This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

NOTA: Los procedimientos en animales se realizaron de acuerdo con el Comité de Ética Institucional de Cuidado de Animales en Ginebra, Suiza y la Oficina Veterinaria Cantonal. Número de Autorización de GE / 63/14. 1. Preparación de una suspensión de células individuales a partir de médula ósea <ol><li> Sacrificio del ratón (8-12 semanas de edad) por dislocación cervical. Esterilizar patas traseras con etanol al 70%. Retire la piel de las patas traseras. </li><li> Diseccionar fémures y tibias del ratón y eliminar el tejido de las piernas con un bisturí. Enjuague piernas con etanol al 90% y el lugar en una placa de cultivo lleno de PBS. </li><li> Bajo el capó cultura, pañuelo de papel limpio de fémur y la tibia con un bisturí y se separan en articulación de la rodilla. Cortar los extremos de los huesos. </li><li> Médula ósea Flush de cada hueso utilizando una aguja 23G y una jeringa de 1 ml llena con medio de preparación (fenol DMEM / F12 sin rojo, 1% de suero de rata), a unos 50 ml tornillo tubo. Interrumpir agregados celulares por delicadeza pases a través de la ne 23Gedle y una jeringa de 1 ml. Traiga volumen total de 25 ml con medio de preparación. </li><li> Centrífuga 250 xg, 5 min, 4 ° C. Sobrenadante Descartar. Pellet Resuspender en 1 ml de glóbulos rojos tampón de lisis RBLC (8,3 g NH 4 Cl, 1 g de NaHCO 3, 1 ml de EDTA (100 mM), completa a 1 litro con agua destilada, filtro de 0,22 micras) en unos 15 ml tornillo tubo. Incubar 30 segundos en hielo. </li><li> Añadir 10 ml de medio de preparación y centrífuga 250 xg, 5 min, 4 ° C. Desechar el sobrenadante y resuspender en 2 ml de medio de preparación. </li></ol> 2. neutrófilos Enriquecimiento por selección negativa <ol><li> Añadir 150 l de ratón cóctel enriquecimiento de neutrófilos (kit de plataforma de aislamiento de células tales como EasySep). Mezclar e incubar en hielo durante 10 min. </li><li> Añadir 10 ml de fenol DMEM libre de rojo. Invertir una vez y centrifugar 250 xg, 3 min, 4 ° C. </li><li> Sobrenadante Descartar. Resuspender pellet en 1,75 ml de medio de preparación en 5 ml de poliestireno ronda tubos de fondo. Añadir 250 l BiotCóctel en Selección. Mezclar e incubar en hielo durante 10 min. </li><li> Vortex las partículas magnéticas (del kit de plataforma de aislamiento de células) durante 30 segundos para volver a suspender las partículas. Añadir 500 partículas magnéticas mu l a la suspensión celular. Mezclar e incubar en hielo durante 10 min. </li><li> Coloque el tubo en el imán. Espere 3 min. </li><li> Invertir el imán a un nuevo 5 ml de poliestireno ronda tubos de fondo para recoger las células no marcadas deseadas. No tome la última gota que cuelga en el tubo. Las células no deseadas se mantendrán en el tubo en el interior del imán. Deseche este tubo en un peligros biológicos. </li><li> Coloque el nuevo tubo en el imán. Espere 3 min. </li><li> Invertir el imán a un nuevo tubo de 15 ml de tornillo para recoger las células no marcadas deseadas. No tome la última gota que cuelga en el tubo. </li><li> Volumen completa a 5 ml con rojo fenol libre de DMEM. </li></ol> 3. El etiquetado de los neutrófilos <ol><li> Añadir 0,5 l de un tinte de color naranja como la célula Rastreador de Orange (concentración de trabajo1 M, CMRA - clorometil-rodol-acetato, madre 10 mM en DMSO). Incubar 2 minutos a 37 ° C. </li><li> Añadir 2,5 ml de medio de preparación. Centrífuga 250 xg, 5 min, 4 ° C. </li><li> Sobrenadante Descartar. Resuspender pellet en 1 ml de medio de preparación en un tubo de 1,5 ml. </li><li> Tomar una alícuota de contar con hematocitómetro. </li><li> Incubar 5 minutos a 37 ° C. Centrífuga 250 xg, 5 min, 4 ° C. </li><li> Sobrenadante Descartar. Resuspender pellet en 1 ml de PBS. Centrifugar 250 xg, 5 min, 4 ° C para lavar. </li><li> Sobrenadante Descartar. Resuspender pellet como 10x10 6 células por 100 l de PBS. Mantenga en hielo hasta que la inyección iv. NOTA: Las células tienen que ser iv inyecta lo más rápidamente posible. 6-12x10 6 neutrófilos / ratón se obtienen por lo general. </li></ol> 4. Preparación del ratón por Microscopia intravital NOTA:.. Paso 4.1) y 4.2) se debe realizar al comienzo del experimento para evitar tiempo de espera prolongado de los neutrófilos marcados en icmi. <ol><li> Pese la CX 3 CR1 ratón gfp (8-12 semanas de edad). </li><li> Preparar 800 l de anestésicos ketamina (60 mg / kg, xilazina 4,5 mg / kg, acepromazone 1,75 mg / kg en PBS). </li><li> Anestesiar el ratón. Ip inyección con 200 l / 20 g de ratón. Anestesia de control por los pies pellizcos y marcando la frecuencia respiratoria. </li><li> Inyectar neutrófilos (10x10 6 células en 100 l de PBS) por vía intravenosa utilizando las venas de la cola laterales o del seno retro-orbital, a conveniencia del experimentador. </li><li> Ponga el ratón en el cojín de calefacción y proteger los ojos con el gel ocular. </li><li> Un cubreobjetos de vidrio (35 mm de diámetro) se coloca en una placa de cultivo tisular de 10 cm, que tiene un agujero de diámetro 30 mm. Uso de aceite es mantener la placa de cultivo de tejido en contacto con el cubreobjetos. </li><li> Incisión en la piel del abdomen con unas tijeras para exponer el peritoneo. Incisión en el peritoneo con tijeras para exponer intestino. </li><li> Añadir 200 l de PBS (pre-calentada a 37 ° C) en el pcavidad eritoneal. Tome el ratón en la mano y ponlo boca abajo, de modo que el intestino se sale de la cavidad peritoneal con una suave presión. Coloque el ratón en la placa de cultivo tisular. </li><li> Deroll suavemente el intestino con bastoncillos de algodón esterilizado en el cubreobjetos con el fin de exponer los vasos mesentéricos. </li><li> Cortar pañuelos de papel en bandas y mojar con 37 ° C PBS calentado. Inmovilizar el intestino con las piezas de pañuelos de papel para disminuir los movimientos resultantes de la peristalsis. </li><li> Coloque la placa de cultivo de tejidos y el ratón dentro de la cámara de termostato 37 ° C en el escenario de la bandeja a medida del microscopio invertido. Introducir una jeringa que contiene anestésicos sc en la pata trasera. NOTA: Además, el ratón puede recibir oxígeno (0,5 L / min) con una máscara. </li><li> Ratón de control para la anestesia por pellizco del dedo del pie y el control de la frecuencia respiratoria cada 30 min. Si es necesario, proporcionar un impulso de anestésicos (20 l) para mantener adecuadamente la anestesia. NOE: El secado de la vasculatura debe ser evitado. Por lo tanto, su humedad se mantiene mojando regularmente los pañuelos de papel utilizados para inmovilizar el intestino con 37 ° PBS calentado-C. </li></ol> 5. Medición de neutrófilos y monocitos Comportamiento en buques inflamadas in vivo utilizando Microscopia intravital <ol><li> Láseres Set de 488- y 561- en la potencia más baja necesaria para evitar fototoxicidad inducida por láser. En nuestros experimentos, es inferior a 0,5%. La intensidad de las buenas prácticas agrarias en monocitos y tinte de color naranja en los neutrófilos es tan brillante que normalmente 0,3% es suficiente. NOTA: Esto representa una potencia láser de 0,15 a 0,25 mW con nuestro sistema. </li><li> Adquirir imágenes de 512 x 512 píxeles (es decir, 635 micras) en 2,2 segundos con un agujero de alfiler abierto con el objetivo seco del microscopio invertido 20X. Use un z-profundidad entre 10 y 20 micras, que asegura la señal suficiente con las potencias bajas láser. NOTA: Por lo general, realizamos experimentos con un z-profundidad entre 12 y 1656; m. En el caso de cifras presentadas y películas, el Z-profundidad es de 20 micras. Contraste de fase permite la identificación de las paredes endoteliales y las imágenes se realiza en las venas mesentéricas. Dado que las células que patrullan mueven muy lentamente con una velocidad de 5 a 10 micras de / min 6, la grabación de un campo de interés cada 20 seg es satisfactoria. Con los ajustes descritos, la etapa motorizada permite la grabación de hasta 7 campos de interés al mismo tiempo. </li><li> Coloca el ratón y los vasos mesentéricos en la cámara de termostato del microscopio se les permite recuperarse de la preparación durante 30 minutos antes de la adquisición de la primera película. Durante este tiempo, elegir varios campos de interés. Centrarse en la superficie luminal del vaso más cercana al objetivo. Si es necesario, se aprovechan de la leve autofluorescencia del endotelio para ajustar el enfoque. NOTA: La cirugía durante la preparación del ratón destaca ligeramente el endotelio. En consecuencia, laminados de los neutrófilos y/ o monocitos se pueden observar en la primera después de la preparación del ratón 15 min. </li><li> Grabar una primera película de 30 minutos bajo condiciones de estado estacionario, una imagen cada 20 segundos. NOTA: El software adquiere cada campo de interés en 2,2 segundos y la platina motorizada se mueve automáticamente de un campo a otro. Se remonta a la primera posición dentro de 20 segundos durante los 30 minutos de la película de grabación. </li><li> Para iniciar la inflamación de los vasos de la sangre, añadir una gota de 20 l de PBS que contenía 100 mg de TLR2 / TLR1 agonista Pam3CSK4 12 directamente sobre los vasos fotografiados. </li><li> Controlar el enfoque para cada campo de interés. NOTA: Durante el curso de adquisición, siempre hay pequeños cambios de enfoque en el eje z. A pesar de 10 a 20 micras z la profundidad, esto puede resultar en una disminución de la intensidad de fluorescencia. Por lo tanto, reorientar manualmente cada campo de interés al final de cada película, si es necesario. Grabar películas durante 30 min períodos y hasta 3 horas paraTal después de la iniciación de la inflamación. </li><li> Al final del experimento, sacrificar ratón anestesiado por dislocación cervical. </li></ol> 6. Generación de archivos de película y Seguimiento de leucocitos NOTA: El software de adquisición de Nikon genera .nd2 archivos, pero el siguiente procedimiento sería similar con otro software y marcas. <ol><li> Exportar archivos como serie tiff. </li><li> Ir al software ImageJ <a href="http://rsb.info.nih.gov/ij/">(http://rsb.info.nih.gov/ij/).</a> Importe la serie tiff. Cada canal como un archivo diferente. </li><li> Aplicar un filtro de mediana con un radio de 2,0 píxeles para los canales verde y rojo para reducir el ruido de fondo (Proceso&gt; Filtros&gt; La mediana ...). </li><li> Convertir archivos en binario (Proceso&gt; Binary&gt; Hacer binario). Habilitar el software para calcular el umbral para cada imagen. </li><li> Combinar canales en una sola serie de imágenes (Imagen&gt; Color&gt; Combinar Canales ...). Seleccione los monocitos en el gradon canal, los neutrófilos en el canal rojo y la luz transmitida en el canal gris. </li><li> Convertir imágenes apiladas en colores RGB (Imagen&gt; Color&gt; pila para RGB). </li><li> Guardar archivo como .avi </li><li> Los datos se importan y se compilan en el software Imaris (Bitplane). Los movimientos de los monocitos y los neutrófilos son entonces rastreados mediante el Asistente de Seguimiento Spot. </li></ol>

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The authors declare that they have no competing financial interests.

Las metodologías descritas en este manuscrito proporcionan un enfoque coherente para estudiar de manera eficiente los monocitos y el comportamiento de los neutrófilos en las venas mesentéricas bajo condiciones de estado estacionario e inflamatorias. El paso crucial de la preparación es la inmovilización del intestino con tejidos contacto con el medio PBS. Si se realiza correctamente, los vasos mesentéricos están muy bien expuestos en el cubreobjetos para la adquisición de imágenes. ...

<html> Los neutrófilos y los monocitos son las células del sistema inmune innato que circulan continuamente en la sangre. Tras la lesión o infección, inducen señales inflamatorias diapedesis de leucocitos en los tejidos dañados y infectadas, en el presente documento iniciar una respuesta inmune celular a 1-3. La rapidez de la movilización de leucocitos determina el resultado positivo de las respuestas inmunes. Estos procesos complejos se basan en moléculas específicas (por ejemplo, selectinas, quimiocinas endotelio unida) presentes en el endotelio inflamado que ayudan para el establecimiento de contactos adhesivas entre leucocitos circulantes y el endotelio 1-3 .Para obtener ideas sobre las moléculas implicadas en los leucocitos cascada de reclutamiento, es importante para visualizar la cinética de reclutamiento de células y para rastrear el comportamiento de cada célula / población. Un método eficaz consiste en microscopía intravital en ratones transgénicos que expresan las proteínas fluorescentes en las células de interés. contenido &quot;&gt; Hasta la fecha, varios enfoques mediante microscopía intravital se desarrollaron para la imagen de la vasculatura 4,5. Por ejemplo, se utilizó la imagen de la dermis del oído vasculatura o venas mesentéricas por microscopía confocal intravital para identificar el comportamiento de patrullaje de Ly6C murino monocitos bajos y humana CD14 dim monocitos CD16 + en el lado luminal de los vasos sanguíneos en condiciones de estado estacionario 6-8. El modelo vasculatura cremáster ratón se utiliza a menudo para monitorear el comportamiento de neutrófilos o monocitos Ly6c inflamatoria altos en condiciones inflamatorias o isquémicas en ratones transgénicos. En ese caso, cremáster se estimula a través de la inyección de intraescrotal IL1β, CCL2, TNFß o fMLP. Después de 2-4 horas, tejidos se exteriorizan y se analizaron por microscopía confocal intravital 9-11 quirúrgicamente. El siguiente protocolo describe un método para monitorear monocitos y neutrófilos, al mismo tiempo con cualquierde fluorescencia invertida microscopio confocal. Por otra parte, nuestro método detalla cómo reproducir el mismo recipiente antes (condición de estado estable) y después de la inflamación y cómo seguir la cinética de reclutamiento de leucocitos. Para este fin, se utiliza el CX 3 CR1 ratón gfp / peso, cuya monocitos expresar eGFP, iv inyectado con una naranja neutrófilos murinos marcados con colorante. El uso de un microscopio confocal invertido, es posible (1) para rastrear y analizar los monocitos bajos Ly6c patrullaje bajo condiciones de estado estacionario y (2) para seguir el reclutamiento de monocitos y neutrófilos ambos en el mismo recipiente después de la inflamación local. Aquí creamos las condiciones inflamatorias mediante el agonista TLR2 / TLR1 Pam3CSK4 12. Además, tal formación de imágenes puede ayudar a determinar el papel de las moléculas específicas de interés en las diversas etapas de la cascada de reclutamiento de leucocitos si se realiza en ratones knock-out específicas o en presencia de anticuerpos bloqueantes 6,9,13.

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

Respuesta inmune eficiente depende de la rápida movilización de leucocitos de la sangre al sitio de la infección o lesión. La investigación de la migración de leucocitos in vivo es crucial para entender la base molecular de la migración transendotelial de leucocitos y la interacción con el endotelio vascular. Un enfoque poderoso implica microscopía intravital en ratones transgénicos que expresan las proteínas fluorescentes en las células de interés. Aquí se presenta un protocolo para los monocitos de imágenes y neutrófilos en el CX 3 CR1 gfp / peso iv ratón inyectado con neutrófilos marcado tinte de color naranja con un microscopio confocal invertido. Películas Time-lapse recogidos de 30 minutos a varias horas de imágenes de permitir el análisis del comportamiento de leucocitos en las venas mesentéricas, tanto en condiciones de estado estacionario e inflamatorias. También describe los pasos para inducir localmente inflamación de los vasos de sangre con TLR2 / TLR1 agonista Pam3SK4 y supervisar el subsequent reclutamiento de neutrófilos y monocitos. La técnica presentada también se puede utilizar para controlar otras poblaciones de leucocitos e investigar moléculas implicadas en el reclutamiento de leucocitos o el tráfico que utilizan otros estímulos o ratones transgénicos.

El manuscrito describe un protocolo optimizado para controlar fácilmente el comportamiento de los monocitos y neutrófilos en las venas mesentéricas de ratones anestesiados en tiempo real. El uso de una cámara termostatizada-C 37 ° es obligatoria para mantener la temperatura del ratón y también debido al movimiento dependiente de la temperatura de los leucocitos. Preparación del ratón se muestra en la Figura 1. La Figura 2 muestra toda la zona visto bajo el microscopio. Luz tran...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K vistas.  University of Geneva. We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Video: Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Seguimiento de Rastreo de neutrófilos intraluminal, la migración transendotelial y la quimiotaxis de los tejidos por microscopía de vídeo intravital

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Investigación

Inmunología e Infección

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Imágenes en tiempo real de heterotypic plaquetas neutrófilos Interacciones en el endotelio activado durante la inflamación vascular y la formación de trombos en ratones vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

El estudio de las interacciones de<em&gt; Staphylococcus aureus</em&gt; Con neutrófilos mediante citometría de flujo y microscopía Tiempo Lapso

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Muy resuelto Microscopía intravital rayas-iluminación de centros germinales

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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 ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Largo Plazo intravital Multifotónica de imágenes de microscopía de células inmunitarias en sanos y enfermos de hígado utilizando ratones CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Interacciones intravital microscopía de leucocitos endoteliales y plaquetas de leucocitos en mesenterial venas en ratones

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imágenes en vivo de la actividad antifúngica por los neutrófilos humanos primarios y los monocitos en respuesta a las<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Proyección de imagen de interacción de células en Mucosa traqueal durante la infección del Virus de la Influenza usando microscopia Intravital de dos fotones

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Infiltración de leucocitos del músculo cremaestro en ratones evaluado por microscopía intravital

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...