Este trabajo fue apoyado por el Instituto Nacional de Alergias y Enfermedades Infecciosas de Grant R01AI079253 (a BHS) y por el Instituto Nacional del Cáncer Centro del Cáncer de subvención de apoyo CA016056 al Instituto del Cáncer Roswell Park.

Todos los procedimientos realizados en animales en este estudio fueron aprobados por el Comité de Cuidado de Animales y el empleo en el Instituto del Cáncer Roswell Park y cumplan con todo el estado, federal, y los Institutos Nacionales de la normativa de salud. 1. In vivo de macrófagos Etiquetado Nota: PHK26 es un colorante lipófilo que se tomará por las células fagocíticas, tanto en la circulación y en los tejidos cuando se administra por vía intravenosa (iv). PKH26 se utilizó para el etiquetado in vivo para minimizar la cantidad de manipulación posterior a la cosecha de los macrófagos. PKH-26 tiene la ventaja de acumular de manera estable en los macrófagos alveolares durante periodos prolongados. Esperar 10 días después de la administración permite la limpieza de todas las células de la etiqueta de la circulación dejando sólo macrófagos alveolares de forma estable marcada con PKH26 8, 9. PKH26 es un fluorocromo rojo que tiene de excitación (551 nm) y emisión (567 nm) características compatiblescon los sistemas de detección de rodamina o ficoeritrina. Cualquier procedimiento de etiquetado puede introducir artefactos, incluyendo la pérdida de viabilidad. Sin embargo, puesto que utilizamos el mismo enfoque para el etiquetado de los macrófagos en diferentes genotipos de ratón (por ejemplo, WT frente a la NADPH-oxidasa deficiente), los efectos de estos artefactos serían uniforme. <ol><li> Diluir la solución madre de PKH26 (1 x 10 -3 M en etanol) 1:05 en diluyente C proporcionada por el fabricante. </li><li> De carga 100 l de PKH26 diluida en una jeringa de 1/2 ml de insulina (29 g x 1/2 &quot;) para cada ratón. </li><li> Administrar a los ratones por inyección iv (vena de la cola o bien retro-orbital) al menos 10 días antes de la cosecha. </li></ol> 2. Recolección de los macrófagos alveolares de lavado broncoalveolar (BAL) El número de ratones a utilizar para un experimento puede variar. Un rendimiento típico de un ratón no estimulada está en el intervalo de 3-5 x 10 5 macrófagos alveolares, sin embargo este número puede variar en gran medida based de factores tales como el tamaño del ratón, la experiencia de la persona que realiza el lavado, y el número de instilaciones de líquido de lavado y el volumen retirado de los pulmones. En este protocolo de lavado pulmonar se realiza con una cavidad torácica cerrada que junto con repetidos lavados de 1 ml ayuda a aumentar el rendimiento de células. <ol><li> La eutanasia del ratón mediante la administración de una dosis letal de anestésico Avertin (12,5 mg) por vía intraperitoneal (IP) usando condiciones asépticas para mantener la solución estéril. </li><li> Pulverizar ratón a fondo con etanol al 70%. </li><li> Retire la piel desde el extremo craneal de animales exponiendo el tórax (Figura 1A). <ol><li> Hacer un pequeño corte en la piel justo debajo del diafragma. </li><li> Inserte el dedo en el corte y tire de la piel lejos del extremo craneal del animal (Figura 1A). </li><li> Empuje ligeramente hacia arriba en la cabeza, que se extiende la parte ventral del cuello (fig. 1A). </li></ol></li><li> Corte un pequeño agujero a través de la caja torácicaen el lado derecho del tórax para desinflar los pulmones y también permitir la visualización de los pulmones durante los siguientes pasos. </li><li> Busque la tráquea, y cuidadosamente diseccionar el tejido circundante. <ol><li> Retire las glándulas salivales, los músculos esternohioideo y laringe, etc con pinzas curvas. </li><li> Levante con cuidado con unas pinzas curvas y cortar con tijeras el tejido delgado que cubre la tráquea (adventicia) que revela la estructura del cartílago anillado subyacente. </li></ol></li><li> Inserte catéter en la tráquea. <ol><li> Con unas pinzas curvas, colocar una sutura detrás (dorsal a) la tráquea y tirar de una longitud de 10 cm a través de la abertura. </li><li> Comenzando por encima del cricoides (el anillo engrosado del cartílago) guiar cuidadosamente el catéter 22 G por la tráquea parando donde la tráquea se desaparece detrás del esternón. </li><li> Firmemente atar sutura alrededor de la tráquea y el catéter para un ajuste perfecto. Retire la aguja del catéter. </li></ol></li><li> Ensamble de 4 vías llave de paso. <ol><li> Llenaruno 6 ml de jeringa con DPBS, 1% de FBS y se unen a 4 vías llave de paso tal como se indica en la Figura 1B. </li><li> Adjuntar un vacío 6 ml jeringa en la posición indicada en la 4 vías llave de paso (Figura 1B). </li><li> Conecte el puerto abierto en el 4 vías llave de paso para catéter (Figura 1B). Tenga cuidado de no desconectar el catéter de la tráquea. </li></ol></li><li> Recoger el líquido del lavado de los pulmones. Nota: Los investigadores pueden optar por usar menores volúmenes de líquido de lavado para reducir al mínimo la posibilidad de daños en el tejido. Es importante que el mismo enfoque puede aplicarse de manera coherente en todos los grupos de ratones. <ol><li> Gire la manija de la llave de paso por lo que la jeringa vacía se cierra y se inyecte lentamente ~ 1 ml de DPBS, 1% de SFB para inflar los pulmones. Observar la inflación de pulmón a través del agujero en el paso 2.4. </li><li> Tenga cuidado de no inflar demasiado los pulmones. </li><li> Gire la manija de la llave de paso para que la jeringa llena se cierra, y retirar con cuidado la gripe<html> Identificación de los pulmones. </li><li> Observar los pulmones se desinflan a través del agujero cortado en el paso 1.4. El catéter puede necesitar ser movido ligeramente hacia atrás o hacia delante para conseguir un buen sorteo. Tomar la precaución especial para mantener el catéter dentro de la tráquea. </li><li> Repetir los pasos 2.8.1-2.8.4 5-6x hasta que todo el DPBS, 1% de FBS ha sido inyectado y retirado de los pulmones. Nota: la recuperación del líquido pulmonar no será del 100% del fluido inyectado. </li></ol></li><li> Vaciar la jeringa con el líquido de lavado en un tubo cónico de 50 ml. Mantener las células a temperatura ambiente. </li><li> Precipitar las células por centrifugación a 300 xg durante 5 min a temperatura ambiente y se vierte el sobrenadante. Esto puede ser guardado para el análisis de citoquinas si se desea. </li><li> Resuspender las células en 1-5 ml de RPMI 1640 con 10% de FBS y contar en un hemocitómetro. En el ratón sin estimular,&gt; 95% de las células recogidas por BAL se espera que sean los macrófagos, que pueden ser confirmados mediante citología. </li></ol> 3. El cultivo y cosecha Hongo &quot;&gt; El Aspergillus fumigatus utilizada en este protocolo expresa GFP en todas las etapas de su ciclo de vida:. Esporas, hifas y esporofitos Esta cepa fúngica fue proporcionada por el Dr. Margo Moore, de la Universidad Simon Fraser, Burnaby, BC, Canadá. <ol><li> Placa de conidios de una cepa de Aspergillus fumigatus que expresan GFP en Sabouraud infusión de cerebro corazón (BHI) agar tubos inclinados con cloranfenicol (0,05 g / L) y gentamicina (0,5 g / L). </li><li> Incubar en la oscuridad taparon sin apretar durante 7 a 10 días a temperatura ambiente. </li><li> Cosecha conidios por inclinación lavado con 10 ml de 0,01% de Tween 20 en DPBS. Ligeramente raspar el hongo con el STRIPETTE para aflojar. Enjuague la inclinación varias veces para aumentar el rendimiento. </li><li> Pase suspensión de conidios a través de un filtro de células de 100 micras en una cónica 50 ml. </li><li> Pellet conidios por centrifugación a 400 xg durante 10 min. </li><li> Conidios Resuspender en 50 ml de DPBS y contar utilizando un hemocitómetro. </li><li> Lavar dos veces con DPBS y diluir a concentraciones deseadastración. </li></ol> 4. Fungal Challenge y microscopía confocal Para investigar el papel de la NADPH oxidasa de los macrófagos en la defensa del huésped, se evaluó la capacidad de los macrófagos alveolares aislados de ratones de tres genotipos para controlar el crecimiento de esporas de Aspergillus fagocitados. Los genotipos utilizados en este estudio incluyen; 1) ratones de tipo salvaje, 2) NCF1 * / * Mn-ratones con una mutación NCF1 de origen natural (NCF1 codifica para p47 phox, un componente necesario de la NADPH oxidasa de los fagocitos) dejándolos deficiente de NADPH oxidasa, y 3) NCF1 * / * Mn + ratones transgénicos (MN indica la transgén albergar NCF1 de tipo salvaje bajo el control del promotor de CD68 humano), donde la actividad de la NADPH oxidasa se ​​ha reconstituido en la población de monocitos / macrófagos 10, 11. La microscopía confocal es el más adecuado para este ensayo debido a la presenciay el crecimiento de los conidios no fagocitado en las diapositivas. El microscopio confocal permitirá la visualización de los planos individuales dentro del eje Z, y permitir la diferenciación entre hongo que crece alrededor en lugar de en el interior de los macrófagos. Todas las imágenes de confocal se adquieren utilizando una lente de inmersión en aceite de 63X. Z-pilas se adquieren con un factor de zoom electrónico de 2,5. El espesor de una pila Z se determina utilizando DAPI y las imágenes de campo claro para localizar la parte superior y la parte inferior del campo. Z-pilas variaron de 14 a 24 micras con rodajas individuales ~ 1 m de espesor. Para la cuantificación de los macrófagos, las exploraciones individuales de zoom electrónico 1X se realizaron en 10 campos por genotipo. Se identificaron macrófagos intacta en virtud de PKH26 y tinción DAPI de la membrana y el núcleo, respectivamente. Los conidios fueron identificados sobre la base de la expresión FITC y la imagen de campo brillante. <ol><li> Preparar cubreobjetos y placas estériles <ol><li> En virtud de una cabina de seguridad biológica, remoje 22 x 22 mm cubreobjetos de vidrio en el 70% etanol durante 5-10 min. </li><li> Enjuague cubreobjetos en PBS estéril. </li><li> Usando una pinza estéril, coloque suavemente un único portaobjetos en la parte inferior de cada pocillo de una placa de 6 pocillos estériles envasados ​​individualmente. </li></ol></li><li> Semilla ~ 1 x 10 6 cosechado PKH26 etiquetado macrófagos alveolares en suspensión en 300 l de RPMI 1640, 10% de FBS a cada cubreobjetos. El número exacto puede variar dependiendo del rendimiento de la cosecha. </li><li> Permitir que los macrófagos se adhiera mediante la incubación a 37 ° C con 5% de CO 2 durante la noche. </li><li> A la mañana siguiente, añadir aproximadamente 1 x 10 7 GFP-expresando A. fumigatus conidias suspendió en 100 l pre-calentado (37 ° C) de RPMI 1640, 10% de FBS. </li><li> Placa de Regreso a 37 ° C incubadora con 5% de CO 2. </li><li> Las células deben ser fijados por un mínimo de 2 horas a temperatura ambiente o durante la noche a 4 ° C. A los 3, 7, y 14 hr células de arreglo mediante la adición de un volumen igual de 2% de formaldehído a cada pocillo (concentración final 1% de formaldehído). Coloque las placas para elmomentos de la hora temprana a 4 ° C durante la noche. Para el punto de tiempo final (14 horas), fijar las células a temperatura ambiente durante 2 horas. </li><li> Después de la fijación, retire suavemente cubreobjetos con unas pinzas y enjuague en DPBS. </li><li> Eliminar el exceso de líquido colocando el borde del cubreobjetos sobre un Kimwipe doblado. </li><li> Aplicar medio de montaje 6 l de fluorescencia con DAPI a un portaobjetos de microscopio de vidrio. </li><li> Coloque un borde del cubreobjetos sobre el portaobjetos y colocar suavemente con las células hacia abajo en el medio de montaje. Medio de montaje se extienda y formar una capa uniforme debajo del cubreobjetos. No hay necesidad de presionar sobre el cubreobjetos. </li><li> Selle los bordes del cubreobjetos con esmalte de uñas transparente y dejar secar al aire. </li><li> Tienda prepara diapositivas en una caja de diapositivas (protegido de la luz) a temperatura ambiente hasta que se vaya a analizar por microscopía confocal. </li></ol>

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Los autores no tienen nada que revelar.

El uso de este enfoque para el análisis ex vivo de la actividad antifúngica de los macrófagos junto con in vivo de exposición fúngica, Hemos demostrado anteriormente que la NADPH oxidasa en macrófagos desempeña un papel importante en la defensa contra A. fumigatus 4. El uso de los macrófagos alveolares aislados nos permite centrarse en su actividad antifúngica en ausencia de otras células inmunes (por ejemplo, los neutrófilos reclutados) que defienden contra ...

Los macrófagos alveolares son las células fagocíticas de primera línea que se encuentran con microbios inhalados. Los macrófagos reconocen motivos Aspergillus por los receptores de reconocimiento de patógenos, ingieren y limitan el crecimiento de las esporas inhaladas (conidios), e inician la respuesta inflamatoria. La NADPH oxidasa de los fagocitos convierte oxígeno molecular para el anión superóxido y intermediarios reactivos del oxígeno aguas abajo (Rois). La enfermedad granulomatosa crónica (CGD) es un trastorno hereditario de la NADPH oxidasa se caracteriza por infecciones bacterianas y fúngicas graves y por las respuestas inflamatorias excesivas. Aunque la NADPH oxidasa es crítico para la defensa del huésped mediada por neutrófilos 1 - 3, la importancia de la NADPH oxidasa en macrófagos no está bien definido. Estudios anteriores han demostrado que los macrófagos alveolares ingerir y destruir las esporas de Aspergillus, mientras que los neutrófilos se dirigen principalmente a la etapa de las hifas 5. Sin embargo, ha habido Resul en conflictoTS como para el papel de la NADPH oxidasa en macrófagos en el control del crecimiento de A. fumigatus esporas 6, 7. El objetivo de este método era para delinear el papel específico de la NADPH oxidasa en los macrófagos en la mediación de la defensa del huésped frente a A. esporas fumigatus. Se encontró que la NADPH oxidasa en macrófagos alveolares controla el crecimiento de fagocitado A. fumigatus esporas 4. Para modelar más de cerca la respuesta del huésped a los hongos inhalados, utilizamos los macrófagos alveolares cosechadas por lavado broncoalveolar de los ratones estimulados inmediatamente después del sacrificio. El uso de los macrófagos alveolares aislados nos ha permitido centrarse en su actividad antifúngica en ausencia de otras células del sistema inmune (por ejemplo, neutrófilos reclutados) que defienden contra Aspergillus in vivo. En este método, los macrófagos alveolares se tiñeron in vivo antes de la cosecha a fin de minimizar la cantidad de manipulación posterior a la cosecha. &lt;/ P&gt; Otra ventaja de este protocolo es el uso de un GFP-expresando A. cepa fumigatus. Este hongo expresa GFP desde la etapa de conidios por la etapa de hifas y no tiene necesidad de una mayor tinción. Para obtener imágenes con mayor detalle, se optó por la microscopía confocal, que permite la reconstrucción de las estructuras tridimensionales a partir de las imágenes obtenidas. Reconstrucción tridimensional da la capacidad de diferenciar entre las hifas en crecimiento alrededor en lugar de en o a través de un macrófago. Las conidias también puede ser precisamente distingue como intracelular frente extracelular.

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			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
		</tr>
	</tbody>
</table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

El pulmón es una interfaz donde células huésped están expuestos rutinariamente a los microbios y productos microbianos. Los macrófagos alveolares son las células fagocíticas de primera línea que se encuentran los hongos y otros microbios inhalados. Los macrófagos y otras células inmunes reconocen motivos de Aspergillus por receptores de reconocimiento de patógenos e iniciar respuestas inflamatorias aguas abajo. La oxidasa NADPH fagocítica genera intermediarios reactivos del oxígeno (ROI) y es fundamental para la defensa del huésped. Aunque la NADPH oxidasa es crítico para la defensa del huésped mediada por neutrófilos 1 - 3, la importancia de la NADPH oxidasa en macrófagos no está bien definido. El objetivo de este estudio fue definir el papel específico de la NADPH oxidasa en macrófagos en la mediación de la defensa del huésped contra A. fumigatus. Se encontró que la NADPH oxidasa en macrófagos alveolares controla el crecimiento de fagocitado A. fumigatus esporas 4. Aquí se describe un método para evaluar la capacidad de un ratónmacrófagos lveolar (AMS) para controlar el crecimiento de las esporas de Aspergillus fagocitados (conidios). Los macrófagos alveolares se tiñeron in vivo y diez días después aisladas de ratones por lavado broncoalveolar (BAL). Los macrófagos se cultivaron sobre cubreobjetos de vidrio, y luego sembraron con la proteína verde fluorescente (GFP) que expresan A. esporas fumigatus. En momentos determinados, las células se fijan y el número de macrófagos intactas con esporas fagocitados se evaluaron por microscopía confocal.

<html> Recogida de los macrófagos alveolares por BAL de ratones no estimulados se traducirá en una población&gt; 95% de pureza basado en citología. El 3 y 7 horas (Figura 2) puntos de tiempo preceden a la etapa de hifas de crecimiento de hongos y permiten una comparación de la eficiencia fagocítica de los conidios entre los genotipos. El punto de tiempo de 14 horas es donde genotipos se pueden comparar con respecto a la capacidad para inhibir la transición de conidios fagocitado a la etapa de hifa...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Un método para evaluar la capacidad de los macrófagos alveolares aislados de ratón para controlar el crecimiento de esporas de Aspergillus fagocitados por microscopía confocal.

La evaluación de la actividad anti-fúngica de aislados macrófagos alveolares por microscopía confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K vistas.  Roswell Park Cancer Institute.  Un método para evaluar la capacidad de los macrófagos alveolares aislados de ratón para controlar el crecimiento de esporas de Aspergillus fagocitados por microscopía confocal. 

Video: Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital del bazo: Análisis cuantitativo de la movilidad del parásito y el flujo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Investigación

Inmunología e Infección

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imágenes en tiempo real de las células mieloides en Dinámica<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores de Spinning disco microscopía confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfección altamente eficiente de humanos THP-1 macrófagos por Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteómico de perfiles de los macrófagos por electroforesis 2D

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterización fenotípica de los macrófagos a partir de riñón de rata por Citometría de Flujo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Aislamiento, caracterización y purificación de los macrófagos de los tejidos afectados por la inflamación relacionados con la obesidad

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualización de macrófagos trampas extracelular mediante Microscopía Confocal

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Proyección de imagen 3D Time-lapse de la fagocitosis por macrófagos de ratón

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...