Name: selective harvesting of marginating-pulmonary leukocytes
Uploaded: 2016-03-11T15:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Los autores y este trabajo fueron apoyados por NIH / NCI subvención # R01CA172138 (a SBE).

Los procedimientos que implican sujetos animales han sido aprobados por el Comité de Cuidado y Uso de Animales Institucional (IACUC) de la Universidad de Tel-Aviv. 1. Protocolo de rata <ol><li> Preparativos <ol><li> Organizar 2 mariposa 21 G agujas. Opcionalmente, se preparan 2 mariposa 21 G-agujas romas filo presentando el borde afilado de la aguja. </li><li> Esterilizar instrumentos quirúrgicos: 2 pares de tijeras, pinzas afiladas embotado, pinzas hemostáticas, pinzas de dientes de tejidos esterilizados en autoclave a 121 ° C durante al menos 30 minutos en el establecimiento de la gravedad (en seco). </li><li> Preparar heparinizada PBS (30 unidades / ml) mediante la adición de 30 unidades de heparina por ml de solución salina tamponada con fosfato (1x PBS). Utilizar a temperatura ambiente. 35 ml es el volumen mínimo necesario por animal. </li><li> Transmitir heparinizada PBS en las líneas de bombas peristálticas y las agujas de mariposa para evitar burbujas de aire. </li></ol></li><li> La perfusión de los pulmones y la recolección de MP-leucocitos <ol><li> La eutanasia a los animales por una sobredosis de 8% de isoflurano. Confirmar la eutanasia por el cese de monitoreo de respiración. </li><li> Tras el cese de la respiración, de inmediato abrir las cavidades peritoneales y el pecho con las tijeras y pinzas estériles diente-tejido. Realizar una incisión abdominal en la línea media a lo largo del abdomen hasta el proceso xifoides. </li><li> Levantar el esternón utilizando los fórceps, el corte con cautela la caja torácica en ambos lados, sin perforar cualquier órgano interno o grandes vasos sanguíneos. Sujetar una pinza hemostática en el esternón, y rostral plegar la caja torácica para exponer el complejo cardiopulmonar. </li><li> Insertar una aguja de mariposa en el ventrículo derecho del corazón, y dentro de aproximadamente un minuto recoger el máximo volumen de sangre en una jeringa (~ 6 ml de un animal de 250 g). </li><li> Abrazadera de la vena cava con una pinza hemostática para evitar la perfusión hacia atrás del hígado (véase la discusión). </li><li> Mantenga la aguja mariposa dentro del ventrículo derecho, mientras que la sustitución de la jeringa que contiene la sangre con el tubo de salida de la peristaltic bomba. </li><li> Inserte una segunda aguja de mariposa conectada a una jeringa de recolección 5 ml en el ventrículo izquierdo, evitando la penetración del septo interventricular. </li><li> Encienda la bomba peristáltica a una velocidad de aproximadamente 5 ml / min y recoger suavemente en la jeringa los primeros mililitros de líquido de perfusión que están contaminadas con sangre. Continuar hasta que el color se vuelve líquido de perfusión de oscuro a rojo pálido. (Descartar el perfuste contaminada con sangre). </li><li> Sin cese de la bomba peristáltica, sustituir rápidamente la jeringa de recolección 5 ml con una jeringa de 20 ml cosecha y recoger 20 ml de líquido de perfusión pulmonar que emplea una mayor velocidad de perfusión (hasta 7 ml / min). Monitorear continuamente el flujo de perfundido en la jeringa de recogida, evitando al mismo tiempo la formación de vacío. </li><li> Cese el flujo de la bomba peristáltica. </li></ol></li><li> Extracción leucocitos <ol><li> Centrifugar el perfundido durante 10 minutos a 400 x g. </li><li> Aspirar el líquido. </li><li>Añadir 10 ml de PBS o medio, de centrifugación a 400 xg durante 10 min, y aspirar el líquido. Repita este paso dos veces. El uso de PBS o medio depende de la consiguiente uso de la muestra. </li><li> Añadir 20 ml de PBS o medio, de centrifugación a 400 xg durante 10 min, y aspirar el líquido. </li><li> Reconstituir las células a la concentración deseada, con base en el uso de la muestra (FACS, PCR, etc.). </li></ol></li></ol> 2. Protocolo de ratón <ol><li> Preparativos <ol><li> Organizar 2 mariposa 25 G agujas. Opcionalmente, se preparan 2 mariposa 25 G-agujas romas filo presentando el borde afilado de la aguja. </li><li> Esterilizar instrumentos quirúrgicos: 2 pares de tijeras, pinzas afiladas embotado, pinzas hemostáticas, pinzas de dientes de tejidos esterilizados en autoclave a 121 ° C durante al menos 30 minutos en el establecimiento de la gravedad (en seco). </li><li> Preparar heparinizada PBS (30 unidades / ml) mediante la adición de 30 unidades de heparina por ml de solución de 1x PBS. Utilizar a temperatura ambiente. 25 ml es el volumen mínimo necesario por animal. </li><li> Stream heparinizada PBS en las líneas de bombas peristálticas y las agujas de mariposa para evitar burbujas de aire. </li></ol></li><li> La perfusión de los pulmones y la recolección de MP-leucocitos <ol><li> La eutanasia a los animales por una sobredosis de 8% de isoflurano. Confirmar la eutanasia por el cese de monitoreo de respiración. </li><li> Tras el cese de la respiración, de inmediato abrir las cavidades peritoneales y el pecho con las tijeras y pinzas estériles diente-tejido. Realizar una incisión abdominal en la línea media a lo largo del abdomen hasta el proceso xifoides. </li><li> Levantar el esternón utilizando los fórceps, el corte con cautela la caja torácica en ambos lados, sin perforar cualquier órgano interno o grandes vasos sanguíneos. Sujetar una pinza hemostática en el esternón, y rostral plegar la caja torácica para exponer el complejo cardiopulmonar. </li><li> Abrazadera de la vena cava con una pinza hemostática para evitar la perfusión hacia atrás del hígado (véase la discusión). </li><li> Insertar una aguja de mariposa conectada a la tubería de salida de la bomba peristáltica a la derecha ventricle del corazón, y una segunda aguja de mariposa conectada a una jeringa de recolección 2 ml en el ventrículo izquierdo del corazón, avoidingpenetration del tabique interventricular. </li><li> Encienda la bomba a una velocidad de aproximadamente 2 ml / min y recoger los primeros ml de líquido de perfusión contaminada con sangre en la jeringa de recolección hasta que el líquido de perfusión se convierte de oscuro a rojo pálido (Descartar el perfuste contaminada con sangre). </li><li> Sin cese de la bomba peristáltica, sustituir rápidamente la jeringa de recolección 2 ml con una jeringa de 10 ml de cosecha y recoger 10 ml de líquido de perfusión pulmonar que emplea una mayor velocidad de perfusión (hasta 4 ml / min). Monitorear continuamente el flujo de perfundido en la jeringa de recogida, evitando al mismo tiempo la formación de vacío. </li><li> Cese el flujo de la bomba peristáltica. </li></ol></li><li> Extracción leucocitos <ol><li> Centrifugar el perfundido durante 10 minutos a 400 x g. </li><li> Aspirar el líquido. </li><li> Añadir 10 ml de PBS o medio de centrifugación a 400,xg durante 10 min, y aspirar el líquido. Repita este paso tres veces. El uso de PBS o medio depende de la consiguiente uso de la muestra. </li><li> Añadir 10 ml de PBS o medio, de centrifugación a 400 xg durante 10 min, y aspirar el líquido. </li><li> Reconstituir las células a la concentración deseada, con base en el uso de la muestra (FACS, PCR, etc.). </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, 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+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, 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=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

Los autores no tienen nada que revelar.

El método presentado en este documento permite la recogida selectiva y el estudio de la población única de MP-leucocitos. En comparación con los leucocitos esplénicos o circulante, la población MP se caracteriza por una composición distinta de subpoblaciones de leucocitos, los niveles de activación más altos, mayor liberación de varias citoquinas, y más altos niveles de ARNm de pro- y citocinas antiinflamatorias 2,3. Específicamente, hemos demostrado que las células MP-NK son más citotóxicos qu...

Los leucocitos se adhieren a los capilares de los pulmones (es decir, marginando-pulmonar (MP) leucocitos) 1 se mostraron para exhibir composición de leucocitos distinto y actividad única en comparación con los leucocitos de otros compartimentos inmunes (por ejemplo, la circulación, el bazo, la médula ósea) 2- 4. Por ejemplo, exhiben MP-leucocitos superior natural killer (NK) citotoxicidad frente a varias células tumorales, en comparación con circulantes y esplénicas células NK, así como los niveles diferenciados de ARN mensajero (ARNm) y aumento de la secreción de citoquinas pro- y anti-inflamatorias. La composición de células también se diferencia de leucocitos circulantes como MP-leucocitos tienen una relación más alta de la inmunidad innata / adaptativa en comparación con los leucocitos circulantes (50% vs. 30%, respectivamente). El objetivo del método presentado en este documento es para permitir la recolección selectiva de MP-leucocitos, con el fin de estudiar este compartimiento inmune importante y única (población de células), y para elucidate el impacto de las diversas manipulaciones (por ejemplo, la activación inmune) en estas células específicas. Para comprender el significado de esta población única, es importante tener en cuenta que el sistema inmune puede controlar las células tumorales, las micrometástasis, y la enfermedad residual circula a través de las funciones in vivo de la inmunidad mediada por células (CMI). Esta capacidad es evidente a pesar del fracaso precedente del sistema inmunológico para controlar el tumor primario, y apoyado por amplia en pruebas in vivo en pacientes con cáncer y en modelos animales 5. Es importante destacar que estos resultados son a menudo incompatibles con los estudios in vitro y ex vivo, que informan que la mayoría de las células tumorales autólogas son resistentes a la citotoxicidad mediante la circulación de leucocitos en muestras de sangre de seres humanos y animales (medido por ensayos de citotoxicidad) 6,7. Esta discrepancia puede ser atribuida a la existencia in vivo de poblaciones de leucocitos distintos, tales como los antesmencionada población de leucocitos MP, y concretamente su subpoblación de células NK activadas 3. De hecho, las células tumorales (singénicos MADB106), que se encontraron ser resistente a la circulación y leucocitos esplénicos, se demostró que la lisis por las células NK MP-3,8. Por lo tanto, las células supuestamente &#39;NK-resistentes&#39; MADB106 que hacen metástasis a los pulmones de fischer344 ratas (F344) están controlados por las células MP-NK, pero no por las células NK esplénicas, que comúnmente se estudian dada su facilidad de acceso o circulante. Purificado y activos MP-leucocitos son inaccesibles a través de los métodos de cosecha estándar de leucocitos de los pulmones, que se basan en la molienda tejido pulmonar o la degradación biológica 9. Nuestro enfoque tiene dos ventajas importantes en comparación con los enfoques de procesamiento de tejidos. En primer lugar, el enfoque de perfusión cosecha selectivamente MP-leucocitos, separándolas de otras células que se originan en el parénquima pulmonar, intersticial, y compar bronco-alveolartments. En segundo lugar, la técnica de perfusión mejor conserva la integridad y el medio fisiológico de MP-leucocitos, a diferencia de la molienda y el procesamiento biológico se acerca que dañan las células, alterar su morfología, e inducen la producción y liberación de diversos factores que modulan la actividad inmune y específicamente suprimen NK 10 citotoxicidad. Los pulmones son un órgano principal objetivo para la metástasis del cáncer y para diversas enfermedades infecciosas. Todas las células malignas circulantes y células infectadas pasan a través de los capilares pulmonares, donde tienen que deformarse e interactuar con las células endoteliales de los capilares y leucocitos residentes. En estas condiciones, las células circulantes pueden ser fácilmente dirigidos por residentes MP-leucocitos. Por tanto, parece biológicamente ventajoso tener leucocitos activados en este compartimiento inmune, y es importante para estudiar este MP-población única en diferentes entornos biológicos, experimentales y clínicos. Es digno de notar que syactivación stemic inmune por varias-respuesta modificadores biológicos (por ejemplo, ácido poliinosínico-policitidílico (poli I: C) o oligodesoxinucleótidos tipo-C CpG (CpG-C ODN)) han demostrado para activar MP-leucocitos más de leucocitos circulantes 3, 8,11.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

La exposición MP-compartimiento de una composición de leucocitos subconjunto diferente en comparación con los leucocitos circulantes. Utilizando el análisis de citometría de flujo, se identificaron y se cuantificaron las subpoblaciones de leucocitos para caracterizar la composición de ambos leucocitos circulantes y MP. Granulocitos y linfocitos fueron identificados en base a primero y la cara dispersa. Dentro de los linfocitos, se identificaron NKRP-1 células brillantes...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

La recolección selectiva de leucocitos marginando-pulmonar

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K vistas.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Video: Selective Harvesting of Marginating-pulmonary Leukocytes

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Activación de los macrófagos alveolares murinos recolección y evaluación de celular inducida por nanopartículas polianhidrido

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Investigación

Bioingeniería

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Inmunología e Infección

Selective Harvesting of Marginating-hepatic Leukocytes

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

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

La recolección selectiva de leucocitos marginando-hepáticas

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

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

Cosecha de las células endoteliales de las puntas del globo de catéteres de Swan-Ganz después de cateterismo cardíaco derecho

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Medicina

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Análisis citométrico de flujo para la identificación de las células inmunes innatas y adaptativas del pulmón murino

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>Aislamiento de células inmunitarias y no inmunitarias de pulmones de ratón para citometría de flujo

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>Aislamiento de poblaciones vivas de células mieloides y epiteliales del pulmón de ratón

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

Aislamiento de poblaciones vivas de células mieloides y epiteliales del pulmón de ratón

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Establecimiento de cultivos in vitro de LLEC mediante selección magnética de células

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Autor destacado: Desvelando las funciones celulares y las posibles implicaciones clínicas de las células endoteliales linfáticas leptomeníngeas 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Recolección y cultivo primario de células endoteliales linfáticas leptomeníngeas

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

La recolección selectiva de leucocitos marginando-pulmonar

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Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Cosecha de las células endoteliales de las puntas del globo de catéteres de Swan-Ganz después de cateterismo cardíaco derecho

Análisis citométrico de flujo para la identificación de las células inmunes innatas y adaptativas del pulmón murino

Aislamiento de poblaciones vivas de células mieloides y epiteliales del pulmón de ratón

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Recolección y cultivo primario de células endoteliales linfáticas leptomeníngeas