Este trabajo fue financiado por los institutos canadienses de investigación salud (148808 y 148792), SE contó con un corazón y un movimiento Fundación, premio personal de la Oficina Provincial de Ontario, March of Dimes, Ted Rogers Centre para la investigación del corazón y el Peter Munk Centro cardíaco. XCC tiene una beca de Banting CIHR. LA sostiene un corazón & movimiento/Richard Lewar beca premio.

Declaración de ética: este protocolo ha sido revisado y aprobado por el Comité de cuidado del Animal en la red de salud universitaria (Toronto, Canadá) y está de acuerdo con el Consejo Canadiense sobre el cuidado Animal.
1. preparación de buffer
<ol><li>Preparar el tampón HBB (de Hank balanceado sal solución (HBSS), suero bovino inactivado por calor 2%, albúmina de suero bovino 0,2%). Añadir 10 mL de calor inactiva el suero bovino y 1 g de albúmina de suero bovino a 500 mL de HBSS. Filtro de esterilización a través de un filtro μm 0.2 y almacenar a 4 ° C.</li>
	<li>Preparar el tampón FACS (tampón de fosfato salino (PBS), 2% calor inactiva el suero bovino, 1 mM de ácido etilendiaminotetraacético (EDTA, pH 8.0)). Añadir inactivado de calor de 10 mL de suero bovino y 1 mL de EDTA de 0.5 M a 500 mL de PBS. Filtro de esterilización a través de un filtro μm 0.2 y almacenar a 4 ° C. Nota: Las soluciones pueden almacenarse a 4 ° C durante 1 mes.</li>
</ol>2. etiquetado de leucocitos que circulan
<ol><li>Preparar asépticamente inyección de CD45 anti ratón con 1 μg de anticuerpo diluido en tampón de fosfato estéril salino (PBS) para un volumen de inyección final de 200 μL/ratón. Volumen de carga en una jeringa de insulina 28,5 G y mantener lejos de la luz. Nota: Utilice dos anticuerpos anti-CD45 marcado con fluorocromo diferente para distinguir el intravascular (teñido a través de la administración de i.v.) de la tinción intracardiaco (manchado después de la digestión; ver paso 4).</li>
	<li>
Caliente previamente ratones (8 – 12 semanas) exponiendo la cola a una lámpara de calor rojo durante 5 minutos. ADVERTENCIA: No recaliente el animal. Mantenga la lámpara a una distancia segura (> 30 cm).<ol>
<li>Refrenar el animal en posición esternal utilizando un dispositivo apropiado. Exponer y estabilizar la cola con la mano no dominante por inmovilizar la base de la cola con el índice y el dedo medio y la región medio-distal de la cola con el pulgar y el dedo anular. PRECAUCIÓN: No aplique demasiada fuerza en la punta de la cola ya que la piel puede ser halada. Inserte la aguja con el bisel hacia arriba en la vena. Mantenga la aguja paralela a la vena en todo momento ya que es fácil punzar la vena. Nota: Sangre en la jeringa puede ocurrir y es un signo de una buena inyección. Doblar la aguja a un ángulo de 90° puede facilitar la administración de i.v.. Confirme con su oficina de seguridad como agujas de flexión pueden representar un peligro.</li>
		<li>Inyectar por vía intravenosa 200 μL en la vena de la cola. El líquido debe fluir fácilmente y eliminar temporalmente la vena. PRECAUCIÓN: No fuerce el líquido en si encuentra resistencia. Esto es signo de mala colocación de la aguja.</li>
		<li>Permiten el anticuerpo a circular durante 5 minutos antes humanamente euthanizing ratón.</li>
	</ol>
</li>
</ol>3. corazón de aislamiento y la digestión
<ol><li>
Eutanasia a ratones con CO2 o por cualquier otro procedimiento de eutanasia institucionalmente aceptado.
	<ol>
<li>Eutanasia de CO2 , coloque el ratón en la cámara de CO2 y la tasa de utilización a 20% del volumen por minuto (es decir, si la cámara es de 10 L, llenado en 2 L/min). Funcionar durante 2 min y monitor ratón hasta que ha dejado de respirar.</li>
		<li>Apague el CO2 y dejar el ratón un adicional 1-2 min Asegúrese de que ha dejado de respirar antes de abrir la cámara y realizar un pellizco del dedo del pie para confirmar que el ratón está muerto antes de proceder a procesar el ratón.</li>
	</ol>
</li>
	<li>Rocíe el ratón con etanol al 70%. Levante la piel en el abdomen y diseccionar el ratón cortando a lo largo del principio de la línea media ventral a nivel de las caderas hasta el esternón. Abrir el abdomen y mover el hígado para exponer el diafragma.</li>
	<li>
Cortar el diafragma y las costillas en ambos lados de la línea media, siendo cuidadoso para evitar pinchar los pulmones y el corazón.
	<ol>
<li>Exponer el corazón por peeling espalda las costillas, luego corte primero las aurículas izquierdas seguidas de la aurícula derecha. PRECAUCIÓN: Tenga cuidado al cortar las aurículas, asegurando arterias ni venas no se lesionan, como esto puede disminuir la eficacia del lavado de la sangre contenida en los órganos.</li>
		<li>Inundar el corazón con 15 – 20 mL de PBS frío utilizando una jeringa de 60 mL con una aguja de 21 G y PBS frío. Suavemente Sujete la base del corazón con unas pinzas e Inserte la aguja en el ventrículo izquierdo cerca del ápice. Repita el procedimiento en el ventrículo derecho. Una correcta inyección resulta en blanco pulmones y hígado pálido. Nota: La perfusión debe realizarse en 5 minutos después de la eutanasia para evitar la coagulación de la sangre, que puede contaminar los órganos de interés con las células circulantes.</li>
	</ol>
</li>
	<li>
Sostenga la base del corazón con unas pinzas y suavemente tire el corazón, luego separar el corazón mediante la reducción de las principales arterias pulmonares y las venas, aorta y el saco pericárdico.
	<ol>
<li>Limpiar el corazón sosteniendo suavemente el corazón entre los dedos del índice y el pulgar en una toalla de papel limpio libre de pelusas y extraiga las aurículas y los restos de las arterias principales y venas y otros contaminantes con el fórceps. Nota: Asegúrese de retira la totalidad de las aurículas y no otros tejidos como el pulmón o los vasos sanguíneos, están presentes como estos tejidos tienen sus propias células inmunes residentes. En particular, el pulmón tiene una población grande de la célula inmune que puede enmascarar a las poblaciones más pequeñas de ésos dentro del miocardio.</li>
	</ol>
</li>
	<li>
Coloque el corazón en un recipiente plástico de 5 cm con 50 μl de PBS frío.
	<ol>
<li>Picar el corazón usando tijeras disección curvas hasta que no hay piezas visibles y tiene una consistencia suave. Nota: Trate de mantener el corazón en un área de contenido del plato en picar para minimizar la pérdida de tejido.</li>
		<li>Transferencia de tejido de thetrituratedheart usando fórceps curvado en el tubo de microcentrífuga de 2 mL con de 800 μl frío Dulbecco modificado Eagle Medium (DMEM).</li>
	</ol>
</li>
	<li>
Añadir 450 U/mL ofcollagenase I, 60 U/mL de hialuronidasa tipo I-S y 60 U/mL de DNasa-I para cada muestra.
	<ol>
<li>Incubar las muestras a 37 ° C en un agitador oscilante a 50 rpm durante 45 – 60 minutos.</li>
	</ol>
</li>
	<li>
Después de la digestión, brevemente muestras de vórtice (20 s) y colocar inmediatamente en hielo para mantener las muestras a 4 ° C.
	<ol>
<li>Utilizando una pipeta de 1 mL, pipetear las muestras hacia arriba y hacia abajo hasta que las muestras de tejido son homogéneas y no obstruir la punta de la pipeta. Nota: Definir un número específico de veces para pipeta puede aumentar la reproducibilidad. Si el corazón no se digiere óptimamente, piezas del miocardio pueden obstruir la pipeta. Pulir suavemente la punta de la pipeta contra el tubo de microcentrífuga ayudará a homogeneizar los trozos más grandes de tejido.</li>
	</ol>
</li>
	<li>
Humedezca previamente un filtro de celular de 40 μm a un tubo cónico de 50 mL con 1 mL de frío HBB.
	<ol>
<li>Añada la muestra homogeneizada en el filtro y lavar a través vertiendo lentamente 12 – 14 mL de frío HBB.</li>
		<li>Transferencia de muestras de filtrado a un tubo cónico de 15 mL con y guardar a 4 ° C.</li>
	</ol>
</li>
	<li>
Centrifugar las muestras a 400 x g durante 5 min a 4 ° C. Aspire de sobrenadante.
	<ol>
<li>Lyse células de sangre rojas añadiendo 1 mL de tampón de lisis de amonio cloruro de potasio (ACK) para cada muestra. Resuspender las muestras con un vórtex e incubar a temperatura ambiente durante 5 minutos.</li>
		<li>Una vez que ha completado la lisis, agregar 5 – 8 mL de frío de Hank balanceado sal solución (HBSS) para detener la hemólisis.</li>
	</ol>
</li>
	<li>Centrifugar las muestras a gfor 400 x 5 min a 4 ° C. Aspire de sobrenadante.</li>
	<li>Añadir 1 mL de tampón FACS a cada muestra y su traslado a un tubo de microcentrífuga de 1,5 mL con. Nota: El protocolo puede hacer una pausa aquí para un máximo de 2 h con las muestras almacenadas a 4 ° C.</li>
</ol>4. flujo Cytometry de tinción y análisis
<ol><li>
Centrifugar las muestras a 400 x g durante 5 min a 4 ° C.
	<ol>
<li>Para reducir el atascamiento no específico anticuerpo, diluido 1: 100 anti-CD16/CD32 y bloqueador de monocitos (1:20) (véase Tabla de materiales) que representa el 50 FACS μl por muestra. Incubar a temperatura ambiente durante 15 minutos.</li>
		<li>Preparar la contabilidad de dilución (1: 250 para cada anticuerpo) del anticuerpo para 50 μl de FACS por muestra. Nota: Un tinte de viabilidad puede incluirse en el cóctel de anticuerpos para excluir las células muertas. Por otra parte, exclusión de tamaño pequeño por análisis de flujo puede ser usado (figura 1C y D).</li>
		<li>Sin lavar, agregar 50 μl de mezcla principal anticuerpo para cada uno de la muestra y mezclar.</li>
		<li>Incubar las muestras a 4 ° C por 30 min en la oscuridad.</li>
	</ol>
</li>
	<li>Añadir 600 μl FACS a cada muestra para lavar y centrifugar las muestras a 400 x g durante 5 min a 4 ° C.</li>
	<li>Aspirar el sobrenadante y resuspender el pellet en 300 μL FACS. Mantener las muestras a 4 ° C en la oscuridad. Nota: Algunos colorantes de viabilidad, como DAPI, requieren tinción dentro de 10 minutos antes de ejecutar la citometría de flujo.</li>
	<li>Analizar mediante citometría de flujo (ver figuras 1 y 2) dentro de lo próximo 3 h. Si más tiempo es necesario, a corto plazo fijación utilizando un paraformaldehido de baja concentración (0.5 – 1% en PBS) se recomienda para preservar la coloración. Nota: Se recomienda filtrar las muestras por un colador de célula 40 μm antes de análisis de citometría de flujo iniciar para evitar la obstrucción de los fluidos de la citometría de flujo.</li>
</ol>

<ol>
	<li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+natural+history+of+human+myocarditis.">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li><li>Epelman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Pinto, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814 (2012).</li><li>Lavine, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+function+of+cardiac+macrophages.">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li><li>Hulsmans, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Nahrendorf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li><li>Clemente-Casares, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD103(+)+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis.">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li><li>Van der Borght, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+Infarction+Primes+Autoreactive+T+Cells+through+Activation+of+Dendritic+Cells.">Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.</a> Cell Reports. 18 (12), 3005-3017 (2017).</li><li>Galkina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+migration+of+effector+CD8++T+cells+into+the+interstitium+of+the+normal+lung.">Preferential migration of effector CD8+ T cells into the interstitium of the normal lung.</a> Journal of Clinical Investigation. 115 (12), 3473-3483 (2005).</li><li>Edelson, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+CD103++dendritic+cells+form+a+unified+subset+developmentally+related+to+CD8alpha++conventional+dendritic+cells.">Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells.</a> Journal of Experimental Medicine. 207 (4), 823-836 (2010).</li></ol>

Los autores no tienen nada que revelar.

Inflamación miocardio o la miocarditis, es una característica de las enfermedades cardiovasculares más. Sin embargo, el miocardio no está desprovista de sus propios componentes inmunes en Estados no patológicos. Durante el estado estacionario, muchas células inmunes residen en el miocardio y desempeñan las funciones esenciales de mantenimiento y protección. La caracterización de estas diversas poblaciones de células no habría sido posible sin métodos como la que se presenta en el presente Protocolo.
<p cl...

Diferentes formas de estrés miocárdico o lesiones, incluyendo isquémico (isquemia reperfusión o infarto de miocardio) y no isquémica (hipertensión o miocarditis), promover el reclutamiento de células inflamatorias con reparadora y protectora, sino también propiedades patógenas. Ya en 1891, Romberg describió por primera vez la presencia de infiltrados celulares en el miocardio de pacientes infectados por el tifus y la fiebre escarlata1. Sin embargo, el estudio detallado de las células inmunes cardiacas requiere el desarrollo de las técnicas más avanzadas de immunophenotyping. En consecuencia, sólo recientemente hemos empezado a entender que una población diversa de células inmunes con papeles de mantenimiento esencial durante el estado estacionario en el miocardio.
La histología ha sido y sigue siendo, el método más común para caracterizar a las células inmunes cardiacas durante la inflamación. Sin embargo, mientras que la histología representa una valiosa herramienta de diagnóstico, su uso en el estudio de las células inmunes cardiacas tiene limitaciones importantes. Las células inmunes que reside en el miocardio representan una proporción muy pequeña de las células totales y son más o menos uniformemente distribuidas en un espacio proporcionalmente enorme. Del mismo modo, varias formas de inflamación cardiaca, como la miocarditis viral, muestran patrones focal de inflamación. Esto significa que un análisis minucioso del sistema inmune del corazón requieren a menudo grados más altos de toma de muestras histológicas, aumentando el costo para reducir el sesgo. Por otra parte, la histología proporciona una cantidad muy limitada de información útil en la identificación de la célula (por ejemplo, el número de parámetros). Con un creciente número de subconjuntos de la célula que requieren la utilización de múltiples marcadores de expresión (incluyendo marcadores de superficie, factores de transcripción o moléculas secretadas), citometría de flujo se ha consolidado como la herramienta más potente para Inmunofenotipificación, debido al bajo costo y alto rendimiento.
El uso de técnicas de immunophenotyping depende estrechamente en el desarrollo de protocolos de digestión eficiente que permitió para el análisis de células individuales de un gran número de células. El desarrollo de exhaustivos protocolos de extracción de la célula abre una nueva ventana de oportunidades en el estudio de la inmunología cardiaca. A través de la combinación de digestión, citometría de flujo y la transcriptómica, las principales poblaciones de macrófagos y células dendríticas que residen en el corazón han sido caracterizados2,3. La población más abundante de las células inmunes cardiacas durante el estado estacionario son CD64++ de MerTK los macrófagos, que pueden dividir más a fondo en su expresión de CCR2 o CD11c2base. CCR2+ macrófagos se originan de la hematopoyesis de la médula ósea adulta, mientras que la mayoría de CCR2 los macrófagos– , que pueden expresar altos o bajos niveles de MHC-II, son principalmente de origen prenatal. Los macrófagos realizan funciones claves como reparación4 y retiro de escombros5 lesiones o apoyando la conectividad eléctrica6 en estado estacionario. Durante diferentes formas de la inflamación se producen una importante afluencia de Ly6CHola MHC-IIlo CD64int monocitos, que más tarde se diferencian en Ly6CHola CCR2+ los macrófagos2,7 . Una población perceptiblemente más pequeña, pero importante, de las células que residen en el miocardio de los individuos sanos se compone de células dendríticas8,9. Los dos subconjuntos principales de DCs convencionales cardiacas (CDC) se han caracterizado recientemente: cDC1 (CD103+ DCs) y cDC2 (CD11b+ DCs). DCs desempeñan papeles importantes en la defensa contra la infección8 pero también pueden promover uno mismo-lesión durante la inflamación, particularmente durante el infarto de miocardio9.
Aquí, describimos un método simple para el aislamiento de las células inmunes cardíacas viables de miocardio de ratón. El método combina la digestión enzimática y mecánica con filtración celular-colador para obtener una suspensión unicelular que puede ser analizada, o más purificada, por clasificación de citometría de flujo o enriquecimiento de grano magnético. Las medidas exactas de células del miocardio extravasculares requieren perfusión cardiaca con el fin de eliminar los posibles contaminantes del torrente sanguíneo de la microcirculación cardiaca. Además, presentamos un paso opcional del etiquetado intravascular de células inmunes que pueden utilizarse para distinguir aún más células del miocardio de contaminantes intravasculares, basados en el Galkina et al protocolo10. Por último, presentamos un análisis de citometría de flujo básico para identificar las principales subpoblaciones de macrófagos y cDC.

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse: Batf3-/-</td>
			<td>The Jackson Laboratory</td>
			<td>JAX: 013755</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of extravascular immune cells of the heart

El sistema inmune es un componente esencial de un corazón sano. El miocardio es hogar de una rica población de subconjuntos diferentes de célula inmune con compartimentación funcional durante el estado estacionario y durante diferentes formas de la inflamación. Hasta hace poco, el estudio de las células inmunes en el corazón requiere el uso de la microscopía o protocolos de digestión mal desarrollados, que proporcionaron suficiente sensibilidad durante la inflamación severa pero no pudieron con confianza identifican pequeños — pero clave — las poblaciones de células durante el estado estacionario. Aquí, discutimos una combinación simple método enzimático (colagenasa, hialuronidasa y ADNasa) y digestión mecánica de corazones murino precedido por la administración intravascular de anticuerpos fluorescente etiquetado para diferenciar pequeños pero inevitable contaminantes intravascular de la célula. Este método genera una suspensión de células viables aisladas que pueden ser analizadas por citometría de flujo para identificación, phenotyping y cuantificación, o más purificado con clasificación celular activado por fluorescencia o la separación magnética de grano para transcripcionales estudios análisis o in vitro . Incluimos un ejemplo de un análisis de citometría de flujo de paso a paso para distinguir las poblaciones de células dendríticas del corazón y macrófagos clave. Para un experimento de tamaño medio (10 corazones) la terminación del procedimiento requiere de 2-3 h.

Hasta la fecha, ningún buen método ha sido diseñado para aislar las células inmunes de otras componentes de la célula cardiaca, como cardiomiocitos. Por lo tanto, el análisis de la suspensión de la célula cardiaca mediante citometría de flujo requiere la sincronización con CD45 para identificar las poblaciones de células inmunitarias, seguidas por celular único y pequeño tamaño exclusión bloquea (figura 1A). Alternativamente, u...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

Aislamiento e identificación de las células inmunes Extravascular del corazón

Este protocolo presenta un método sencillo y eficaz para aislar, identificar y cuantificar las células inmunes que reside en el miocardio de ratones durante el estado estacionario o inflamación. El protocolo combina la digestión enzimática y mecánica para la generación de una suspensión unicelular que puede ser más analizada por citometría de flujo.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Research

JoVE Journal

Immunology and Infection

12.4K vistas.  University Health Network (UHN). Este protocolo presenta un método sencillo y eficaz para aislar, identificar y cuantificar las células inmunes que reside en el miocardio de ratones durante el estado estacionario o inflamación. El protocolo combina la digestión enzimática y mecánica para la generación de una suspensión unicelular que puede ser más analizada por citometría de flujo.

Video: Isolation and Identification of Extravascular Immune Cells of the Heart

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Una introducción a las avispas parasitarias de<em&gt; Drosophila</em&gt; Y la respuesta inmune antiparasitario

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagen intravital del timo de ratón utilizando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Diferenciar los roles funcionales de la expresión génica de las células inmunes y no inmunes en la colitis de ratón por Trasplante de Médula Ósea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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 ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Aislamiento de tejido adiposo células inmunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...