NKS is supported by NIH award K08 AI108690.

Se llevaron a cabo todos los estudios que se examina y las directrices estrictas de acuerdo con el Comité Institucional de Cuidado de Animales y el empleo (IACUC) de la Facultad de Medicina de Harvard, que cumple con los estándares veterinarios establecidos por la American Association for Laboratory Animal Science (AALAS). 1. Horizontal transmisión de bacterias a través de co-vivienda (Opcional) <ol><li> Para minimizar la contaminación exógena (sobre todo si el uso de ratones gnotobióticos), practicar una técnica aséptica durante el montaje de las jaulas estériles desechables, el uso de alimentos y agua que ambos se han tratado en autoclave. </li><li> Use punzones para los oídos o etiquetas para marcar de forma individual 6 semanas de edad C57BL / 6 ratones que albergan diferentes microbiotas y alojarlos en la misma jaula. Dado que los ratones son coprofagia y comen bolitas fecales desde el suelo de la jaula, microbios, naturalmente, serán transmitidos horizontalmente entre los ratones. </li><li> los ratones co-Casa en ≥1 semana para dar tiempo a que la transferencia microbiana y el cambio inmunológica antes del análisis. </li></ol&gt; 2. Preparación de una suspensión de células individuales de la capa intraepitelial Small-intestinal y la lámina propia <ol><li> Preparación de las soluciones <ol><li> Preparar los medios de extracción (por intestino delgado): 30 ml de RPMI + 93 l 5% ditiotreitol (DTT) (v / w) + 60 l de EDTA 0,5 M + 500 l de suero bovino fetal (FBS). Añadir la TDT inmediatamente antes de su uso. </li><li> Preparar los medios de digestión (por el intestino delgado): 25 ml de RPMI + 12,5 mg dispasa + 37,5 mg colagenasa II + 300 l de FBS. Añadir la colagenasa y dispasa inmediatamente antes de su uso. </li><li> Para todas las incubaciones se realizan a 37 ° C, soluciones Precaliente a 37 ° C. </li></ol></li><li> La eutanasia a los ratones por asfixia de CO 2 seguido por dislocación cervical. </li><li> Coloque el lado dorsal del ratón y rociar el abdomen con un 70% de etanol. Use las tijeras para realizar una laparotomía cortando secuencialmente la piel y la fascia peritoneal a continuación a lo largo de la línea media ventral lado a otrom la sínfisis púbica a la apófisis xifoides, exponiendo así a la cavidad peritoneal. </li><li> Con unas tijeras para separar el intestino delgado desde el estómago por transección del esfínter pilórico. Retire con cuidado el intestino delgado del peritoneo, las burlas lejos la grasa mesentérica. <ol><li> Para eliminar completamente el intestino delgado, hacer un segundo corte en la unión íleo-cecal. Coloque el intestino delgado aislado en frío (es decir., 4 ° C) RPMI que contiene 10% de FBS para maximizar la viabilidad celular. </li></ol></li><li> Retire con cuidado las grandes trozos de grasa utilizando pinzas curvas. Tenga cuidado para evitar que se rompa el propio tejido intestinal al intentar quitar la grasa. </li><li> Para eliminar el contenido intestinal, los intestinos suavemente a ras con 15 - 20 ml de PBS frío usando una aguja de alimentación 18 G colocada en una jeringa. </li><li> Use las tijeras para extirpar PP, y colocarlos en RPMI frío que contenía FBS al 5%. EPA se encuentra en el lado antimesentérico del intestino delgado y aparecen como un blanco multi-lobuladamasa. Dependiendo de la cepa específica, un ratón normalmente tiene 8 - 12 PP. </li><li> Cortar el intestino delgado hasta 3 - 4 pulgadas segmentos. </li><li> Eliminar la grasa residual enrollando cada segmento del intestino delgado en una toalla de papel humedecida con RPMI, utilizando un bisturí aburrida para provocar la grasa lejos del tejido. </li><li> Girar el tejido dentro por canulación de los segmentos intestinales con pinzas curvas y agarrar el extremo distal del tejido. A continuación, utilice un par de pinzas rectas para eliminar suavemente el segmento de tejido de las pinzas curvas (comenzando en el extremo proximal), lo que resulta en el tejido que se está invertida. </li><li> Coloque los segmentos de tejido en un vaso que contiene 30 ml de medio de extracción y una barra de agitación. Asegure la tapa en el recipiente, y se agita a 500 rpm durante 15 minutos a 37 ° C; agitación debe ser vigorosa pero no turbulento. </li><li> Después de la incubación, utilizar un filtro de acero para separar piezas de tejido a partir del sobrenadante que contiene epitelio, que debe aparecer turbia. Este sobrenadante contiene ilinfocitos ntraepithelial que se pueden analizar aún más, si se desea, proceder a la filtración de la muestra en los pasos 2.18 - 2.23. </li><li> agitar manualmente las piezas de tejido en RPMI para lavar medios de extracción residuales. </li><li> Coloque el tejido en una toalla de papel seca y se tapa de punta a punta varias veces para facilitar la eliminación de moco residuales que no fue liberado por el medio de extracción. </li><li> Coloque los fragmentos de tejido en un tubo de 1,5 ml con 600 l de medio de digestión. </li><li> Use las tijeras para picar el tejido hasta que las piezas ya no se adhieren a las tijeras y la solución parece homogénea. Este paso es crítico para asegurar la digestión enzimática completa del tejido. </li><li> Añadir el intestino delgado picada a una taza que contiene 25 ml de medio de digestión. Se agita a 500 rpm durante 30 min a 37 ° C. A mitad de camino a través de la digestión (es decir., A los 15 min), pipetear arriba y abajo con una pipeta serológica para ayudar a romper cualquier grandes trozos de tejido. </li><li> tejido digerido Filter (o epithELIAL capa de la etapa 2.12 si va a componer de procesamiento) a través de un filtro de células de 100 micras en un tubo de 50 ml. Enjuague el filtro con 20 ml de RPMI que contenía 10% de FBS. </li><li> Centrifugar la solución filtrada a 500 xg durante 10 min a 4 ° C. </li><li> decantar cuidadosamente el sobrenadante y resuspender el pellet en 1 ml de RPMI que contenía 10% de FBS. </li><li> Filtro de células se resuspendió a través de un filtro de 40 micras de células en un tubo de 50 ml. Enjuague filtro con 20 ml de RPMI que contenía 10% de FBS. </li><li> Centrifugar la solución se filtró a 500 xg durante 10 min a 4 ° C. </li><li> decantar cuidadosamente el sobrenadante y pellet se resuspende en 1 ml de RPMI que contenía 2% de FBS. </li></ol> 3. Preparación de una suspensión de células individuales de las placas de Peyer <ol><li> Transferencia extirpó PP a una taza con 25 ml de medio de fermentación y una barra de agitación. Asegure la tapa y centrifugado a 500 rpm durante 10 minutos a 37 ° C. </li><li> Filtrar digerido PP a través de un filtro de 40 micras de células en un tubo de 50 ml. Si cualquier grupo remaen, ellos presione a través del filtro usando el extremo plano del émbolo de una jeringa de 1 ml. </li><li> Enjuague filtro con 10 ml de RPMI que contenía 10% de FBS. </li><li> Centrifugar la solución se filtró a 500 xg durante 10 min a 4 ° C. </li><li> decantar cuidadosamente el sobrenadante y pellet se resuspende en 1 ml de RPMI que contenía 2% de FBS. </li></ol> 4. La tinción de pavimentación de Citometría de Flujo <ol><li> Alícuota volumen apropiado de células (por ejemplo., 200 l de LPL) en una placa de fondo redondo de 96 pocillos. </li><li> Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar el sobrenadante por inversión de la placa. </li><li> Resuspender las células en 90 l de RPMI que contiene FBS al 2% y una dilución 1: 100 de anti-CD16 / 32 (bloque Fc). Incubar durante 10 minutos a 4 ° C. </li><li> Añadir 10 l de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar el sobrenadante por inversión de la placa. </li><li> Resuspender las células en 250 l de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar el sobrenadante porinvirtiendo la placa. </li><li> (Opcional) las células se tiñen con tinte de viabilidad, si se desea, siguiendo el protocolo del fabricante. </li><li> Resuspender las células en 100 l 1% de formalina para fijar las células. Incubar durante 1 hora en la oscuridad a 4 ° C. </li><li> Añadir 200 l de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar el sobrenadante por inversión de la placa. </li><li> Resuspender las células en 250 l de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar el sobrenadante por inversión de la placa. </li><li> Resuspender las células en 200 l de PBS. Analizar las células utilizando un citómetro de flujo. </li></ol>

<ol>
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No conflicts of interest declared.

Se presenta un protocolo para el aislamiento y caracterización de citometría de los linfocitos del intestino delgado, incluyendo la LPL va a componer, y linfocitos en el PP de flujo. Para los interesados ​​en la evaluación de cómo los cambios en la microbiota afectan el sistema inmunológico del intestino delgado, se detallan los pasos sencillos que intervienen en la transmisión horizontal de organismos entre los ratones que albergan diferentes microorganismos involucrados. Aunque este protocolo se centra en el...

La tarea principal del intestino delgado es a menudo considerada como la digestión y absorción de nutrientes 1. Si bien esta función metabólica es claramente esencial, el intestino delgado tiene un papel igualmente importante en la protección del huésped de la descarga continua de antígenos ambientales que se encuentran dentro de la luz 2. El tracto intestinal separa el mundo exterior (por ejemplo., Antígenos luminales) de el ambiente interno del huésped con una capa epitelial que es sólo una única capa de células de espesor. Como tal, el sistema inmunitario del intestino delgado tiene el formidable tarea de equilibrar su umbral para la reactividad, lo que permite antígenos extraños de la dieta y comensales microbios para entrar en la mucosa con un mínimo, en su caso, la respuesta inmune mientras que el montaje de una respuesta sólida contra la invasión de patógenos y otros antígenos &quot;perjudiciales&quot;. Las respuestas inmunitarias excesivas o inapropiadas a estos antígenos pueden conducir a la enfermedad patológica (por ejemplo., Inflammaenfermedad toria del intestino, diabetes tipo I, esclerosis múltiple) y debe evitarse 3-6. En general, se considera que el tracto gastrointestinal para representar el órgano más grande inmune en el cuerpo, que contiene más de 70% de todas las células 7 que secretan anticuerpo. El sistema inmunitario del intestino delgado se compone de 3 compartimentos principales - la lámina propia (LP), la capa intraepitelial, y parches de Peyer (PP) - que cada uno contiene un grupo distintivo de linfocitos 2. Los linfocitos LP (LPLS) son principalmente las células T TCRαβ con células ~ 20% de B; linfocitos intraepiteliales (IEL) contienen muy pocas células B con más células T que TCRγδ + células T TCRαβ; y PP, que son los órganos linfoides secundarios incrustados en la pared del intestino delgado, contienen células B ~ 80%. Aunque cada una de estas regiones anatómicas tiene funciones ligeramente distintas y bases ontológicas, que funcionará de ahmoda armonized para proteger al huésped de insultos patógenos. Por otra parte, existe un creciente reconocimiento de que la microbiota es un determinante crítico para el desarrollo del sistema inmune intestinal, con un creciente reconocimiento de la relación entre los microbios específicos afines y la ontogenia de determinados linajes celulares 8,9. Además, dado que la educación del sistema inmunitario intestinal afecta a las respuestas inmunes en sitios anatómicamente distantes (por ejemplo., La artritis, la esclerosis múltiple, neumonía), se ha hecho evidente que el desarrollo del sistema inmunitario intestinal es relevante para más procesos de la enfermedad de reconocido previamente 10 -12. Como tal, el interés en evaluar cuantitativamente el sistema inmune intestinal ha extendido más allá de las interacciones huésped-patógeno para incluir ahora la interacción huésped-comensal y la patogénesis de muchas enfermedades sistémicas así. Dada la variabilidad de los métodos actuales de laaislamiento de los linfocitos intestinales, un método que se ha optimizado para el rendimiento, la viabilidad, y la consistencia y equilibrar el tiempo requerido es cada vez más crítica. Protocolos que involucran gradientes de Percoll son el tiempo y mano de obra intensiva y potencialmente más propenso a un error humano, dando lugar a un rendimiento variable de 13 y viabilidad. En este documento, se proporciona un protocolo optimizado para el aislamiento y caracterización de linfocitos de los 3 compartimentos inmunes del intestino delgado. Además, dado el aumento de interés en alteraciones de microbios inducida en el sistema inmune de la mucosa, que incluye pasos que se pueden utilizar para permitir la transmisión horizontal de microorganismos entre los ratones para evaluar cómo estos cambios afectan cuantitativamente el sistema inmunitario intestinal.

<table>
	<tbody>
		<tr>
			<td>Sterile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>555092</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile mouse cage</td>
			<td>Innovive</td>
			<td>MS2-AD</td>
			<td>contains lid, cage bottom, and alpha-dri bedding</td>
		</tr>
		<tr>
			<td>metal feeder</td>
			<td>Innovive</td>
			<td>M-FEED</td>
			<td></td>
		</tr>
		<tr>
			<td>water bottle</td>
			<td>Innovive</td>
			<td>M-WB-300</td>
			<td></td>
		</tr>
		<tr>
			<td>card holder</td>
			<td>Innovive</td>
			<td>CRD-HLD-H</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclavable rodent chow (NIH-31M)</td>
			<td>Zeigler</td>
			<td>4131207530</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GemBio</td>
			<td>100-510</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase II</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td>
		</tr>
		<tr>
			<td>collagenase, type II&#160;</td>
			<td>Invitrogen</td>
			<td>17101-015</td>
			<td>the concentration in the protocol is based on an activity level of 245 U/mg</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>feeding needle (18 G, 2&#34; length)</td>
			<td>Roboz</td>
			<td>FN-7905</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (15 blade)</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-purpose cups, 120 ml</td>
			<td>VWR</td>
			<td>89009-662</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>VWR</td>
			<td>58949-062</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-position stir plate, 9-position</td>
			<td>VWR</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel conical strainer, 3 inch&#160;</td>
			<td>RSVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, round-bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32 (Fc block)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>(optional) fixable viability dye eFluor 780</td>
			<td>eBiosciences</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin, neutral buffered</td>
			<td>Thermo Scientific</td>
			<td>5725</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

Los intestinos - que contienen el mayor número de células inmunes de cualquier órgano en el cuerpo - están constantemente expuestos a antígenos extraños, tanto microbiana y la dieta. Dada la comprensión creciente de que estos antígenos luminales ayudan a dar forma a la respuesta inmune y que la educación de las células inmunes en el intestino es crítica para un número de enfermedades sistémicas, se ha aumentado el interés en la caracterización del sistema inmunitario intestinal. Sin embargo, muchos protocolos publicados son arduo y que consume tiempo. Presentamos aquí un protocolo simplificado para el aislamiento de linfocitos de la propria del intestino delgado lámina, capa intraepitelial, y las placas de Peyer que es rápido, reproducible, y no requiere gradientes de Percoll laboriosas. Aunque el protocolo se centra en el intestino delgado, también es adecuado para el análisis de los dos puntos. Por otra parte, se destacan algunos aspectos que pueden necesitar una optimización adicional en función del ques científica específicación. Este enfoque da como resultado el aislamiento de un gran número de linfocitos viables que posteriormente se pueden utilizar para el análisis de citometría de flujo o medios alternativos de caracterización.

Análisis citométrico de flujo suspensiones de células individuales de linfocitos pequeños intestinal debe producir una discreta población de células que tienen similares dispersión frontal y lateral características como esplenocitos (Figuras 1A y 1B). Los linfocitos pueden comenzar a morir si el tejido no se mantiene a 4 ° C durante las etapas iniciales del aislamiento, lo que resulta en la población de linfocitos que tiene una dispersión frontal inferior y se...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

El aislamiento y la citometría de flujo Caracterización de murino Small intestinal linfocitos

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

isolation-flow-cytometric-characterization-murine-small-intestinal

Research

JoVE Journal

Immunology and Infection

27.3K vistas.  Boston Children’s Hospital. There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. 

Video: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Investigación

Inmunología e Infección

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Aislamiento de linfocitos de ratón del tracto genital Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

El aislamiento y la citometría de flujo análisis de las células mononucleares de sangre periférica Glioma infiltrantes

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Aislamiento y Análisis de citometría de flujo, de Human endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Aislar los linfocitos desde el sistema de inmune Intestinal pequeño ratón

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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 ...