Este trabajo fue apoyado por un UNCF-Merck Postgrado de Investigación en Ciencias de Becas Tesis (JLM), Instituto Nacional del Corazón, los Pulmones y la Sangre de subvenciones del Instituto SR1-HL-62228 (JSJ) y RO1 HL-073.990-(JSJ).

1. Preparación de la solución de Hanks balance de sal (HBSS) <ol><li> Autoclave un vaso de vidrio L, 1000 ml cilindro graduado, y una barra de agitación. </li><li> Reconstituir la solución en polvo HBSS en el autoclave un vaso de vidrio L hasta la marca de 900 ml con agua destilada. </li><li> Mezcle la solución HBSS con 3,4 g de sal de Na + Hepes y ajustar el pH a un valor entre 7.35 a 7.41 utilizando HCl concentrado o NaOH. </li><li> Una vez que el pH deseado se ha alcanzado y se mantiene estable, añadir 5 g de albúmina de suero bovino para la solución a temperatura ambiente hasta que la albúmina se disuelva por completo. </li><li> Añadir 10 ml de agua destilada con antibióticos, antimyotic, a la solución y hasta que doble el volumen total es de 1000 ml de la marca. </li><li> Mezcle bien la solución. </li><li> Filtrar la solución HBSS y separarla en 50 ml tubos Falcon para su uso posterior. Tubos adicionales se pueden congelar y usar en una fecha posterior. </li></ol> 2. Animal PREPARACIÓn - La inserción del tubo traqueal <ol><li> Preparar una bandeja con los instrumentos necesarios y material para la inserción de un tubo traqueal (es decir, pinzas, tijeras, tubos traqueales, 3-0 negro sutura trenzada, ventilador de pequeños animales, etc.) </li><li> Una vez que la rata está completamente anestesiado con un cóctel de ketamina / xilazina en la proporción de 2 a 1 la ketamina xilazina, afeitarse el abdomen, el pecho y la garganta de la rata. Para asegurarse de que un profundo estado anestésico que se ha logrado, una pizca pie de la rata y la verificación de la retirada del pedal de la extremidad. Asegúrese de que la rata está profundamente anestesiado antes de hacer las incisiones. </li><li> Lugar a la rata con la cabeza erguida sobre una tabla de cortar que se inclina en un ángulo de 30 ° </li><li> Haga una incisión en la piel de la mitad del abdomen por debajo de la mandíbula inferior. Diseccionar los músculos del triángulo anterior del cuello para exponer la tráquea. </li><li> Coloque un 3-0 sutura trenzada negro en la tráquea. </li><li> Hacer una incisión en la tráquea entre los anillos cartilaginosos und insertar el tubo traqueal. Ate la sutura firmemente para asegurar el tubo traqueal en la tráquea. </li><li> Conecte el tubo traqueal a un ventilador de pequeños animales. </li></ol> 3. Instrumento de preparación para el aislamiento de células inmunes <ol><li> Preparar una bandeja con los instrumentos para el procedimiento de celda de aislamiento inmunológico (es decir, pinzas, tijeras, HBSS, recortado blandas teflón calibre 24 Baxter intravascular sobre la aguja rápida-cath) </li><li> Preparar dos jeringas de 10cc, llenar una jeringa con HBSS para introducir el tampón en el saco pericárdico y reservar el otro para aspirar las células HBSS que contiene. 3.2.1. Reducir la punta de teflón del calibre 24 Baxter intravascular sobre la aguja rápido cateterismo (1,6 cm) por 1 / 2 a 3 / 4 de su longitud original para el procedimiento de aislamiento. </li><li> Coloque todas las jeringas, tampones, y un vacío de 15 ml tubo Falcon (para mantener el líquido extraído una vez haya finalizado el procedimiento) en el hielo. </li></ol> &lt;P CLASS = &quot;jove_title&quot;&gt; 4. Aislamiento de células inmunes <ol><li> Haga una incisión en la pared abdominal (a lo largo de la línea alba) hasta llegar a la apófisis xifoides. </li><li> A partir ligeramente lateral a la apófisis xifoides, hacer incisiones bilaterales a través de la caja torácica continua en un ángulo hacia la axila. Tenga cuidado de no perforar los pulmones, el corazón o pericardio. </li><li> Una vez que las incisiones a ambos lados del esternón se completa, diseccionar el diafragma de las costillas inferiores, de corte lateral a medial. Nota: Tenga siempre consciente de que el pericardio se conecta con el esternón y el diafragma para evitar daños. Utilice el ligamento falciforme como un hito tener en cuenta que el pericardio se adjunta en el lado opuesto del diafragma. Además, si el pericardio se corta, la capacidad de aislar las células se pierde. </li><li> Con cuidado, levante un lado de la caja torácica para exponer la cavidad torácica y el corazón (encapsuladas por el pericardio).</li><li> Tome la jeringa llena de HBSS y encontrar un punto en el saco pericárdico aproximadamente a medio camino entre el esternón y el corazón (de preferencia más rostral del caudal) e inserte la punta de teflón de la acorta rápido cateterismo a través de las dos capas del pericardio. </li><li> Una vez que el catéter está en su lugar, proceder con cuidado llenar el espacio pericárdico con 2 a 3 ml de solución tampón HBBS. Usted sabrá de inmediato si el catéter está bien colocado cuando se observa el espacio pericárdico en expansión. Si no es testigo de relleno, retirar y volver a colocar el quick-cath. </li><li> Cuando el saco pericárdico se llena, tome la jeringa de 10 cc demás y coloque la punta de nuevo en el pericardio y comenzar a aspirar el buffer. El orificio de entrada puede utilizar el mismo u otro puede ser creado. Asegúrese de que la punta no se acercan demasiado a las paredes del saco pericárdico, mientras que proporciona la succión, especialmente en la parte inferior del corazón. Esto podría conducir a la perforación del saco pericárdico y la pérdida de células que contienen célulass. </li><li> Mientras se aspira el tampón, la punta del catéter se puede mover en el interior del saco pericárdico con el fin de recoger de amortiguación tanto como sea posible. </li></ol> Nota: En caso de hacerlo, un poco romper el pericardio y haga un hoyo en la parte superior del corazón más grande que el agujero original, no se preocupe. Siempre y cuando el pericardio rodea todavía lo suficiente del corazón aún puede realizar otro lavado sobre el corazón, aunque con una menor cantidad de buffer. Para la recolección de una cantidad óptima de las células, de tres a cuatro lavados se necesitan. <ol start="9"><li> Coloque el buffer se extrae en los tubos Falcon que se colocaron en hielo. </li><li> Quitar el corazón, los pulmones y / o cualquier otro tejido, si es necesario para estudios posteriores. Después de la recolección de células del sistema inmune y otros tejidos, la rata debe ser rápido y la eutanasia humanitaria. </li><li> Centrifugar las muestras recogidas inmune celular durante 10 minutos a 200 gramos a 4 ° C. Siempre mantener los tubos en hielocuando no esté en uso. </li><li> Eliminar el sobrenadante y reconstituir el precipitado en 1 ml de tampón de Hyclone o HBSS. </li></ol> Nota: Es importante tener en cuenta el número de células del sistema inmune obtenidas de cada corazón después del procedimiento celda de aislamiento inmunológico. 5. Los resultados representativos: Un ejemplo de los mastocitos aislados cardíaca obtenidos con esta técnica y se tiñeron con azul de toluidina y safranina O / azul alcián se presentan en las figuras 2A y 2B, respectivamente. Como se puede observar en la Figura 3, el mástil cardiaco, mantiene su capacidad de liberación de histamina después del tratamiento con activadores conocidos como compuesto 48/80 y la sustancia P como se describe en Morgan et al. 9 diagramas de dispersión Representante de la citometría de flujo de células CD4 + y las células CD8 + se presentan en las figuras 4 y 5, respectivamente. Los linfocitos T son más identificados y fenotipo en una población de células de esta técnica. Figura 1. Diagrama de flujo del procedimiento cardíaco el aislamiento de células inmunes. <img alt="Figura 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . Imágenes de la figura 2 histológico representante de los mastocitos cardiaca se tiñen de la siguiente manera: (A) con azul de toluidina y (B) son safranina O / azul Alcian. <img alt="Figura 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Figura 3. Estudio funcional de las células cardiacas del mástil aislado de la región epicárdica del miocardio y estimuladas con compuesto 48/80 (10μg/mL) y la sustancia P (10μM). <img alt="Figura 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Figura 4. Representante de flujo de citometría de diagramas de dispersión de las células CD4 + aisladas células marcadas con la sustancia P (1:100 dilution). <img alt="Figura 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Figura 5. Citometría de flujo Representante de CD8 + aisladas células marcadas con la sustancia P (dilución 1:100).

<ol>
	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+mast+cell+degranulation+prevents+left+ventricular+remodeling+induced+by+chronic+volume+overload+in+rats.">Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.</a> Journal of Cardiac Failure. 11, 548-556 (2005).</li><li>Chancey, A. L., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mast+cell-mediated+activation+of+gelatinase+and+alteration+of+ventricular+diastolic+function.">Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function.</a> Am. J. Physiol. Heart. Circ. Physiol. 282, H2152-H2158 (2002).</li><li>Levick, S. P., Gardner, J. D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

No tenemos nada que revelar.

Este método de aislamiento de las células inmunes cardíaco permite el análisis de su función, el fenotipo, y su vencimiento sin que la activación no deseada que se produce normalmente durante la tradicional técnica enzimática aislamiento digestión. La media del número total de células que se obtienen con esta técnica es de alrededor de 400.000 por el corazón de rata, y tinción diferencial y el análisis de citometría de flujo de estas células indica población mixta de linfocitos T (~ 70%), los macrófag...

<table border="1"><tbody><tr><td> Nombre de los reactivos y equipos </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Salina equilibrada de Hanks </td><td> Sigma </td><td> H4891 </td><td> Una botella hace un balance de la sal L Hanks Solución. Sal reconstituir Hanks &#39;Balance Solución como parte del procedimiento. </td></tr><tr><td> Antibióticos Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 ml Nalogene filtro de botella </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> De 6 N, agregar volúmenes iguales de HCL a desionizadael agua. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> De 5 N, agregar volúmenes iguales de NaOH al agua desionizada. </td></tr><tr><td> El agua destilada </td><td></td><td></td><td></td></tr></tbody></table> Tabla de Equipos: <table border="1"><tbody><tr><td> Nombre de los reactivos y equipos </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 3-O sutura de seda trenzada </td><td> Ethicon Productos </td><td> SA64H </td><td></td></tr><tr><td> Ventilador de Pequeños Animales </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Ajuste la frecuencia respiratoria a 40,5 respiraciones por minuto. </td></tr><tr><td> Bisel calibre 18 agujas </td><td> BD Biosciences </td><td> 305199 </td><td> Trim aguja de 1 / 2 a 3 / 4 de su longitud original para su uso como un tubo traqueal. </td></tr><tr><td> Consejo de teflón (con aguja retirado) de calibre 24, Baxter rápido cateterismo catéter </td><td> Baxter </td><td> 2N111 </td><td> Reducir la longitud de la punta de teflón de 1 / 2 a 3 / 4. </td></tr><tr><td> Jeringa de 10 cc </td><td> Fisher Scientific </td><td> 14 a 823-16B </td><td></td></tr><tr><td> 15 ml tubos Falcon </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B centrífuga refrigerada </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Recuerde que antes de enfriar y mantener a 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemocitómetro </td><td> Hausser Científico </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardiaco las células inmunes están ganando interés por el papel que desempeñan en el remodelado patológico de muchas enfermedades cardíacas 1-5 Estas células inmunes, que incluyen los mastocitos, las células T y los macrófagos,. Almacenar y liberar una variedad de mediadores biológicamente activos, incluyendo citoquinas y proteasas como la triptasa. 6-8 Estos mediadores han demostrado ser jugadores clave en el metabolismo de la matriz extracelular mediante la activación de metaloproteinasas de la matriz o causar la acumulación de colágeno mediante la modulación de la función de los fibroblastos cardíacos. 9-11 Sin embargo, las técnicas disponibles para el aislamiento de las células inmunes tienen cardiaca sido problemático debido a que utilizan la colagenasa bacteriana para digerir el tejido miocárdico. Esta técnica provoca la activación de las células del sistema inmune y por lo tanto una pérdida de la función. Por ejemplo, los mastocitos se cardiaca significativamente menos sensibles a los compuestos que causan la degranulación. 12 Por tanto, hemos desarrollado una técnica que permite taislamiento de las células cardiacas funcionales inmune que daría lugar a una mejor comprensión del papel de estas células en la enfermedad cardíaca que. 13, 14 Este método requiere una familiaridad con la localización anatómica de xifoides de la rata proceso, la axila y el ligamento falciforme, y el pericardio del corazón. Estas señales son importantes para aumentar el éxito del procedimiento y para asegurar un mayor rendimiento cardíaco de células inmunes. Estas células inmunes aisladas cardíaco puede ser utilizado para la caracterización de la funcionalidad, el fenotipo, la madurez y la cultura co-experimentos con otras células cardíacas para obtener una mejor comprensión de sus interacciones.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Este método para aislar células del sistema inmune funcional del corazón es una alternativa a los métodos convencionales de digestión de colagenasa, que provoca la activación de células inmunes no deseadas, lo que resulta en una disminución de la respuesta de estas células. Nuestro método de aislamiento de los rendimientos de las células inmunes funcional cardiaca, evitando los problemas asociados con la digestión enzimática.

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 ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K vistas.  University of South Carolina- School of Medicine. Este método para aislar células del sistema inmune funcional del corazón es una alternativa a los métodos convencionales de digestión de colagenasa, que provoca la activación de células inmunes no deseadas, lo que resulta en una disminución de la respuesta de estas células. Nuestro método de aislamiento de los rendimientos de las células inmunes funcional cardiaca, evitando los problemas asociados con la digestión enzimática.

Video: Isolation of Functional Cardiac Immune Cells

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 ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

La detección de glucógeno en las células mononucleares de sangre periférica con ácido periódico de Schiff tinción

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Aislamiento de las células dendríticas del tracto reproductivo femenino humano estudios fenotípica y funcional

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Aislamiento de mastocitos de peritoneo derivado y su caracterización funcional con Ca2 +-proyección de imagen y pruebas de degranulación

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...