Los autores desean agradecer a la Srta. Liana Mackiewicz de asistencia técnica. Este trabajo ha sido posible gracias al apoyo del Gobierno del Estado victoriano infraestructura operativa y del Gobierno de Australia National Health and Medical Research Council IRIISS Proyecto de Donación y 1031212.

1. La infección de ratones con P. berghei ANKA- <ol><li> Descongelar una alícuota de criopreservado P. berghei ANKA GRI. </li><li> Reprime un cerebral malaria resistente a ratones BALB / c de ratón donante (8-12 semanas de edad) mediante la técnica de restricción de dos manos. Inyectar ratón con 100-200 l de pRBC utilizando una jeringa de 1 ml de insulina (28G aguja). Rutinariamente, 1-2 ratones donantes se inyectan. </li><li> En 4-5 días post-infección (pi) eliminar ratón donante de la jaula y lo coloca en una estación de trabajo o un paño desechable de trabajo. </li><li> Suavemente contener ratón por el extremo de la cola con unas tijeras pequeñas y cortar la punta de la cola (aproximadamente 1 mm). Alternativamente, una punción pequeña cola usando una aguja de 25 G se pudo realizar. </li><li> Colocar una gota de sangre de la vena de la cola del ratón cerca del final de un portaobjetos de microscopio esmerilado final. </li><li> Coloque una segunda diapositiva (spreader) en un ángulo de 45 ° y una copia en la gota de sangre. Una vez que la sangre se extiende a lo largo del borde, pulse la víspera esparcidor de diapositivasólo en todo el portaobjetos de un microscopio para hacer el frotis de sangre. Deje que la mancha se seque al aire. </li><li> Fijar los frotis de sangre en 100% de metanol durante 30 segundos y luego se tiñen con Giemsa portaobjetos recién preparada durante 10 min. </li><li> Enjuague frotis de sangre con agua corriente durante 1 minuto, se deja secar al aire y examinar portaobjetos bajo el microscopio (100x, aceite de inmersión). Enumerar pRBC a 1.000 eritrocitos y calcular parasitemia por ciento. Los ratones donantes debería alcanzar los niveles de parasitemia entre 2.5-5% antes de que puedan ser sangrados para la infección de animales experimentales. </li><li> Recoger sangre del ratón donante por sangrado retro-orbital utilizando un heparinizada micro-hematocrito tubo capilar. Todos los experimentos se llevan a cabo de conformidad con el Walter y Eliza Hall Institute requisitos comité de ética animal. Dependiendo de las necesidades locales Animal Comité de Ética otros procedimientos sangrado puede ser utilizado. Disolver la cantidad necesaria de sangre en medio RPMI a una concentración final de 1x10 6 pRBC/0.2 ml. Considere la posibilidad de queun hematocrito normal de ratón es ~ 6x10 9 eritrocitos / ml 1. </li><li> Infect experimental C57BL / 6 ratones (8-12 semanas de edad) mediante la inyección por vía intraperitoneal (ip) 1x10 6 pRBC/0.2 ml. </li><li> Para controlar los niveles de parasitemia de ratones infectados siga los pasos 1.3-1.8. La parasitemia debe determinarse cada 2-3 días a partir del día 2 o 3 pi </li><li> El inicio de la malaria severa en C57BL / 6 ratones pueden manifestarse como apariencia encorvada, fallo de piel y baja actividad. Algunos ratones puede recuperar en esta etapa, pero la progresión de la pérdida de reflejo autoadrizable indica enfermedad irreversible y los animales deben ser sacrificados. </li></ol> 2. La perfusión intracardiaca de ratones y de extracción del cerebro <ol><li> Montar un sistema de perfusión manual de gravedad asegurando un depósito de 500 ml a un soporte de columna fijado a la parte superior de un soporte de columna de 60 cm. Conectar un tubo de plástico en el extremo inferior del depósito y garantizar una abrazadera de la tubería para controlar el flujo de tampón. Conecte el tubo a una aguja de 23 G. Fill hasta el depósito con PBS (mantenido a 22 ° C), abrir la abrazadera y permitir tampón para correr a través del tubo para eliminar todas las burbujas de aire. </li><li> P. eutanasia berghei ANKA-ratón infectado por inhalación de CO 2. Confirmar la muerte por la ausencia de pedal, orbital y las respuestas respiratorias. </li><li> Pin ratón sacrificados por las patas traseras y delanteras dorsalmente en un tablero de espuma de poliestireno disección contenida en una bandeja de plástico. Dependiendo de ética locales Animal Comité requisitos anestesia puede administrarse antes de comenzar pefusion intracardial. </li><li> Limpie lado ventral con 70% de etanol. </li><li> Con unas tijeras grandes y pinzas para abrir la piel a lo largo de la línea media para exponer la cavidad torácica. Del pliegue cutáneo y el pin a los lados. </li><li> Mantenga el esternón con unas pinzas finas y cortar el diafragma y a lo largo de ambos lados del esternón cortar las costillas. Tenga cuidado de no dañar los vasos sanguíneos grandes. </li><li> Pin de la caja torácica por el esternón libremente al lado de la cabeza. </li><li> Mantenga ingenio ventrículosh finas pinzas y el atrio derecho incisión cuidadosamente con unas tijeras finas. </li><li> Inserte la aguja 23G conectada al sistema de perfusión por gravedad en el ventrículo izquierdo hacia la aorta ascendente mientras PBS está en marcha. Inserte sólo 0,5 cm de la punta de la aguja. </li><li> Perfundir ratón durante 5 minutos o hasta que el efluente es clara. </li><li> Liberar ratón y lo pondré sobre su abdomen. Limpiar la cabeza con 70% de etanol. </li><li> Con unas tijeras grandes, haga un corte justo por encima del área de la médula espinal cervical. </li><li> El uso de tijeras finas para hacer una mediana caudal-rostral corte a partir de la base del cráneo y la cáscara de la piel para exponer el cráneo. </li><li> Sosteniendo la cabeza, colocar las hojas de una tijera pequeñas en cada cavidad orbital y realizar un corte entre las órbitas. </li><li> Haga un corte longitudinal a lo largo de la sutura sagital y retire con cuidado el cráneo en cada hemisferio del cerebro hacia el exterior. </li><li> Usando una espátula, levante suavemente el cerebro y lo coloca en un tubo de centrífuga de 10 ml que contenía medio RPMI. </li></ol>3. Aislamiento de Brain-secuestrados leucocitos (BSL) <ol><li> Trabajando en una cabina de seguridad, lugar recién cosechado cerebro en la parte superior de un filtro de acero inoxidable de células (40-60 tamaño de malla) en una placa de Petri que contenía 3-5 ml de medio de cultivo RPMI tejido. </li><li> Cortar el tejido cerebral en trozos pequeños. </li><li> Empuje pequeñas piezas de tejido cerebral a través del colador celular usando un émbolo de cristal. </li><li> Transferir homogeneizado de cerebro a un tubo de 10 ml y centrifugar a 250 xg durante 10 min a 4 ° C. </li><li> Disolver sedimento en 3 ml de medio RPMI, que contiene 0,05% de colagenasa D y 2U/ml DNAsa I. </li><li> Girar mezcla durante 30 min a temperatura ambiente. Eliminar los restos celulares por empujar la mezcla a través de un filtro de células de nylon 70 micras y / o mediante la incubación en hielo durante 5 min. </li><li> Lay homogeneizado de cerebro en un cojín 7 ml de Percoll al 30% y se centrifuga a 400 xg (sin frenos) durante 20 min a temperatura ambiente. </li><li> Resuspender el sedimento en 1 ml de tampón de lisis de glóbulos rojos e incubar en hielo durante 5 min alisar pRBC adherente, que no puede ser eliminado desde el cerebro por perfusión intracardial. </li><li> Añadir 9 ml de medio RPMI, lavar las células y centrifugar a 250 xg durante 10 min a 4 ° C. </li><li> Resuspender el pellet que contiene BSL en 50-100 l de medio RPMI y las células de transferencia a un tubo Eppendorf. </li><li> Diluir una alícuota de 5-10 l de la muestra de células 1:2 en azul tripán para la identificación de células viables. </li><li> Contar las células viables bajo el microscopio utilizando un hemocitómetro. A partir del día 6 en adelante, cuando pi P. berghei ANKA de ratones infectados muestran signos neurológicos, 20.000-100.000 BSLs se puede recuperar. </li></ol> 4. Inmunofenotipificación de BSL por citometría de flujo multicolor <ol><li> Centrifugar las células a 250 xg durante 10 min a 4 ° C, aspirar el sobrenadante y resuspender el pellet en 50 l de tampón de tinción (PBS, 1% de suero de ternera fetal (FCS), 2 mM de EDTA). </li><li> Añadir el 1 l de anticuerpo purificado anti-CD16/32 (Fcblock). </li><li> Se incuban las células durante 10 min en hielo. </li><li> Lavar las células con 1 ml de tampón de tinción. Centrifugar las células a 250 xg durante 10 min a 4 ° C y aspirar el sobrenadante. </li><li> Resuspender las células en 50 l de tampón de tinción que contiene predeterminados diluciones óptimas requeridas de anticuerpos fluorescentes, es decir, anti-CD4, anti-CD8, anti-TCR y anti-NK1.1. </li><li> Incubar durante 1 hora en hielo. </li><li> Lavar las células con 1 ml de tampón de tinción. Centrifugar las células a 250 xg durante 10 min a 4 ° C y aspirar el sobrenadante. </li><li> Resuspender las células en 100 l de PBS. </li><li> Transferir las células a un tubo de flujo plástico citometría. </li><li> El uso de un citómetro de flujo, obtener al menos 5,000-10,000 eventos. Adecuados controles de células no teñidas y muestras fluorocromo compensación deben ser incluidas como sea necesario. </li><li> Analizar los resultados utilizando el software apropiado citometría de flujo. </li></ol>

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No hay conflictos de interés declarado.

El aislamiento y el análisis de BSL es un método que permite la caracterización y cuantificación de células inflamatorias que migran hacia el cerebro en respuesta a la lesión tisular o la infección en modelos experimentales de ratón. La introducción de un paso de perfusión intracardiaca para la retirada del compartimento de la sangre antes de la extracción de órganos y posterior aislamiento de las células es útil para prevenir la contaminación de células inflamatorias con no inflamatorias leucocitos circ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td></td><td></td><td></td><td> Soluciones y tampones </td></tr><tr><td> Giemsa azur-eosina-azul de metileno solución </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Dilución 1:10 en agua destilada </td></tr><tr><td> Medio RPMI </td><td></td><td></td><td> Ratón tonicidad </td></tr><tr><td> Ratón tonicidad PBS </td><td></td><td></td><td> 20 mM de fosfato de sodio, 0,149 de NaCl, pH 7,3 </td></tr><tr><td> 0,4% de azul de tripano </td><td> Sigma Aldrich </td><td> T-8154 </td><td> Dilución 1:2 </td></tr><tr><td> La colagenasa D </td><td> Worthington Biochemical </td><td></td><td></td></tr><tr><td> DeoxyribonuClease (DNasa) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> De páncreas bovino </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 30% solución en PBS </td></tr><tr><td> Ultrapura Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Cloruro de amonio (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Red Cell Lysis Buffer </td><td></td><td></td><td> 17 mM Tris, 14nm NH 4 Cl, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA sal disódica </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Anticuerpos y conjugados </td></tr><tr><td> Anti-ratón CD16/CD32 (Fc Block), clon 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 l en 50 l tampón de tinción (0.5mg/50 ml) </td></tr><tr><td> FITC-anti-ratón, clon CD4 H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-ratón NK1.1, clon PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-anti-ratón CD8, clon 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-anti-ratón TCR-β, clon H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-ratón de CXCR3, clon 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinilado anti-ratón de CCR5, clon C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Estreptavidina-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipos y materiales </td></tr><tr><td> SuperFrost portaobjetos de microscopio </td><td> Lomb Menzel-Gläser </td><td></td><td></td></tr><tr><td> Pinzas de disección, tijeras </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml de PBS reservorio </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Caucho tubería </td><td></td><td></td><td></td></tr><tr><td> Aguja 23G </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Disociación celular kit que contiene metal tamiz </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 micras filtro de células de nylon </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Hemocitómetro </td><td> Neubauer GmbH </td><td> 717810 </td><td></td></tr><tr><td> Tubos de citometría de flujo </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Se describe un método para el aislamiento y caracterización de células adherentes inflamatorias de los vasos sanguíneos cerebrales de P. berghei ANKA-ratones infectados. La infección de las cepas susceptibles de ratón con esta cepa del parásito en los resultados de la inducción de la malaria cerebral experimental, un síndrome neurológico que recapitula algunos aspectos importantes de Plasmodium falciparum mediada por paludismo grave en 1,2 seres humanos. Formas maduras de la malaria en etapa de sangre expresar proteínas parasitarias en la superficie de los eritrocitos infectados, lo que les permite unirse a las células endoteliales vasculares. Este proceso induce obstrucciones en el flujo sanguíneo, lo que resulta en hemorragias hipoxia y 3 y también estimula el reclutamiento de los leucocitos inflamatorios en el sitio de secuestro del parásito. A diferencia de otras infecciones, es decir, virus neutrotopic 4-6, ambas parasitados malaria-glóbulos rojos (pRBC) así como Inflamm asociadoAtory leucocitos permanecen secuestrados dentro de los vasos sanguíneos en lugar de infiltrar el parénquima cerebral. Así, para evitar la contaminación de los leucocitos secuestrados con no inflamatorias circulantes, células de perfusión intracardiaca extenso de los ratones infectados antes de la extracción de órganos y procesamiento de tejidos es necesario en este procedimiento para retirar el compartimento de la sangre. Después de la perfusión, los cerebros se cosechan y disecado en trozos pequeños. La estructura del tejido es más interrumpido por tratamiento enzimático con colagenasa D y DNAsa I. El homogeneizado cerebral resultante se centrifugó en un gradiente de Percoll que permite la separación de secuestrados cerebro-leucocitos (BSL) de la mielina y los residuos de otro tejido. Las células aisladas se lavan a continuación, se contaron usando un hemocitómetro y se tiñeron con anticuerpos fluorescentes para su posterior análisis por citometría de flujo. Este procedimiento permite la caracterización fenotípica completa de la migración de los leucocitos inflamatorios en el cerebro en rerespuesta a varios estímulos, incluyendo accidente cerebrovascular, así como infecciones víricas o parasitarias. El método también proporciona una herramienta útil para la evaluación de nuevos tratamientos anti-inflamatorios en modelos animales preclínicos.

Los resultados en la figura. 2 muestran porcentajes y números absolutos de las diferentes poblaciones BSL recuperados de los cerebros de los ratones control perfundidos o unperfused infectadas por la malaria e ingenuo. Aislado BSL fueron teñidas con PE-anti-NK1.1 y anticuerpos APC-anti-TCR-β como se indica en el texto del Protocolo. De acuerdo con resultados anteriores 7-9, αβTCR + células T comprende una elevada proporción de la piscina BSL en cerebros de perfundidos infectadas por la malar...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Un método para el aislamiento de los leucocitos inflamatorios adherentes de los vasos sanguíneos cerebrales de los<em&gt; Plasmodium berghei</em&gt; ANKA de ratones infectados se describe. El método permite la cuantificación así como la caracterización fenotípica de los leucocitos aislados después de la tinción con anticuerpos fluorescentes y el posterior análisis por citometría de flujo.

Aislamiento y análisis de Brain-secuestrados Los leucocitos de<em&gt; Plasmodium berghei</em&gt; ANKA de ratones infectados

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

12.7K vistas.  The Walter and Eliza Hall Institute of Medical Research. Un método para el aislamiento de los leucocitos inflamatorios adherentes de los vasos sanguíneos cerebrales de los Plasmodium berghei ANKA de ratones infectados se describe. El método permite la cuantificación así como la caracterización fenotípica de los leucocitos aislados después de la tinción con anticuerpos fluorescentes y el posterior análisis por citometría de flujo.

Video: Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

El aislamiento de la infiltración de leucocitos cerebro-

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Investigación

Inmunología e Infección

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética y los Oxidativo ráfaga: Los protocolos para el aislamiento y la Evaluación de leucocitos humanos y plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

El análisis de los efectos de las células del estroma en el reclutamiento de leucocitos de Flujo

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Aislamiento de leucocitos de la interfaz humana Materno-fetal

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Aislamiento de leucocitos a partir de los tejidos murinos en la interfase materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Aislamiento de leucocitos infiltrantes de ratón Piel Usando enzimática Digest y Separación de degradado

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

El aislamiento de las células mieloides de ratón de la piel y el drenaje de ganglios linfáticos Siguiendo intradérmica La inmunización con viva atenuada<em&gt; Plasmodium</emLos esporozoitos&gt;

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

La recolección selectiva de leucocitos marginando-hepáticas

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...