Esta labor fue apoyada por los Institutos Nacionales de Salud de subvención R01 AI088209 (a KAH). También agradecemos a la Universidad de Minnesota Flujo de Recursos Citometría.

En este estudio, los adultos - se utilizaron (6 8 semanas) hembra C57BL / 6 ratones. Los ratones se adquirieron en el Instituto Nacional del Cáncer y mantuvieron en condiciones libres de patógenos específicos. La Universidad de Comité de Cuidado y Uso de Animales institucional Minnesota (IACUC) aprobó toda la experimentación animal. 1 Preparación de Instrumentos y búferes <ol><li> Preparar la solución de enzima (medio RPMI 1640 con 0,05% [w / v] de Liberase TH y 100 U / ml de DNasa I), tampón rico en albúmina (1X PBS [Ca 2 + / Mg 2 + libre] con 0,5% de suero bovino mM de ácido etilendiaminotetraacético albúmina y 2 [EDTA]), tampón FACS (PBS 1X [Ca 2 + / Mg 2 + libre] que contiene suero bovino fetal al 1% [FBS] y 0,02% NaN 3), FACS clasificación tampón (PBS 1X [Ca 2 + / Mg 2 + libre] que contiene 2% de FBS), y medio RP10 (medio RPMI-1640 suministrado con FBS al 10%). </li><li> Preparar 10 placas paneo. Preparar la solución de recubrimiento 40 ml (0,01 mg / ml de Gavena anti-IgG de rata en 0,1 M NaHCO3 Buffer), y añadir 4 ml de solución de revestimiento en cada placa de 100 mm x 15, remolino e incubar a 4 ° CO / N. Vierta la solución de anticuerpo y enjuagar la placa con 5 ml de 1x PBS durante 2 horas, agite vigorosamente entre enjuagues. Guarde las placas preparadas a 4 ° C. </li></ol> 2. Cosecha de Timo Tejido <ol><li> La eutanasia de ratón en una cámara de CO 2. Coloque el animal sobre su espalda sobre una estera de disección, fijar los pies en el felpudo y humedecer la piel mediante pulverización con etanol al 70%. </li><li> Hacer una incisión en la línea media a través de la piel, a partir de por encima de la abertura de la uretra hacia arriba a la mandíbula inferior con una tijera y extender la incisión hacia abajo para las rodillas en ambos lados. La incisión se ve como un revés de &quot;Y&quot;. Tire de la piel suelta de nuevo en los lados, y el pin a la lona disección. </li><li> Perforar el diafragma del xifoides, cortar las costillas en cada lado hasta alrededor de la clavícula, luego levante las costillas confórceps y el pin a la lona disección. El timo es justo debajo de las costillas, y se ve como dos lóbulos blancas finas que recubren el corazón (Figura 1). Tejido conectivo que rodea Desconecte el timo, tirar y eliminar el par de lóbulos del timo con pinzas dentadas curvadas suavemente. Lóbulos del timo lugar en una placa de 6 pocillos que contenía 5 ml de medio RPMI-1640. </li></ol> 3. Preparación de las células del estroma tímico <ol><li> Recortar cualquier tejido graso y conectivo circundante y transferir los lóbulos del timo a un nuevo pocillo de la placa de 6 pocillos que contiene 5 ml de solución de enzima recién preparan. Hacer pequeñas incisiones en los lóbulos del timo con tijeras finas, y se incuba la placa a 37 ° C durante 20 min. </li><li> Pipetear suavemente digerir tejido del timo arriba y abajo 2-3 veces usando la pipeta 5 ml para la disociación del tejido. Recoger la fracción de sobrenadante en un tubo de 50 ml que contiene 10 ml de tampón de albúmina rica en frío para neutralizar enzimas. Mantener el tubo de recogida en hielo. Añadir 2,5solución de enzima ml para el tejido restante y se incuba la placa a 37 ° C durante 15 min. </li><li> Realizar agitación mecánica suave con una jeringa de 3 ml y 18 g de aguja para romper los agregados. Piscina sobrenadante en el tubo de recogida. Añadir 2,5 ml de solución de enzima para el tejido restante y se incuba la placa a 37 ° C durante 15 min. </li><li> Repita la agitación mecánica suave con una jeringa de 3 ml y una aguja de 25 G, y se incuba la placa adicional de 5 - 10 min. </li><li> Después de la digestión completa, piscina el sobrenadante en el tubo de recogida. Centrifugar el tubo de recogida a 400 xg, 4 ° C durante 8 min para recoger las células. Aspirar el líquido y suspender las células sedimentadas en 10 ml de tampón de albúmina-rica y filtrar la muestra a través de malla de 100 mm. Contar las células utilizando un hemocitómetro. A continuación, realice el análisis de flujo cytometic (Sección 4) o el enriquecimiento TEC (Sección 5). </li></ol> 4. Identificación del TEC por Citometría de Flujo </p&gt; <ol><li> Preparar las células para una concentración final de 4 x 10 6 células por 100 l en tampón de FACS para la tinción en una placa de ensayo de pocillos de fondo redondo 96 y mantener la placa en hielo. Añadir 20 l solución de bloqueo Fc (-anticuerpo anti CD16 / CD32 a 10 mg / ml en tampón FACS) a las células e incubar las células durante 10 min en hielo. Haga girar la placa a 400 xg, 4 ° C durante 3 min. Deseche el líquido de los pozos con un movimiento rápido de la cara de la placa hacia abajo en un fregadero. </li><li> Suspender las células sedimentadas en 100 l cóctel de anticuerpos (anti-CD45, -EpCAM, clase MHC II, y -Ly51 anticuerpos, y la lectina UEA-1 marcado con diferentes fluorocromos en fabricante recomienda o probado concentraciones óptimas de cada anticuerpo en tampón FACS ), y se incuban las células en hielo durante 20 min en la oscuridad. Para la compensación, manchar 1 x 10 6 células con cada fluorocromo individual. </li><li> Añadir 100 l de tampón de FACS a cada pocillo y girar la placa a 400 xg, 4 ° C durante 3 min. Repita el paso de lavado setiempo y, a continuación, volver a suspender las células en tampón de FACS 100 l. Transferir las células a un tubo de FACS de fondo redondo de 12 x 15 mm lleno de 300 l de tampón FACS. Adquirir las muestras en el citómetro de flujo y análisis de datos utilizando el software de análisis FACS. Estrategia gating TEC se muestra en la Figura 2. </li></ol> 5. Enriquecimiento del TEC por Toma panorámica <ol><li> Centrifugar las células del timo (que se formulan en la sección 3) a 400 xg, 4 ° C durante 5 min. Aspirar el líquido y suspender las células sedimentadas en tampón de clasificación de FACS en una concentración de 2 x 10 8 células por ml en un tubo cónico de 15 ml o 50 ml. Añadir anticuerpo anti-CD90.2 (Clon 30-H12) (o anti-CD45) a una concentración final de 2,5 mg / ml de suspensión celular y se incuban las células suavemente en un mezclador que gira a 4 ° C durante 30 min. Después de la incubación, lavar con un 5 - 10 veces el volumen de medio RP10 y se centrifuga el tubo a 400 xg, 4 ° C durante 5 min. </li><li> Aspirar el líquido y resuspender las células sedimentadas enRP10 medio a una concentración de 1 x 10 7 por ml. Añadir 5 ml de suspensión celular a cada placa recubierta panning (preparado en la Etapa 1.3), remolino y se incuba durante 30 min a TA. Agitar vigorosamente; transferir los sobrenadantes en un tubo cónico. Enjuague la placa dos veces con medio RP10 y agrupar los sobrenadantes en el tubo de recogida. </li><li> Centrifugar el tubo de recogida a 400 xg, 4 ° C durante 5 min. Aspirar el líquido y resuspender las células sedimentadas en medio RP10 5 ml. Añadir la suspensión celular a una nueva placa de paneo, agitar e incubar durante 30 minutos a temperatura ambiente. Recoger las células enjuagar la placa y piscina para ellos en un tubo cónico. Una vez TEC se enriquecen, de girar las células a 400 xg, 4 ° C durante 5 min y la suspensión de células en 5 ml de tampón FACS clasificación. </li></ol> 6. Purificación del TEC por la clasificación de células <ol><li> Contar las células usando un hemocitómetro y preparar la suspensión de células a una concentración de 4 x 10 7 células en tampón de clasificación FACS. Añadir anticuerpos described en la Sección 4.4 en solución de células e incubar las células suavemente en un mezclador que gira a 4 ° C durante 30 min. Después de la tinción, lavar y suspender las células a una concentración de 10 x 10 6 células por ml de tampón de clasificación FACS. </li><li> Ordenar subconjuntos TEC través de una boquilla 100 mM usando la clasificación de células activadas por fluorescencia. Ordenado de células se recogen en 30% (v / v) de FBS en medio RPMI-1640). Una vez que la clasificación de células se realiza, centrifugar las células a 400 xg, 4 ° C durante 5 min y se preparan para análisis de aguas abajo. </li></ol>

<ol>
	<li>Rothenberg, E. V., Moore, J. E., Yui, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Launching+the+T-cell-lineage+developmental+programme.">Launching the T-cell-lineage developmental programme.</a> Nat. Rev. Immunol. 8, 9-21 (2008).</li><li>Anderson, G., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+epithelial+cells:+working+class+heroes+for+T+cell+development+and+repertoire+selection.">Thymic epithelial cells: working class heroes for T cell development and repertoire selection.</a> Trends Immunol. 33, 256-263 (2012).</li><li>Klein, L., Hinterberger, M., Wirnsberger, G., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+in+the+thymus+for+positive+selection+and+central+tolerance+induction.">Antigen presentation in the thymus for positive selection and central tolerance induction.</a> Nat. Rev. Immunol. 9, 833-844 (2009).</li><li>Starr, T. K., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+selection+of+T+cells.">Positive and negative selection of T cells.</a> Annu. Rev. Immunol. 21, 139-176 (2003).</li><li>Takahama, Y., Takada, K., Murata, S., Tanaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta5t-containing+thymoproteasome:+specific+expression+in+thymic+cortical+epithelial+cells+and+role+in+positive+selection+of+CD8.">beta5t-containing thymoproteasome: specific expression in thymic cortical epithelial cells and role in positive selection of CD8.</a> T cells. Curr. Opin. Immunol. 24, 92-98 (2012).</li><li>Xing, Y., Wang, X., Igarashi, H., Kawamoto, H., Sakaguchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+subunit+G5PR+that+regulates+the+JNK-mediated+apoptosis+signal+is+essential+for+the+survival+of+CD4+and+CD8+double-positive+thymocytes.">Protein phosphatase subunit G5PR that regulates the JNK-mediated apoptosis signal is essential for the survival of CD4 and CD8 double-positive thymocytes.</a> Mol. Immunol. 45, 2028-2037 (2008).</li><li>Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treg+cells,+life+history,+and+diversity.">Treg cells, life history, and diversity.</a> Cold Spring Harb. Perspect. Biol. 4, a007021 (2012).</li><li>Pobezinsky, L. 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=Clonal+deletion+and+the+fate+of+autoreactive+thymocytes+that+survive+negative+selection.">Clonal deletion and the fate of autoreactive thymocytes that survive negative selection.</a> Nat. Immunol. 13, 569-578 (2012).</li><li>Stritesky, G. L., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+self-reactive+T+cells+in+the+thymus.">Selection of self-reactive T cells in the thymus.</a> Annu. Rev. Immunol. 30, 95-114 (2012).</li><li>Xing, Y., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+tolerance:+central+and+peripheral.">T-cell tolerance: central and peripheral.</a> Cold Spring Harb. Perspect. Biol. 4, (2012).</li><li>Anderson, G., Lane, P. J., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+intrathymic+microenvironments+to+establish+T-cell+tolerance.">Generating intrathymic microenvironments to establish T-cell tolerance.</a> Nat. Rev. Immunol. 7, 954-963 (2007).</li><li>Xing, Y., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoproteasome+subunit-beta5T+generates+peptide-MHC+complexes+specialized+for+positive+selection.">Thymoproteasome subunit-beta5T generates peptide-MHC complexes specialized for positive selection.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 6979-6984 (2013).</li><li>Petrie, H. T., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoned+out:+functional+mapping+of+stromal+signaling+microenvironments+in+the+thymus.">Zoned out: functional mapping of stromal signaling microenvironments in the thymus.</a> Annu. Rev. Immunol. 25, 649-679 (2007).</li><li>Rezzani, R., Bonomini, F., Rodella, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+and+molecular+overview+of+the+thymus+as+site+for+T-cells+development.">Histochemical and molecular overview of the thymus as site for T-cells development.</a> Prog. Histochem. Cytochem. 43, 73-120 (2008).</li><li>Rodewald, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+organogenesis.">Thymus organogenesis.</a> Annu. Rev. Immunol. 26, 355-388 (2008).</li><li>Griffith, A. V., 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=Spatial+mapping+of+thymic+stromal+microenvironments+reveals+unique+features+influencing+T+lymphoid+differentiation.">Spatial mapping of thymic stromal microenvironments reveals unique features influencing T lymphoid differentiation.</a> Immunit. 31, 999-1009 (2009).</li><li>Guerder, S., Viret, C., Luche, H., Ardouin, L., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+processing+of+self-antigens+by+subsets+of+thymic+stromal+cells.">Differential processing of self-antigens by subsets of thymic stromal cells.</a> Curr. Opin. Immunol. 24, 99-104 (2012).</li><li>Hogquist, K. A., Xing, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+CD8++T+cells+need+diversity+when+growing+up.">Why CD8+ T cells need diversity when growing up.</a> Immunit. 32, 5-6 (2010).</li><li>Jenkinson, E. J., Parnell, S., Shuttleworth, J., Owen, J. J., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+ability+of+thymic+epithelial+cells+to+mediate+positive+selection+does+not+require+expression+of+the+steroidogenic+enzyme+p450scc.">Specialized ability of thymic epithelial cells to mediate positive selection does not require expression of the steroidogenic enzyme p450scc.</a> J. Immunol. 163, 5781-5785 (1999).</li><li>Murata, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD8++T+cell+development+by+thymus-specific+proteasomes.">Regulation of CD8+ T cell development by thymus-specific proteasomes.</a> Science. 316, 1349-1353 (2007).</li><li>Chidgey, A. P., Pircher, H., MacDonald, H. R., Boyd, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adult+thymic+stromal-cell+suspension+model+for+in+vitro+positive+selection.">An adult thymic stromal-cell suspension model for in vitro positive selection.</a> Dev. Immunol. 6, 157-170 (1998).</li><li>Izon, D. J., Nieland, J. D., Godfrey, D. I., Boyd, R. L., Kruisbeek, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+reveals+unexpected+shared+antigens+between+histologically+defined+populations+of+thymic+stromal+cells.">Flow cytometric analysis reveals unexpected shared antigens between histologically defined populations of thymic stromal cells.</a> Int. Immunol. 6, 31-39 (1994).</li><li>Jenkinson, E. J., Anderson, G., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+T+cell+maturation+on+defined+thymic+stromal+cell+populations+in+vitro.">Studies on T cell maturation on defined thymic stromal cell populations in vitro.</a> J. Exp. Med. 176, 845-853 (1992).</li><li>Klein, L., Klugmann, M., Nave, K. A., Tuohy, V. K., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+of+the+autoreactive+T-cell+repertoire+by+a+splice+variant+of+self+protein+expressed+in+thymic+epithelial+cells.">Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells.</a> Nat. Med. 6, 56-61 (2000).</li><li>Shortman, K., Vremec, D., D'Amico, A., Battye, F., Boyd, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+of+the+thymocytes+associated+with+dendritic+cells+and+macrophages+in+thymic+rosettes.">Nature of the thymocytes associated with dendritic cells and macrophages in thymic rosettes.</a> Cell. Immunol. 119, 85-100 (1989).</li><li>Volkmann, A., Zal, T., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-presenting+cells+in+the+thymus+that+can+negatively+select+MHC+class+II-restricted+T+cells+recognizing+a+circulating+self+antigen.">Antigen-presenting cells in the thymus that can negatively select MHC class II-restricted T cells recognizing a circulating self antigen.</a> J. Immunol. 158, 693-706 (1997).</li><li>Yang, S. J., Ahn, S., Park, C. S., Choi, S., Kim, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+subpopulations+of+thymic+epithelial+cells+by+flow+cytometry+using+a+new+specific+thymic+epithelial+marker,+Ly110.">Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110.</a> J. Immunol. Method. 297, 265-270 (2005).</li><li>Gray, D. H., Chidgey, A. P., Boyd, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thymic+stromal+cell+populations+using+flow+cytometry.">Analysis of thymic stromal cell populations using flow cytometry.</a> J. Immunol. Method. 260, 15-28 (2002).</li><li>Gray, D. H., 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=Unbiased+analysis,+enrichment+and+purification+of+thymic+stromal+cells.">Unbiased analysis, enrichment and purification of thymic stromal cells.</a> J. Immunol. Method. 329, 56-66 (2008).</li><li>Williams, K. 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=Single+cell+analysis+of+complex+thymus+stromal+cell+populations:+rapid+thymic+epithelia+preparation+characterizes+radiation+injury.">Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury.</a> Clin. Transl. Sci. 2, 279-285 (2009).</li><li>Kanof, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+T+cell+subpopulations.">Purification of T cell subpopulations.</a> Curr. Protoc. Immunol. 7 (Unit 7.3), (2001).</li><li>Mage, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+T+cells+and+B+cells+using+panning+techniques.">Fractionation of T cells and B cells using panning techniques.</a> Curr. Protoc. Immunol. 3 (Unit 3.5), (1991).</li><li>Wekerle, H., Ketelsen, U. P., Ernst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+nurse+cells.+Lymphoepithelial+cell+complexes+in+murine+thymuses:+morphological+and+serological+characterization.">Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization.</a> The Journal of experimental medicin. 151, 925-944 (1980).</li><li>Kyewski, B. A., Kaplan, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoepithelial+interactions+in+the+mouse+thymus:+phenotypic+and+kinetic+studies+on+thymic+nurse+cells.">Lymphoepithelial interactions in the mouse thymus: phenotypic and kinetic studies on thymic nurse cells.</a> J Immuno. 128, 2287-2294 (1982).</li><li>Nakagawa, Y., 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=Thymic+nurse+cells+provide+microenvironment+for+secondary+T+cell+receptor+alpha+rearrangement+in+cortical+thymocytes.">Thymic nurse cells provide microenvironment for secondary T cell receptor alpha rearrangement in cortical thymocytes.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 20572-20577 (2012).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J. Immunol. Method. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of immunological method. 385, 23-34 (2012).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

En el protocolo, los pasos críticos son la preparación de las células del estroma del timo (Sección 3) y el enriquecimiento de los TEC (Sección 4). Se recomienda firmemente que la solución de enzima fresca se prepara cada vez y el tejido se trata tan pronto como sea posible. Para timos agrupado, se requiere optimizar el volumen de solución de enzima en función del número de timos. Si cualquier residuo de tejido que queda después de la transformación, la adición de más solución de enzima y la extensión del...

Temprano en el desarrollo de células T, la médula ósea madre hematopoyéticas progenitoras multipotentes derivadas de células son reclutados a la corteza del timo, someterse compromiso de linaje T y se convierten en precursores de células T 1. En la corteza, precursores de células T CD4 y CD8 doble negativos (DN) timocitos se expanden y se diferencian en células CD8 doble positiva (DP) timocitos inmaduros CD4 y, formando un gran grupo de progenitores con receptores de células T altamente variable 1. Sólo un selecto subconjunto restringido por MHC de las células DP se convertirá en único positivos (SP) timocitos CD4 o CD8, migran a la médula del timo, y se diferencian en células T maduras funcionalmente competentes, un evento que se conoce como selección positiva 2 6. En contraste, los clones de los timocitos autorreactivos se someten a selección negativa y se eliminan a través de la apoptosis, convertido a las células T reguladoras para la auto-tolerancia, o desviado a los linfocitos intraepiteliales para los propósitos que aún no están claras 3,7-10. En el timo, las células estromales tímicas forman un microambiente único que proporciona señales para estos diversos destinos de desarrollo de células T 5,11,12. Células del estroma tímico se componen de células epiteliales del timo (TEC) - incluyendo TEC corticales (CTEC) y TEC medulares (mTECs), células dendríticas, macrófagos, fibroblastos, células endoteliales, pericitos derivadas de la cresta neural y otras células mesenquimales 13-15. Entre estos, TEC son cruciales en las diversas etapas de desarrollo de células T 1,2,16,17. Sin embargo, la falta de una manera robusta para aislar TEC ha obstaculizado una comprensión global de sus funciones 16. En particular, CTEC, que forman una red tridimensional que rodea a los progenitores en la corteza, son esenciales para la selección positiva 13,18,19 por razones que aún no están claras. Estudios anteriores proporcionaron pistas sobre la heterogeneidad y el papel de los TEC, sobre todo contando con herramientas morfológicas e histológicas 13 </sup&gt;. Recientemente las funciones únicas de subconjuntos TEC fueron abordados por enfoques genéticos en modelos de ratón 12,20. Una manera robusta y reproducible para aislar TEC es fundamental para lograr la caracterización objetiva de subconjuntos TEC, la evaluación cuantitativa y cualitativa de las funciones TEC, y la aclaración de los mecanismos de cómo CTEC apoyan la selección positiva. Debido a la rareza de TEC en el timo y las interacciones apretados forman en el órgano intacto, el aislamiento de TEC ha sido desafiante. El protocolo descrito aquí se basa en descubrimientos anteriores, actualmente reactivos disponibles, las técnicas y el conocimiento de la estructura y composición del estroma del timo. Hace casi dos décadas, se reportaron varios procedimientos para desagregar los tejidos del timo 21-27, en el que se utilizaron diferentes enzimas durante la digestión, incluyendo tripsina, colagenasa y dispasa. Gray et al. comparado esas enzimas en su precedure 28, e informó de una mejora de la metanfetaminaod con la digestión de múltiples pasos de cócteles enzimáticos 29 que se hicieron ampliamente utilizado 20,30. Sin embargo, este método implica un largo tiempo de preparación y los pasos y los resultados de digestión complejos en el número de células y proporciones de TEC última variable, incluso en la misma cohorte murino 29,30. Hace varios años, Liberase enzima grado de investigación, con adición altamente purificada de colagenasa y proteasa neutra se empezó a utilizar en la disociación del tejido del timo 30. A continuación, describimos un protocolo basado en la digestión-Liberase, con procedimientos de separación mecánica optimizados, que produce un alto número de TEC viables a partir de tejidos de timo de ratón. . Para enriquecer las células del estroma disociadas, estudios previos han utilizado tanto los gradientes de densidad o la separación de cuentas magnéticas 21,29. Sin embargo, ambos métodos causan severa perdida de ciertas poblaciones de células del estroma del timo, en particular la población de CTEC 29. Eliminación citotóxica y técnicas de paneo <shasta&gt; 31,32 han sido ampliamente utilizados para el agotamiento o la separación de los linfocitos en el campo inmunológico 12,31. Después de la comparación de estas técnicas, hemos establecido el protocolo actual de panoramización para el enriquecimiento de TEC. La condición suave durante el procedimiento de enriquecimiento conduce a una menor muerte celular e imparcial y una mayor recuperación de TEC. La suspensión de células aisladas del timo se describe en la Sección 3 se puede aplicar directamente a análisis citométrico de flujo para la identificación y caracterización de subconjuntos TEC y células dendríticas. Sección 4 describe una forma sencilla y útil para identificar subconjuntos TEC utilizando citómetro de flujo multiparamétrica. Para los experimentos que buscan obtener CTEC purificados o mTECs, enriquecimiento TEC y clasificación de células procedimientos se pueden encontrar en la sección 5 y 6.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

El timo es un órgano vital para el desarrollo de linfocitos T. De las células del estroma tímico, células epiteliales del timo (TEC) son particularmente cruciales en múltiples etapas de desarrollo de células T: compromiso de las células T, la selección positiva y selección negativa. Sin embargo, la función de TEC en el timo sigue siendo incompleta entendido. En el artículo, se proporciona un método para aislar los subconjuntos TEC de timo de ratón fresco con una combinación de alteración mecánica y digestión enzimática. El método permite que las células del estroma tímico y timocitos a ser liberados de manera eficiente de célula-célula y célula-matriz conexiones extracelular y para formar una suspensión de una sola célula. Uso de las células aisladas, citometría de flujo multiparamétrica se puede aplicar a la identificación y caracterización de TEC y las células dendríticas. Debido TEC son una rara población de células en el timo, también se describe una forma eficaz para enriquecer y purificar TEC por el agotamiento de los timocitos, el tipo de célula más abundante en el timo. Folloala el enriquecimiento, el tiempo de clasificación de células se puede disminuir de modo que la pérdida de viabilidad celular puede reducirse al mínimo durante la purificación de TEC. Las células purificadas son adecuados para diversos análisis aguas abajo como el Real Time-PCR, Western blot y perfiles de expresión génica. El protocolo promoverá la investigación de la función de TEC y así como el desarrollo de la reconstitución in vitro de células T.

Usando este protocolo, un órgano timo fue retirado de un ratón adulto (Sección 2) y una suspensión de células del timo se preparó como se describe en la Sección 3. La suspensión celular obtenida constaba de timocitos, células estromales hematopoyéticas derivadas de las células del estroma y no hematopoyéticas. CD45 es un marcador pan-hematopoyético expresó en ambos timocitos y células estromales hematopoyéticas tales como macrófagos y células dendríticas. En contraste, los TEC no son de origen hematop...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Aquí se describe un método eficiente para el aislamiento, identificación y purificación de ratón las células epiteliales del timo (TEC). El protocolo puede ser utilizado para estudios de la función del timo para el desarrollo normal de células T, la disfunción del timo, y la reconstitución de células T.

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

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

isolation-identification-purification-murine-thymic-epithelial

Research

JoVE Journal

Immunology and Infection

31.7K vistas.  University of Minnesota.  Aquí se describe un método eficiente para el aislamiento, identificación y purificación de ratón las células epiteliales del timo (TEC). El protocolo puede ser utilizado para estudios de la función del timo para el desarrollo normal de células T, la disfunción del timo, y la reconstitución de células T. 

Video: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

La aplicación de un ratón ligado Ensayo placas de Peyer asa intestinal para evaluar la absorción por las células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Investigación

Inmunología e Infección

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, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

El aislamiento, tratamiento y análisis de células gingivales murinos

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Aislamiento de células estromales murinas en nódulos linfáticos

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

A gran escala de purificación de porcino o bovino fotorreceptor Outer Segmentos para Fagocitosis ensayos sobre células pigmento retiniano epitelial

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Promoción de 3-D agregación de células epiteliales del timo FACS purificada con EAK 16-II / EAKIIH6 auto-montaje de hidrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificación de ADN Viral para la identificación de las proteínas virales y celulares asociadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.