Agradecemos al Dr. Itzjak Oschry y el Dr. Orit Sagi-Asif, por su ayuda con la clasificación de FACS. Esta investigación fue apoyada por las subvenciones a NE del Programa Europeo sobre el Séptimo Programa Marco de la Unión (FP7/2007-2013) en el marco del acuerdo de subvención n º [276890], de la Asociación de Cáncer de Israel (# 20110078), y de la Israel Cancer Research Fund (Investigación Career Development Award).

1. La disección del tejido mamario o la piel de ratones <ol><li> Antes de diseccionar el tejido deseado de los ratones, preparar los siguientes materiales y reactivos: Suministros: <ul><li> Herramientas de cirugía (lavado con detergente y después se sumergieron en 70% de etanol). </li><li> Superficie de espuma de poliestireno para descansar el ratón durante la disección. </li><li> Alfileres o agujas para sujetar ratones durante la disección. </li><li> Tarro de cristal con tapa para la digestión, que contiene una barra de agitación magnética (autoclave). </li><li> Baño de agua a 37 ° C, que contiene un agitador magnético sumergible y agua suficiente para sumergir el frasco de digestión, mientras descansa sobre la placa de agitación. </li></ul> Reactivos: <ul><li> Tampón FACS I: (4 ° C): Fosfato de Dulbecco salina tamponada con CMF (magnesio calcio libre) + 0,5% de BSA </li><li> Solución de colagenasa (hecha inmediatamente antes de su uso): 0,05 g de colagenasa II 0,05 g de colagenasa IV 0,01 g Deoxyribonucarrendar 20 ml tampón FACS I. Mantener la solución de colagenasa en hielo hasta su uso. </li><li> PharmLyse (Cloruro de Amonio Reactivo de Lisis). </li><li> Tampón FACS II: Fosfato de Dulbecco salina tamponada con CMF + 1% de FCS. </li></ul></li><li> La disección de las glándulas mamarias (véase también 17). <ul><li> Eutanasiar ratones hembras que utilizan dióxido de carbono (CO 2) inhalación. </li><li> En una superficie de espuma de poliestireno, coloque el ratón sobre la espalda y el pin con firmeza los cuatro miembros que utilizan agujas de calibre 25 (Figura 1A). Pulverizar el ratón generosamente con 70% de etanol. </li><li> Con unas pinzas, tire hacia arriba de la piel abdominal en la línea media y hacer una pequeña incisión con tijeras afiladas. </li><li> A partir de la incisión, corte la piel hasta el cuello del animal mientras que evita la perforación de las cavidades abdominales o torácicas. </li><li> Cortar la piel de las patas traseras de la incisión de línea media hacia la pierna, lo que resulta en una &quot;forma de Y&quot;. </li><li> Tire de la piel lejos del mouse cuerpo y fijar abajo, exponiendo las glándulas mamarias unidos a la parte inferior de la piel (Figura 1B). </li><li> Utilizar Q-consejos para separar suavemente las glándulas mamarias de la piel durante el corte del tejido conjuntivo, con pequeñas tijeras afiladas para separar la glándula mamaria hacia la espina dorsal del ratón. </li><li> Las glándulas abdominales (# 4, 5) son los mejores para este protocolo. También puede utilizar la glándula mamaria 2 º, pero hay que tener en cuenta que estas glándulas están anatómicamente situado en las proximidades del músculo pectoral que pueden ser difíciles de separar, lo que resulta en células de origen del tejido mixto. </li><li> Nota: Cuando la disección de tejido tumoral, se recomienda eliminar regiones necróticas. </li></ul></li><li> La disección de tejido de la piel: La forma más fácil de obtener tejido de la piel, sin tener que lidiar con la eliminación del vello es usar orejas de ratón. Si una mayor cantidad de tejido es necesario, puede afeitarse el pelo de ratón torso y diseccionar la piel. <ul><li> Eutanasiar ratones ucantar inhalación de CO 2. </li><li> Usando unas tijeras afiladas, corte ambos oídos y el lugar inmediatamente en el tarro de digestión que contiene un volumen pequeño (~ 0,5 ml) de PBS en hielo. </li></ul></li></ol> 2. Preparación de la glándula mamaria / piel suspensión de células individuales <ul><li> Coloque glándulas mamarias / piel en el tarro de digestión que contiene un pequeño volumen de PBS en hielo. Si el tejido es lavado con sangre x2 con PBS, y luego descartar el exceso de PBS. </li><li> Pique a fondo con tijeras curvas. </li><li> Añadir (nota: esta cantidad de solución de colagenasa de manera eficiente se disocian aproximadamente 5 g de tejido mamario o del tumor y las orejas de un máximo de 15 ratones) 20 ml de solución de colagenasa. </li><li> Coloque inmediatamente en una placa de agitación a 37 ° C baño de agua, y se incuba durante 15 minutos mientras se agitaba a velocidad media. </li></ul> Nota: Los tiempos óptimos de incubación puede variar si la digestión de otros tejidos. <ul><li> Para detener la reacción, agregar 30 ml de DMEM frío + 10% FCS. </li><li> Filtrar la mezcla de tejido / medios de comunicación a través de un filtro de 70 micras celda colocada en la parte superior de un tubo cónico de 50 ml. </li><li> Centrifugar el tubo cónico durante 5 min a 450 xg, a 4 ° C. </li></ul> 3. Lisis de los glóbulos rojos Nota: este paso no es necesario si los ratones eran de corazón perfundido con PBS antes de ser sacrificado. <ul><li> Aspirar el sobrenadante utilizando una pipeta de vidrio unido a vacío, sin alterar el sedimento celular. </li><li> Para lisar RBC, se resuspende el sedimento celular en 10 ml de solución PharmLyse e incubar durante 5 min a temperatura ambiente. </li><li> Neutralizar la solución PharmLyse añadiendo aproximadamente 40 ml de tampón FACS I. </li><li> Girar el tubo cónico con las células durante 5 min a 450 xg, a 4 ° C. </li><li> Aspirar el sobrenadante </li></ul> 4. El bloqueo de Fc Endógeno <ul><li> Opcional: contar las células para determinar el volumen adecuado para la siguiente steps. </li><li> Resuspender las células en 1.3 ml de tampón FACS I (según el número de células y la cantidad de anticuerpos que desea utilizar). </li><li> Añadir FcBlock (anti-ratón CD16/CD32) para alcanzar una dilución 1:50 (10 mg / ml), e incubar en hielo durante 10-20 &#39;. No hay necesidad de lavar FcBlock. </li></ul> 5. La tinción <ul><li> Sacar 100 ml de alícuotas a tubos eppendorf que se utilizó como control un-manchado y controles individuales de color (de acuerdo con el número de anticuerpos fluorescentes utilizados). </li><li> Para fibroblastos etiqueta: añadir anti-PDGFR (directamente conjugado con fluoróforo) a una dilución de 1:50 (10 mg / ml). </li><li> Para etiquetar las otras poblaciones de células (por ejemplo células del sistema inmune, macrófagos, células endoteliales, etc): añadir apropiados anticuerpos conjugados con fluorescencia para marcadores de superficie, a una dilución de 1:50. </li></ul> Nota: aunque PDGFR es un marcador robusto para los fibroblastos, se recomienda incluir anticuerpos para icélulas mmune y de células epiteliales con el fin de excluir cualquier poblaciones positivas dobles y por lo tanto mejorar la pureza de los fibroblastos aislados. <ul><li> Añadir 2 l de cada anticuerpo para cada uno de los individuales de control de color tubos eppendorf. </li><li> Se incuban las células con anticuerpos de 1 hora en hielo en la oscuridad. </li><li> Para lavar: llenar los tubos con tampón FACS I y girar durante 5 minutos a 450 xga 4 ° C. </li><li> Aspirar el sobrenadante, resuspender las células en tampón FACS I a una concentración de aproximadamente 2 x 10 6 células / ml, o cualquier concentración más adecuada para la clasificación con su clasificador FACS disponible. </li></ul> Opcional: Las células pueden ser resuspendidas en tampón FACS II para la duración de la clasificación. Esto puede mejorar la viabilidad celular. Sin embargo, la exposición a factores en suero pueden tener una expresión génica efecto, que debe ser probado, y tenerse en cuenta. <ul><li> Transfiera las células marcadas en tubos FACS con filtro superior: filtros de células en los tubos paraevitar los agregados celulares. </li><li> Para excluir las células muertas, añadir 7AAD (0,5 g / ml) o yoduro de propidio (PI) para muestras (excepto para el control sin manchas). </li></ul> Nota: Para evitar la pérdida de las células, usted puede agregar 7AAD/PI a la muestra sin manchas después de grabar en el FACS, y utilizar de nuevo como 7AAD de sólo control. <ul><li> Mantener las muestras en hielo durante el análisis. </li></ul> 6. Aislamiento de células por FACS (Nota: esta parte del protocolo requiere conocimientos previos en el funcionamiento de un clasificador FACS, por ejemplo, BD FACS AriaII, o la ayuda de un técnico especializado). <ul><li> Utilizando el software clasificador FACS (BD FACSDiva), preparar parcelas de análisis adecuados para analizar dispersión frontal (FSC), dispersión lateral (SSC), 7AAD, FITC, PE y cualquier otro fluoróforo utilizado para marcar células (Figura 2). </li><li> Antes de cargar cada muestra para el clasificador de células, vórtice brevemente para resuspender las células. </li><li> Comience por analiING la muestra sin manchar con el fin de establecer la activación periódica de la población total de células, a la puerta de salida restos de células, y para determinar la autofluorescencia o la tinción de fondo. </li><li> Analizar la muestra sin teñir + 7AAD para determinar y puerta de la población de células vivas de la población celular total. </li><li> Analizar cada control de un solo color para determinar y calibrar los parámetros tales como la compensación. </li><li> Analizar la muestra teñida para definir la posición de las puertas para la clasificación. </li><li> Una vez que los parámetros y las puertas para las poblaciones de células deseadas se han establecido, empezar a clasificar las poblaciones celulares marcadas en tubos eppendorf o tubos de 15 ml cónicos que contienen medio de cultivo o tampón de lisis de ARN (por ejemplo, tampón RLT o Trizol). </li></ul> Nota: Si la clasificación de un gran número de células, no se recomienda para ordenar directamente en tampón de lisis de ARN, como el gran volumen de fluido de vaina que acompaña a las células, se diluirá el tampón de lisis y reducen el rendimiento. <ul><li> Spin celdas hacia abajo inmediatamente después de su clasificación ha terminado, y la transferencia a un medio fresco o tampón de lisis de ARN, dependiendo del propósito de su experimento. </li><li> Si la clasificación con el fin de cultivar células aisladas, realice clase estéril. Para una mejor recuperación de las células, el uso de rojo fenol libre de DMEM + 5% de FCS en lugar de tampón FACS II y recoger las células en medio de cultivo con FCS al 20%. </li><li> Si el cultivo de fibroblastos ordenados, alwaysplate en placas recubiertas de colágeno y nunca directamente sobre plástico, ya que resulta en la activación de fibroblastos que producen cambios en su expresión génica. </li></ul>

<ol>
	<li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Seminars in cell &amp; developmental biology. 21, 19-25 (2010).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stromal+fibroblasts+in+tumors.">Heterogeneity of stromal fibroblasts in tumors.</a> Cancer Biol. Ther. 6, 618-619 (2007).</li><li>Ostman, A., Augsten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+and+tumor+growth--bystanders+turning+into+key+players.">Cancer-associated fibroblasts and tumor growth--bystanders turning into key players.</a> Current opinion in genetics &amp; development. 19, 67-73 (2009).</li><li>Allinen, 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=Molecular+characterization+of+the+tumor+microenvironment+in+breast+cancer.">Molecular characterization of the tumor microenvironment in breast cancer.</a> Cancer Cell. 6, 17-32 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Orimo, A., 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=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Experimental cell research. 316, 1324-1331 (2010).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner.</a> Cancer Cell. 17, 135-147 (2010).</li><li>Sugimoto, H., Mundel, T. M., Kieran, M. W., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fibroblast+heterogeneity+in+the+tumor+microenvironment.">Identification of fibroblast heterogeneity in the tumor microenvironment.</a> Cancer Biol. Ther. 5, 1640-1646 (2006).</li><li>Neumann, 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=Cell+culture+and+passaging+alters+gene+expression+pattern+and+proliferation+rate+in+rheumatoid+arthritis+synovial+fibroblasts.">Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts.</a> Arthritis research &amp; therapy. 12, R83 .</li><li>Sherr, C. J., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+mitotic+clock+or+culture+shock.">Cellular senescence: mitotic clock or culture shock.</a> Cell. 102, 407-410 (2000).</li><li>DeNardo, D. 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=Leukocyte+complexity+in+breast+cancer+predicts+overall+survival+and+functionally+regulates+response+to+chemotherapy.">Leukocyte complexity in breast cancer predicts overall survival and functionally regulates response to chemotherapy.</a> Cancer Discovery. 1, 54-67 (2011).</li><li>Pahler, J. C., 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=Plasticity+in+tumor-promoting+inflammation:+impairment+of+macrophage+recruitment+evokes+a+compensatory+neutrophil+response.">Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response.</a> Neoplasia. 10, 329-340 (2008).</li><li>Pietras, K., Pahler, J., Bergers, G., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+paracrine+PDGF+signaling+in+the+proangiogenic+tumor+stroma+revealed+by+pharmacological+targeting.">Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.</a> PLoS Med. 5, e19 (2008).</li><li>Quante, 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=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer Cell. 19, 257-272 (2011).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>Xouri, G., Christian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+tumor+stroma+fibroblasts.">Origin and function of tumor stroma fibroblasts.</a> Seminars in cell &amp; developmental biology. 21, 40-46 (2010).</li></ol>

No hay conflictos de interés declarado.

Mientras que los experimentos realizados en cultivo de tejidos puede ser informativo y sugieren principios funcionales que se pueden verificar in vivo, se sabe que ocurren grandes cambios en la expresión génica de las células en cultivo 11,12. Con el fin de evitar una etapa de cultivo de tejidos cuando el perfil de expresión de genes en fibroblastos, hemos desarrollado un protocolo que permite el aislamiento de normales, así como los asociados al cáncer de fibroblastos de ratón fresco o tejido...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nombre del reactivo</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentarios (opcional)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Industrias Biológicas</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Colagenasa II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; La colagenasa IV</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Desoxirribonucleasa</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Cell filtro de 70 micras</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Purificado anti-ratón CD16/CD32</td&gt;<td&gt; BD Pharmingen</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; A través de la sonda (7AAD)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-ratón CD140a-PE (PDGFRA)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-ratón FITC F4/80-</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM w / o rojo fenol</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Colágeno tipo I</td&gt;<td&gt; BD Biosciences</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Asociados con el cáncer fibroblastos (CAF) son el tipo celular más importante en el estroma tumoral de muchos tipos de cáncer, en particular, carcinoma de mama, y su presencia prominente menudo está asociada con mal pronóstico 1,2. CAFs son una subpoblación de fibroblastos estromales activados, muchas de las cuales expresan el marcador de miofibroblastos α-SMA 3. CAF se originan a partir de los fibroblastos del tejido local, así como de derivados de médula ósea células reclutadas en el tumor en desarrollo y adoptar un fenotipo CAF bajo la influencia del microambiente del tumor 4. CAF se muestra para facilitar la iniciación del crecimiento del tumor y la progresión a través de señalización que promueve la proliferación de células tumorales, angiogénesis, invasión y 5-8. Hemos demostrado que la CAF estimular el crecimiento tumoral mediante la mediación de tumor que promueven la inflamación, a partir de las primeras etapas de pre-neoplásicas 9. A pesar de la creciente evidencia de las CAF papel clave que desempeñar para facilitar el crecimiento del tumor,CAF estudian ha sido un reto en curso debido a la falta de marcadores específicos de CAF y la gran heterogeneidad de estas células, con muchos subtipos co-existentes en el microambiente tumoral 10. Además, el estudio de fibroblastos in vitro se ve obstaculizada por el hecho de que su perfil de expresión de genes a menudo se altera en cultivo de tejidos 11,12. Para abordar este problema y para permitir la expresión de genes de perfiles imparcial de fibroblastos de ratón fresco y tejidos humanos, hemos desarrollado un método basado en los protocolos anteriores para la clasificación de células activadas por fluorescencia (FACS) 13,14. Nuestro enfoque se basa en la utilización de PDGFR como un marcador de superficie para aislar fibroblastos de ratón fresco y el tejido humano. PDGFR se expresa abundantemente por ambos fibroblastos normales y cafés 9,15. Este método permite el aislamiento de poblaciones puras de fibroblastos normales y cafés, incluyendo, pero no limitado a α-SMA + activado miofibroblastos. Fibroblastos aislados pueden entonces serutilizado para la caracterización y la comparación de la evolución de la expresión génica que se produce en los CAF durante la tumorigénesis. De hecho, nosotros y otros informaron de perfiles de expresión de los fibroblastos aislados por clasificación celular 16. Este protocolo se realizó con éxito para aislar y crear un perfil poblaciones altamente enriquecidas de fibroblastos de tejidos de piel, páncreas mamaria, y pulmón. Además, este método también permite el cultivo de células clasificadas, con el fin de realizar los experimentos funcionales y para evitar la contaminación por las células tumorales, lo cual es a menudo un gran obstáculo cuando se trata de los CAF de cultivo.

Uso de PDGFR como un marcador para los resultados de fibroblastos en el aislamiento de poblaciones altamente enriquecidas de los fibroblastos del tejido. El nivel de pureza después de la clasificación fue del 99%, cuantificada por análisis post-especie (Figura 2A). Estimando el porcentaje de contaminación de las células de fibroblastos no por la expresión relativa de los genes de control específicos de la célula (Figura 2B) muestra típicamente 0.1-0.6% de contaminación. Este n...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cáncer de fibroblastos asociados (CAF) facilitar la iniciación del crecimiento del tumor y la progresión a través de señalización que promueve la proliferación, la angiogénesis y la inflamación. Aquí se describe un método para aislar poblaciones puras de los fibroblastos normales y CAFS de ratón fresco y tejidos humanos por clasificación de células, usando PDGFR como un marcador de superficie.

Aislamiento de fibroblastos normales y el cáncer asociado-de los tejidos frescos por Clasificación células activadas por fluorescencia (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

isolation-normal-cancer-associated-fibroblasts-from-fresh-tissues

Research

JoVE Journal

Biology

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

35.5K vistas.  Tel Aviv University. Cáncer de fibroblastos asociados (CAF) facilitar la iniciación del crecimiento del tumor y la progresión a través de señalización que promueve la proliferación, la angiogénesis y la inflamación. Aquí se describe un método para aislar poblaciones puras de los fibroblastos normales y CAFS de ratón fresco y tejidos humanos por clasificación de células, usando PDGFR como un marcador de superficie.

Video: Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Investigación

Biología

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Fluorescencia de células activadas por la clasificación de los protoplastos de la planta

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) El etiquetado y posterior clasificación de células activadas fluorescencia para la Cultura independiente de la identificación de carbono orgánico disuelto degradación bacterioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

El aislamiento de fibroblastos portales rata por<em&gt; In situ</em&gt; Hígado de perfusión

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análisis específico de célula de<em&gt; Arabidopsis</em&gt; Hojas usando citometría de flujo

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Microaspiración de células únicas como estrategia alternativa a la clasificación celular activada por fluorescencia para la separación de mezclas de virus gigantes

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Clasificación celular activada por fluorescencia para el aislamiento de las poblaciones celulares escleractinianas

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Aislamiento y caracterización de exosomas de fibroblastos del músculo esquelético

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...