Ce travail a été financé par les Instituts nationaux de la santé Grant R01 AI088209 (à KAH). Nous remercions également l&#39;Université du Minnesota cytométrie en flux de ressources.

Dans cette étude, des adultes (6-8 semaines) femelles C57BL / 6 ont été utilisés. Les souris ont été achetées auprès du National Cancer Institute et maintenues dans des conditions libres d&#39;agents pathogènes spécifiques. L&#39;Université de Comité de protection et d&#39;utilisation des animaux institutionnel Minnesota (IACUC) a approuvé toute expérimentation animale. 1 Préparation des instruments et tampons <ol><li> Préparer la solution d&#39;enzyme (milieu RPMI 1640 avec 0,05% [p / v] de Libérase TH et 100 U / ml de DNase I), un tampon d&#39;albumine riche (1X PBS [Ca2 + / Mg2 + exempt] avec 0,5% de sérum bovin l&#39;albumine et 2 mM d&#39;acide éthylènediaminetétraacétique [EDTA]), un tampon FACS (PBS 1X [Ca2 + / Mg2 + exempt] contenant du sérum bovin fœtal à 1% [FBS] et 0,02% de NaN3), tampon de tri FACS (PBS 1X [Ca2 + / Mg2 + exempt] contenant 2% de FBS) et RP10 milieu (milieu RPMI-1640 fourni avec 10% de FBS). </li><li> Préparer 10 plaques panoramiques. Préparer la solution de revêtement de 40 ml (0,01 mg / ml de Gavoine anti-IgG de rat dans 0,1 M de tampon NaHCO 3), et ajouter 4 ml d&#39;une solution de revêtement dans chaque plaque de 100 x 15 mm, tourbillon et incuber à 4 ° CO / N. Verser la solution d&#39;anticorps et rincer la plaque en utilisant 5 ml de PBS 1x pour 2 fois, agiter vigoureusement entre les rinçages. Conserver les boîtes préparées à 4 ° C. </li></ol> 2. récolte de Thymus tissus <ol><li> Euthanasier souris dans une chambre de CO 2. Mettre l&#39;animal sur son dos sur un tapis de dissection, épinglez les pieds sur le tapis et mouiller la fourrure par pulvérisation d&#39;éthanol à 70%. </li><li> Faire une incision sur la ligne médiane à travers la peau, à partir d&#39;au-dessus de l&#39;ouverture de l&#39;urètre vers le haut de la mâchoire inférieure avec des ciseaux et s&#39;étendent vers le bas à l&#39;incision du genou des deux côtés. L&#39;incision ressemble à une tête en bas &quot;Y&quot;. Tirez la peau lâche en arrière sur les côtés, et la broche à la natte de dissection. </li><li> Percer la membrane de la pointe du sternum, couper les côtes de chaque côté jusqu&#39;à environ la clavicule, puis soulevez les côtespince et la broche à la natte de dissection. Le thymus est juste sous les côtes, et ressemble à deux lobes blancs minces recouvrant le coeur (Figure 1). Débranchez le tissu conjonctif qui entoure le thymus, tirez doucement et retirer la paire de lobes du thymus avec des pinces dentelées courbes. Lieu lobes du thymus dans une plaque à 6 puits contenant 5 ml de RPMI-1640. </li></ol> 3 Préparation des cellules stromales thymiques <ol><li> Coupez tout le tissu adipeux et conjonctif environnant et transférer les lobes du thymus à un nouveau puits de la plaque de 6 puits contenant 5 ml fraîchement préparer la solution d&#39;enzyme. Faire de petites incisions dans les lobes du thymus avec des ciseaux fins, et incuber la plaque à 37 ° C pendant 20 min. </li><li> Pipette doucement digérer thymus jusqu&#39;à tissulaire et bas 2 - 3 fois avec 5 ml pipette pour la dissociation des tissus. Recueillir la fraction surnageant dans un tube de 50 ml contenant 10 ml de tampon d&#39;albumine riche froide pour neutraliser les enzymes. Garder le tube de collecte sur la glace. Ajouter 2,5ml de solution d&#39;enzyme dans le tissu restant et incuber la plaque à 37 ° C pendant 15 min. </li><li> Effectuer une agitation mécanique douce avec une seringue de 3 ml et une aiguille 18 G de briser les agrégats. Piscine surnageant dans le tube de collecte. Ajouter 2,5 ml de solution enzymatique au tissu restant et incuber la plaque à 37 ° C pendant 15 min. </li><li> Répétez agitation mécanique douce avec une seringue de 3 ml et une aiguille G 25, et incuber la plaque supplémentaire de 5 - 10 min. </li><li> Après digestion complète, en commun le surnageant dans le tube de collecte. Centrifuger le tube de collecte à 400 xg, 4 ° C pendant 8 min pour recueillir les cellules. Aspirer le liquide et suspendre le culot cellulaire dans 10 ml de tampon d&#39;albumine riche et filtrer l&#39;échantillon à 100 mm maille. Compter les cellules en utilisant un hématimètre. Puis effectuer l&#39;analyse des flux cytometic (section 4) ou à l&#39;enrichissement TEC (section 5). </li></ol> 4 Identification des TEC par cytométrie en flux </p&gt; <ol><li> Préparer les cellules à une concentration finale de 4 x 10 6 cellules par 100 pi de tampon pour FACS pour la coloration dans une plaque de puits d&#39;essai à fond rond de 96 et de maintenir la plaque sur de la glace. Ajouter 20 ul de solution de bloc Fc (anti-CD16 / CD32 anticorps à 10 pg / ml dans du tampon FACS) aux cellules et on incube les cellules pendant 10 min sur de la glace. Faire tourner la plaque à 400 x g, 4 ° C pendant 3 min. Jeter le liquide des puits en tapotant la surface de la plaque vers le bas dans un évier. </li><li> Suspendre le culot cellulaire dans 100 pi de cocktail d&#39;anticorps (anti-CD45, -EpCAM, classe -MHC II, et -Ly51 anticorps, et l&#39;UEA-1 lectine marquées avec des fluorochromes à recommandé par le fabricant ou testé des concentrations optimales de chaque anticorps dans un tampon FACS ), et incuber les cellules sur de la glace pendant 20 min dans l&#39;obscurité. Pour la compensation, tacher 1 x 10 6 cellules avec chaque fluorochrome. </li><li> Ajouter 100 ul de tampon FACS à chaque puits et faire tourner la plaque à 400 x g, 4 ° C pendant 3 min. Répétez la seule étape de lavagetemps, puis remettre en suspension les cellules dans du tampon FACS 100 ul. Transférer les cellules dans un tube à fond rond de FACS 12 x 15 mm remplie avec 300 pi de tampon FACS. Acquérir des échantillons sur cytomètre en flux et analyser les données en utilisant un logiciel d&#39;analyse FACS. TEC stratégie de déclenchement est représentée sur la Figure 2. </li></ol> 5. enrichissement des TEC par Panoramique <ol><li> Isoler les cellules du thymus (préparés à la section 3) à 400 xg, 4 ° C pendant 5 min. Aspirer le liquide et en suspension les culots cellulaires dans du tampon de tri FACS à une concentration de 2 x 10 8 cellules par ml dans un tube conique de 15 ml ou 50 ml. Ajouter l&#39;anticorps anti-CD90.2 (clone H12-30) (ou anti-CD45) à une concentration finale de 2,5 pg / ml de suspension de cellules et incuber les cellules doucement sur un agitateur rotatif à 4 ° C pendant 30 min. Après l&#39;incubation, laver avec un 5 - le volume 10x de milieu RP10 et centrifuger le tube à 400 xg, 4 ° C pendant 5 min. </li><li> Aspirer le liquide et remettre en suspension le culot cellulaire dansmilieu RP10 à une concentration de 1 x 10 7 par ml. Ajouter 5 ml de suspension de cellules à chaque plaque revêtue de panoramique (préparé à l&#39;étape 1.3), tourbillon et incuber pendant 30 min à température ambiante. Agiter vigoureusement; transférer les surnageants dans un tube conique. Rincer la plaque deux fois en utilisant un milieu de RP10 et mettre en commun les surnageants dans le tube de collecte. </li><li> Centrifuger le tube de prélèvement à 400 xg, 4 ° C pendant 5 min. Aspirer le liquide et remettre en suspension le culot cellulaire dans 5 ml de milieu de RP10. Ajouter la suspension cellulaire à une nouvelle plaque panoramique, agiter et incuber pendant 30 min à température ambiante. Recueillir les cellules rincer la plaque et mettre en commun les dans un tube conique. Une fois que les TEC sont enrichis, centrifuger les cellules à 400 xg, 4 ° C pendant 5 min et suspendre les cellules dans 5 ml FACS tampon de tri. </li></ol> 6 Purification des TEC par Tri cellulaire <ol><li> Compter les cellules à l&#39;aide d&#39;un hémocytomètre et préparer la suspension de cellules à une concentration de 4 x 10 7 cellules dans du tampon de tri FACS. Ajouter des anticorpsdécrite à l &#39;article 4.4 en solution de cellules et incuber les cellules doucement sur un agitateur rotatif à 4 ° C pendant 30 min. Après la coloration, les cellules laver et suspendre à une concentration de 10 x 10 6 cellules par ml de tampon de tri FACS. </li><li> Trier les sous-ensembles TEC à travers une buse 100 uM en utilisant la fluorescence par tri cellulaire activé. Cellules triées sont collectées dans 30% (v / v) de FBS dans du RPMI-1640). Une fois que le tri de cellules, des cellules se fait de centrifugation à 400 xg, 4 ° C pendant 5 minutes et la préparation pour l&#39;analyse en aval. </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>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Dans le protocole, les étapes critiques sont la préparation de cellules stromales thymiques (section 3) et l&#39;enrichissement des TEC (section 4). Il est fortement recommandé que la solution de l&#39;enzyme fraîche est préparée à chaque fois et le tissu est traité dès que possible. Pour thymus commun, l&#39;optimisation du volume de solution d&#39;enzyme est nécessaire, selon le nombre de thymus. Si un résidu de tissu est laissé après le traitement, en ajoutant davantage de solution d&#39;enzyme et l&#39;...

Au début du développement de cellules T, de la moelle osseuse souches hématopoïétiques progénitrices multipotentes dérivées de cellules sont recrutés dans le cortex du thymus, subir engagement à T lignée et de devenir des précurseurs de cellules T 1. Dans le cortex, précurseurs des lymphocytes T CD4 et CD8 à double négatifs (DN) thymocytes se dilatent et se différencient en immatures CD4 et CD8 positif double (DP) thymocytes, la formation d&#39;une grande piscine avec des progéniteurs très variable T récepteurs cellulaires 1. Seul un sous-ensemble de sélection restreinte au CMH de cellules DP deviendra unique positifs (SP) thymocytes CD4 ou CD8, migrer vers la moelle du thymus, et se différencier en cellules T matures fonctionnellement compétentes, un événement qui est visé à la sélection positive 2 6. En revanche, les clones de thymocytes auto-réactifs sont soumis à une sélection négative et sont évacués par l&#39;apoptose, convertis en cellules T régulatrices pour l&#39;auto-tolérance, ou dérivé de lymphocytes intra-épithéliaux à des fins qui ne sont pas encore clairement 3,7-10. Dans le thymus, les cellules stromales thymiques forment un microenvironnement unique, pour fournir des signaux destin 5,11,12 ces différents de développement des cellules T. Les cellules stromales thymiques sont composés de cellules épithéliales thymiques (CET) - y compris TEC corticales (CTEC) et TEC médullaires (mTECs), les cellules dendritiques, les macrophages, les fibroblastes, les cellules endothéliales, les péricytes de la crête neurale et d&#39;autres cellules mésenchymateuses 13-15. Parmi ceux-ci, les TEC sont essentiels aux différents stades de développement des cellules T 1,2,16,17. Cependant, l&#39;absence d&#39;un moyen efficace d&#39;isoler TEC a entravé une compréhension globale de leurs fonctions 16. En particulier, CTEC, qui forment un réseau tridimensionnel progéniteurs dans le cortex environnant, sont essentiels pour la sélection positive 13,18,19 pour des raisons qui ne sont pas encore claires. Des études antérieures fournies indices quant à l&#39;hétérogénéité et le rôle des TEC, la plupart s&#39;appuyant sur ​​des outils morphologiques et histologiques 13 </sup&gt;. Récemment, les rôles uniques de sous-ensembles TEC ont été abordés par des approches génétiques dans des modèles murins 12,20. Une façon robuste et reproductible pour isoler TEC est fondamentale pour atteindre la caractérisation objective de sous-ensembles de TEC, une évaluation quantitative et qualitative des fonctions de TEC, et la clarification des mécanismes comment CTEC soutenir la sélection positive. En raison de la rareté des TEC dans le thymus et les interactions serrés qu&#39;ils forment dans l&#39;organe intact, l&#39;isolement des TEC a été difficile. Le protocole décrit ici est basé sur les découvertes précédentes, actuellement réactifs disponibles, les techniques et les connaissances de la structure du thymus et de la composition du stroma. Il ya près de deux décennies, plusieurs procédures ont été signalés à désagréger les tissus du thymus 21-27, dans lequel les différentes enzymes ont été utilisés lors de la digestion, y compris la trypsine, la collagénase et Dispase. Gray et al. rapport ces enzymes dans leur precedure 28, et fait état ​​d&#39;une amélioration de la méthamphétamineod, avec une digestion multi-étape de cocktails d&#39;enzymes 29 qui sont devenus largement utilisé 20,30. Cependant, cette méthode implique un temps de préparation longs et complexes étapes de digestion et entraîne dernière variable du nombre de cellules et les proportions des TEC, même dans la même cohorte de souris 29,30. Il ya plusieurs années, Libérase enzyme de qualité de la recherche, hautement purifié contenant la collagénase et la protéase neutre commencé à être utilisé dans le tissu du thymus dissociation 30. Ici, nous décrivons un protocole à base de Libérase digestion, avec des procédés de séparation mécanique optimisés, qui donne un grand nombre de dispositifs de refroidissement thermoélectriques viables à partir de tissus de thymus de souris. . Pour enrichir les cellules stromales dissociés, des études antérieures ont utilisé soit des gradients de densité ou séparation magnétique de perles 21,29. Cependant, les deux méthodes provoquent de graves perdu de certaines populations de cellules stromales thymiques, en particulier la population de CTEC 29. Élimination cytotoxique et techniques panoramique <sjusqu&#39;à&gt; 31,32 ont largement été utilisés pour l&#39;épuisement ou la séparation des lymphocytes dans le domaine immunologique 12,31. Après comparaison de ces techniques, nous avons établi le protocole de panoramique actuel pour l&#39;enrichissement des TEC. La condition douce au cours de la procédure d&#39;enrichissement conduit à moins de la mort cellulaire et impartiale et une récupération accrue de TEC. La suspension de cellules de thymus isolé décrit dans la section 3 peut être directement appliquée à l&#39;analyse par cytométrie de débit pour l&#39;identification et la caractérisation des sous-populations de cellules dendritiques et TEC. La section 4 décrit une méthode simple et utile pour identifier des sous-TEC à l&#39;aide de flux multiparamétrique cytomètre. Pour les expériences qui cherchent à obtenir CTEC purifiés ou mTECs, l&#39;enrichissement de TEC et procédures de tri cellulaire peut être trouvé dans la section 5 et 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

Le thymus est un organe vital pour le développement des lymphocytes T. Des cellules stromales thymiques, les cellules épithéliales thymiques (CET) sont particulièrement cruciales à plusieurs étapes du développement des cellules T: l&#39;engagement des cellules T, de sélection positive et négative sélection. Toutefois, la fonction de refroidissement thermoélectriques dans le thymus reste incomplètement comprise. Dans cet article, nous proposons une méthode pour isoler des sous-ensembles TEC de thymus de souris frais en utilisant une combinaison de rupture mécanique et la digestion enzymatique. Le procédé permet aux cellules thymocytes et stromales thymiques être efficacement libérés à partir de la cellule-cellule et des connexions de la matrice extracellulaire et de cellules pour former une suspension de cellules isolées. Utilisation de cellules isolées, de cytométrie de flux à paramètres multiples peut être appliqué à l&#39;identification et la caractérisation des TEC et des cellules dendritiques. Parce que les TEC sont une population de cellules rares dans le thymus, nous décrivons également un moyen efficace d&#39;enrichir et de purifier les TEC en appauvrissant thymocytes, type cellulaire le plus abondant dans le thymus. Follol&#39;aile enrichissement, le temps de tri de cellule peut être réduite de sorte que la perte de la viabilité des cellules peut être réduite au minimum au cours de la purification de TEC. Cellules purifiées sont adaptés à diverses analyses en aval comme PCR en temps réel, Western blot et profil d&#39;expression génique. Le protocole de promouvoir la recherche de la fonction TEC et ainsi que le développement de la reconstitution in vitro des lymphocytes T.

En utilisant ce protocole, un organe thymus a été enlevée d&#39;une souris adulte (section 2) et une suspension de cellules du thymus a été préparé comme décrit à la section 3 La suspension cellulaire obtenue est composée de thymocytes, les cellules stromales hématopoïétiques dérivés et les cellules stromales non-hématopoïétiques. CD45 est un marqueur pan-hématopoïétique exprimé sur les thymocytes et les cellules stromales hématopoïétiques telles que les macrophages et les cellules dendritiques....

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

Nous décrivons ici un procédé efficace pour l&#39;isolement, l&#39;identification et la purification des cellules épithéliales du thymus de souris (CET). Le protocole peut être utilisé pour l&#39;étude de la fonction du thymus d&#39;un développement normal des lymphocytes T, un dysfonctionnement du thymus et de reconstitution de la cellule T.

Isolement, identification et la purification de cellules murines thymique épithéliales

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 vues.  University of Minnesota.  Nous décrivons ici un procédé efficace pour l'isolement, l'identification et la purification des cellules épithéliales du thymus de souris (CET). Le protocole peut être utilisé pour l'étude de la fonction du thymus d'un développement normal des lymphocytes T, un dysfonctionnement du thymus et de reconstitution de la cellule T. 

Vidéo: 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.

Application d&#39;une souris ligaturés Assay Peyer Patch anse intestinale pour évaluer absorption par les cellules bactériennes M

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

Recherche

Immunologie et infection

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.

Isolation par cytométrie en flux des cellules primaires murins épithéliales alvéolaires de type II pour les études fonctionnelles et moléculaires

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.

Isolation, traitement et analyse des cellules gingivales murins

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.

Isolement des cellules dendritiques myéloïdes et les cellules épithéliales du thymus humain

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.

Isolement de cellules murines ganglions lymphatiques stromales

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.

Purification à grande échelle d&#39;origine porcine ou bovine photorécepteur Outer Segments pour phagocytose essais sur les cellules épithélium pigmentaire rétinien

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.

L&#39;isolement et la transplantation de différents âgées murins thymiques greffes.

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.

Promotion 3-D agrégation de cellules FACS purifiées thymique épithéliales avec EAK 16-II / EAKIIH6 auto-assemblage Hydrogel

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.

Purification de l’ADN Viral pour l’Identification des protéines virus et cellulaires associées

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.