y en la edad adulta para mantener la homeostasis 1 , 2 . Las células apoptóticas necesitan ser eliminadas rápidamente porque inducen inflamación en los tejidos circundantes liberando materiales intracelulares inmunogénicos si no se eliminan por completo 3 . Para facilitar la remoción rápida, las células apoptóticas presentan las denominadas señales de comer-me que son reconocidas por los receptores de engulfment de los fagocitos, y son eliminadas por fagocitosis 3 , 4 , 5 , 6 . Así, la fagocitosis desempeña un papel crucial en el mantenimiento de la homeostasis del huésped, y por lo tanto, elucidar la molécula<html> Los mecanismos subyacentes a la fagocitosis de las células apoptóticas son importantes. Los mecanismos responsables de la fagocitosis de las células apoptóticas parecen ser evolutivamente conservadas entre las especies en los nematodos, moscas y ratones [ 7] . Actualmente se dispone de varios ensayos de fagocitosis para evaluar la absorción de células apoptóticas en estos animales modelo. En C. elegans , 131 células somáticas sufren muerte celular programada durante el desarrollo, y los cadáveres de células son phagocytosed por las células vecinas, que son no fagocitos profesionales [ 8] . Por lo tanto, contar el número de cadáveres de células restantes en C. elegans indica el nivel de fagocitosis in vivo . En la búsqueda de mutantes de nematodos que muestran un mayor número de células muertas, varios genes necesarios para la fagocitosis han sido identificados y caracterizados genéticamente 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . Los ensayos de fagocitosis ex vivo con fagocitos de cultivo primario, generalmente macrófagos, se usan a menudo en ratones. Las células apoptóticas se preparan usando líneas celulares tales como células Jurkat, y se mezclan con fagocitos primarios. Después de una incubación durante varias horas, se cuenta el número total de fagocitos y de fagocitos engullantes para evaluar el nivel de fagocitosis. Como una modificación sofisticada de este método, el grupo de Nagata desarrolló un ensayo de fagocitosis ex vivo con células que expresan un ICAD resistente a caspasa (inhibidor de la DNasa activada por caspasa), en el que las células apoptóticas no sufren fragmentación apoptótica del ADN, Las células son fagocitadas. Cuando estas células se usan como dianas apoptóticas en un ensayo de fagocitosis, sólo el ADN de las células apoptóticas engullidas está fragmentado y teñido por marcado de extremos dUTP mediados por TdT (TUNEL). Por lo tanto, el leveL de la absorción de células apoptóticas se mide por el recuento de señales TUNEL en una mezcla de fagocitos y células apoptóticas [ 13] . En D. melanogaster , los fagocitos profesionales denominados hemocitos, los macrófagos de Drosophila , son responsables de la fagocitosis de las células apoptóticas 14 , 15 . Además de los ensayos de fagocitosis in vitro con líneas celulares de cultivo, se dispone de ensayos de fagocitosis in vivo con embriones enteros de Drosophila . Los embriones de Drosophila son una poderosa herramienta para examinar el nivel de engrosamiento de células apoptóticas debido a que muchas células sufren apoptosis y son fagocitadas por hemocitos durante el desarrollo embrionario 14 , 15 , 16 . Un ejemplo de un ensayo de fagocitosis in vivo es el método desarrollado por el grupo de Franc. En su método, los hemocitos sonProtegido por la inmunotinción de peroxidasina, un marcador de hemocito, las células apoptóticas se tiñen utilizando el tinte nuclear, 7-amino actinomicina D en embriones enteros de Drosophila , y el número de células positivas dobles se cuenta como una señal de fagocitosis 17 . Otro ejemplo de un ensayo de fagocitosis en embriones se basa en el concepto de método de Nagata descrito anteriormente; Sin embargo, la fagocitosis in vivo se evalúa utilizando los embriones de dCAD ( Drosophila caspasa-DNasa activada) moscas mutantes [ 18 , 19] . Estos ensayos de fagocitosis in vivo son útiles para observar directamente la fagocitosis in situ . Sin embargo, las dificultades están asociadas con la exclusión de cualquier sesgo posible en el paso de contar células fagocitosas porque es difícil observar todos los fagocitos y células apoptóticas en embriones enteros debido a su grosor. Para superar esta limitación, desarrollamosUn nuevo ensayo de fagocitosis en embriones de Drosophila . En nuestro método, para poder contar fácilmente los hemocitos fagocitados, se homogeneizan embriones enteros para preparar células embrionarias dispersas. Los fagocitos son detectados por la inmunotinción de un marcador de fagocitos, y las células apoptóticas son detectadas por TUNEL con estas células embrionarias dispersas. El uso de células embrionarias dispersas nos permite medir los niveles de fagocitosis in vivo como si se realizara un ensayo de fagocitosis in vitro que cuantificara con precisión el nivel de fagocitosis. Todos los genotipos de moscas pueden emplearse en este ensayo si se desarrollan hasta la etapa 16 de los embriones 20 , la etapa de desarrollo en la que la depuración de las células apoptóticas por fagocitosis es la más abundante. Este método tiene la ventaja de evaluar cuantitativamente el nivel de fagocitosis y, por tanto, puede contribuir a la identificación de nuevas moléculas implicadas en la fagocitosis de células apoptóticas in vivo .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>whole swine serum</td>
			<td>MP Biomedicals</td>
			<td>55993</td>
			<td>For bloking</td>
		</tr>
		<tr>
			<td>Treff micro test tube(easy fit)&#160; Dnase, Rnase free tube, 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 4625. 9. 01</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>pellet mixer&#160; 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 7339. 9. 03</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C-0130</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher SCIENTIFIC</td>
			<td>27250-018</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Kpl Anti-Rat IgG (H+L) Ab MSA, AP</td>
			<td>KPL</td>
			<td>475-1612</td>
			<td>secondary antibody for 
			stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro- 
			3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td>BCIP, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>nitro blue tetrazolium</td>
			<td>Roche</td>
			<td>11383213001</td>
			<td>NBT, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Anti-Croquemort antibody</td>
			<td></td>
			<td></td>
			<td>described previously in Manaka et al, J. Biol. Chem., 279, 48466-48476</td>
		</tr>
		<tr>
			<td>&#160;Anti-GFP 
			from mouse IgG1&#954; (clones 7.1 and 13.1)&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG-AP Conjugate</td>
			<td>Bio-Rad</td>
			<td>170-6520</td>
			<td>secondary antibody for stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>Apop Tag Peroxidase In Situ Apoptosis Detection Kit</td>
			<td>Millipore</td>
			<td>S7100</td>
			<td>For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.</td>
		</tr>
		<tr>
			<td>3,3&#39;-diaminobenzidine 
			tetrahydrichloride</td>
			<td>nacalai tesque</td>
			<td>11009-41</td>
			<td>DAB, For staining of apoptoitc cells</td>
		</tr>
		<tr>
			<td colspan="2">Table of Fly Strains</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td></td>
			<td></td>
			<td>Control flies, described in Freeman et al., Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>drpr&#916;5</td>
			<td></td>
			<td></td>
			<td>drpr mutant, described in Freeman et al, Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>Itgbn2</td>
			<td></td>
			<td></td>
			<td>Itgbn mutant, described in Devenport et al., Development, 131, 5405-5415</td>
		</tr>
		<tr>
			<td>srpHemoGAL4 UAS-EGFP</td>
			<td></td>
			<td></td>
			<td>described in Br&#252;ckner et al., Dev. Cell., 7, 73-84</td>
		</tr>
		<tr>
			<td>UAS-drpr-IR</td>
			<td>VDRC</td>
			<td>4833</td>
			<td>-</td>
		</tr>
		<tr>
			<td>UAS-Itgbn-IR</td>
			<td>NIG-fly</td>
			<td>1762R-1</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

phagocytosis assay for apoptotic cells in drosophila embryos

Los mecanismos moleculares subyacentes a la fagocitosis de las células apoptóticas necesitan ser aclarados con más detalle debido a su papel en las enfermedades inmunes e inflamables intratables. En este documento se desarrolló un método experimental para investigar la fagocitosis cuantitativamente utilizando la mosca de la fruta Drosophila , en el que la red de genes que controla las reacciones de engulfment se conserva evolutivamente de los mamíferos. Con el fin de detectar con precisión y contando engullir y no engolfar los fagocitos con animales enteros, embriones de Drosophila se homogeneizaron para obtener células dispersadas incluyendo fagocitos y células apoptóticas. El uso de células embrionarias dispersas nos permite medir los niveles de fagocitosis in vivo como si se realizara un ensayo de fagocitosis in vitro en el que es posible observar todos los fagocitos y células apoptóticas en embriones enteros y cuantificar con precisión el nivel de fagocitosis. Se confirmó que este método reproduce los de estudios previosQue identificó los genes necesarios para la fagocitosis de las células apoptóticas. Este método permite analizar la absorción de células muertas y, cuando se combina con la poderosa genética de Drosophila , revelará las complejas reacciones fagocíticas que comprenden la migración, el reconocimiento, el engolfamiento y la degradación de las células apoptóticas por los fagocitos.

Con el fin de examinar la fagocitosis de las células apoptóticas, Drosophila embriones de desarrollo etapa 16 fueron recogidos y preparados como células dispersas. Los hemocitos, macrófagos de Drosophila , se tiñeron por inmunocitoquímica para el marcador de hemocito &quot;Croquemort&quot; 17 , 22 usando un anticuerpo específico 19 , 21 , y las c?...

Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

En el presente documento se describe un ensayo de fagocitosis utilizando las células embrionarias dispersas de Drosophila . Nos permite cuantificar con facilidad y precisión los niveles de fagocitosis in vivo , e identificar nuevas moléculas necesarias para la fagocitosis de las células apoptóticas.

Ensayo de Fagocitosis para Células Apoptóticas en<em&gt; Drosophila</em&gt; Embriones

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

phagocytosis-assay-for-apoptotic-cells-in-drosophila-embryos

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Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Unit 1

Cell Death

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9.8K vistas.  Kanazawa University.  En el presente documento se describe un ensayo de fagocitosis utilizando las células embrionarias dispersas de Drosophila . Nos permite cuantificar con facilidad y precisión los niveles de fagocitosis in vivo , e identificar nuevas moléculas necesarias para la fagocitosis de las células apoptóticas. 

Video: Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi detección de los factores del huésped implicados en Vaccinia Infección del virus utilizando Drosophila Celdas

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Investigación

Inmunología e Infección

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

La observación directa de la fagocitosis y la formación de NET-por los neutrófilos en los pulmones infectados con dos fotones Microscopía

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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

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

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

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

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

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Cuantitativo<em&gt; In vitro</em&gt; Ensayo para medir la adhesión de neutrófilos al Activado Primaria células endoteliales microvasculares humanas en condiciones estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

A gran escala de purificación de porcino o bovino fotorreceptor Outer Segmentos para Fagocitosis ensayos sobre células pigmento retiniano epitelial

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

El uso de una monocapa de monocitos Ensayo para evaluar la fagocitosis mediada por el receptor Fc gamma

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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

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

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

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

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

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Fagocitosis del macrófago alveolar y remoción de bacterias en los ratones

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Evaluar el Cellular la respuesta inmune de la mosca de la fruta, Drosophila melanogasterutilizando un ensayo de fagocitosis en Vivo

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Ensayo de Fagocitosis para Células Apoptóticas en

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