Questo lavoro è stato supportato dal National Institutes of Health di Grant R01 AI088209 (a KAH). Ringraziamo anche l&#39;Università del Minnesota Citometria a flusso di risorse.

In questo studio, adulto - sono stati utilizzati (6 8 settimane) femmina C57Bl / 6 topi. I topi sono stati acquistati dal National Cancer Institute e mantenuti in particolari condizioni di libero patogeni. L&#39;Università del Minnesota Istituzionale Cura e uso Comitato degli animali (IACUC) ha approvato tutti sperimentazione animale. 1 Preparazione degli strumenti e tamponi <ol><li> Preparare la soluzione enzimatica (media RPMI1640 con il 0,05% [w / v] di Liberase TH e 100 U / ml di DNasi I), tampone ricchi di albumina (1X PBS [Ca 2 + / Mg 2 + -free] con 0,5% di siero bovino albumina e 2 mM acido Etilendiammina tetraacetico [EDTA]), tampone di FACS (1X PBS [Ca 2 + / Mg 2 + -free] siero fetale bovino 1% contenente [FBS] e il 0,02% NaN 3), FACS ordinamento tampone (PBS 1X [Ca 2 + / Mg 2 + -free] contenente il 2% FBS), e media RP10 (media RPMI-1640 fornito con 10% FBS). </li><li> Preparare 10 piatti panning. Preparare la soluzione di rivestimento 40 ml (0,01 mg / ml Gavena contro Rat-IgG in 0.1 M NaHCO3 Buffer), e aggiungere 4 ml di soluzione di rivestimento in ciascuna 100 x 15 mm piastra, turbolenza e incubare a 4 ° CO / N. Eliminare la soluzione di anticorpi e lavare la piastra con 5 ml di PBS 1x per 2 volte, agitare vigorosamente tra risciacqui. Conservare le piastre preparate a 4 ° C. </li></ol> 2 Raccolta di Timo Tissue <ol><li> Euthanize del mouse in una camera di CO 2. Mettere l&#39;animale sulla schiena su una stuoia di dissezione, appuntare i piedi sul tappetino e bagnare la pelliccia a spruzzo con il 70% di etanolo. </li><li> Eseguire un&#39;incisione mediana attraverso la pelle, partendo da sopra l&#39;apertura uretrale dritto fino alla mascella inferiore con le forbici ed estendere l&#39;incisione verso il basso per le ginocchia su entrambi i lati. L&#39;incisione sembra un testa in giù &quot;Y&quot;. Tirare la pelle flaccida indietro sui lati, e il pin al tappeto dissezione. </li><li> Forare la membrana dalla xifoide, tagliare le costole su entrambi i lati fino a circa la clavicola, quindi sollevare le costole conpinza e il pin al tappeto dissezione. Il timo è appena sotto le costole, e si presenta come due lobi bianchi sottili sovrapposti cuore (Figura 1). Tessuto connettivo che circonda Scollegare il timo, tirare delicatamente e rimuovere la coppia di lobi del timo con pinze dentellate curve. Lobi Luogo timo in una piastra da 6 pozzetti contenenti 5 ml di RPMI-1640. </li></ol> 3 Preparazione di cellule stromali timica <ol><li> Tagliare qualsiasi tessuto adiposo e connettivo circostante e trasferire i lobi del timo ad un nuovo pozzo della piastra 6 pozzetti contenenti 5 ml di fresco preparano soluzione enzimatica. Fare piccole incisioni nei lobi del timo con le forbici sottili, e incubare la piastra a 37 ° C per 20 min. </li><li> Pipettare delicatamente digerire il tessuto del timo su e giù 2 - 3 volte con 5 ml pipetta per la dissociazione dei tessuti. Raccogliere il surnatante in una provetta da 50 ml contenente 10 ml di tampone ricchi di albumina freddo per neutralizzare gli enzimi. Tenere il tubo di raccolta sul ghiaccio. Aggiungere 2.5soluzione enzimatica ml al tessuto rimanente e incubare la piastra a 37 ° C per 15 min. </li><li> Eseguire agitazione meccanica con una siringa da 3 ml e ago da 18 G per rompere gli aggregati. Pool surnatante nel tubo di raccolta. Aggiungere 2,5 ml della soluzione enzimatica al tessuto rimanente e incubare la piastra a 37 ° C per 15 min. </li><li> Ripetere agitazione meccanica con una siringa da 3 ml e un ago da 25 G, e incubare la piastra supplementare di 5 - 10 min. </li><li> Dopo aver completato la digestione, piscina il surnatante nel tubo di raccolta. Centrifugare la provetta di prelievo a 400 xg, a 4 ° C per 8 minuti per raccogliere le cellule. Aspirare il liquido e sospendere le cellule pellet in 10 ml di tampone ricchi di albumina e filtrare il campione attraverso maglie di mm 100. Contare le cellule utilizzando un emocitometro. Quindi eseguire il flusso cytometic analisi (sezione 4) o TEC arricchimento (sezione 5). </li></ol> 4 Identificazione delle TEC da Citometria a flusso </p&gt; <ol><li> Preparare le cellule ad una concentrazione finale di 4 x 10 6 cellule per 100 microlitri di buffer di FACS per la colorazione in una piastra di prova ben a fondo rotondo da 96 e tenere la piastra su ghiaccio. Aggiungere 20 microlitri soluzione di blocco Fc (anti-CD16 / CD32 anticorpo a 10 mg / ml in tampone per FACS) alle cellule e incubare le cellule per 10 minuti in ghiaccio. Gira la piastra a 400 xg, a 4 ° C per 3 min. Eliminare il liquido dai pozzi muovendo la faccia piastra giù in un lavandino. </li><li> Sospendere le cellule pellet in 100 ml di cocktail di anticorpi (anti-CD45, -EpCAM, classe -MHC II, e -Ly51 anticorpi, e la UEA-1 lectina marcato con fluorocromi diversi a produttori raccomandati o testato concentrazioni ottimali di ciascun anticorpo in tampone FACS ), e incubare le cellule in ghiaccio per 20 minuti al buio. Per compensazione, macchiare 1 x 10 6 cellule con ogni singolo fluorocromo. </li><li> Aggiungere 100 ml di tampone FACS a ciascun bene e far girare la piastra a 400 xg, a 4 ° C per 3 min. Ripetere una fase di lavaggiotempo, e poi risospendere le cellule in 100 microlitri di buffer FACS. Trasferire le cellule di un fondo rotondo tubo FACS 12 x 15 mm riempita con 300 ml FACS buffer. Acquisire campioni sul citofluorimetro e analizzare i dati utilizzando il software di analisi FACS. Strategia di gating TEC è mostrato nella Figura 2. </li></ol> 5. Arricchimento della TEC da Panoramica <ol><li> Centrifugare le cellule del timo (preparati nella Sezione 3) a 400 xg, a 4 ° C per 5 min. Aspirare il liquido e sospendere le cellule pellet nel tampone FACS ordinamento ad una concentrazione di 2 x 10 8 cellule per ml in una provetta conica da 15 ml o 50 ml. Aggiungere anticorpo anti-CD90.2 (clone H12-30) (o anti-CD45) ad una concentrazione finale di 2,5 mg / ml di sospensione cellulare e incubare le cellule delicatamente su un agitatore rotante a 4 ° C per 30 min. Dopo l&#39;incubazione, lavare con un 5 - 10 volte il volume di RP10 medie e centrifugare la provetta a 400 xg, a 4 ° C per 5 min. </li><li> Aspirare il liquido e risospendere le cellule in pelletMezzo RP10 ad una concentrazione di 1 x 10 7 per ml. Aggiungere 5 ml di sospensione cellulare per ogni piastra rivestita panning (preparata al punto 1.3), turbolenza e incubare per 30 minuti a temperatura ambiente. Agitare vigorosamente; trasferire il surnatante in una provetta conica. Sciacquare la piastra due volte utilizzando medie RP10 e piscina surnatanti nel tubo di raccolta. </li><li> Centrifugare la provetta di raccolta a 400 xg, a 4 ° C per 5 min. Aspirare il liquido e risospendere le cellule pellet in 5 ml di mezzo RP10. Aggiungere la sospensione cellulare di una nuova piastra panning, agitare ed incubare per 30 minuti a temperatura ambiente. Raccogliere le cellule lavare il piatto e piscina in un tubo conico. Una volta TEC sono arricchiti, centrifugare le cellule a 400 xg, a 4 ° C per 5 minuti e sospendere le cellule in 5 ml FACS buffer di smistamento. </li></ol> 6. Purificazione di TEC di classificare cellulare <ol><li> Contare le cellule utilizzando un emocitometro e preparare sospensione cellulare ad una concentrazione di 4 x 10 7 cellule in tampone FACS ordinamento. Aggiungi anticorpi desCribed nella Sezione 4.4 nella soluzione di cellule e incubare le cellule delicatamente su un agitatore rotante a 4 ° C per 30 min. Dopo la colorazione, lavare e sospendere le cellule ad una concentrazione di 10 x 10 6 cellule per ml di tampone FACS ordinamento. </li><li> Ordina sottoinsiemi TEC attraverso un ugello 100 micron utilizzando la fluorescenza attivato l&#39;ordinamento delle cellule. Cella ordine sono raccolti in 30% (v / v) di FBS in RPMI-1640). Una volta che l&#39;ordinamento delle cellule è fatto, le cellule centrifugare a 400 xg, a 4 ° C per 5 minuti e prepararsi per l&#39;analisi a valle. </li></ol>

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	<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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Nel protocollo, punti critici sono la preparazione di cellule stromali del timo (sezione 3) e l&#39;arricchimento di TEC (sezione 4). Si raccomanda vivamente che la soluzione enzimatica fresca viene preparata ogni volta e il tessuto viene trattato il più presto possibile. Per thymi studiata, ottimizzando il volume della soluzione enzimatica è richiesta a seconda del numero di thymi. Se eventuali residui di tessuto viene lasciato dopo l&#39;elaborazione, aggiungendo più soluzione enzimatica e l&#39;estensione del temp...

All&#39;inizio lo sviluppo delle cellule T, midollo osseo di cellule staminali ematopoietiche derivate da progenitori multipotenti sono reclutati alla corteccia del timo, sottoposto impegno a T lignaggio e diventare precursori delle cellule T 1. Nella corteccia, precursori delle cellule T CD4 e CD8 doppio negativi (DN) timociti espandersi e differenziarsi in immaturo CD4 e CD8 doppio positivi (DP) timociti, formando una grande piscina di progenitori ai ricettori cellulari altamente variabile T 1. Solo una select sottoinsieme MHC-ristretto di cellule DP diventerà singolo positivi (SP) timociti CD4 o CD8, migrare verso il midollo del timo, e differenziarsi in cellule funzionalmente competenti T maturi, un evento che si riferisce alla selezione come positivo 2 6. Al contrario, cloni di timociti auto-reattivi sottoposti a selezione negativa e vengono rimossi tramite apoptosi, convertito in cellule T regolatorie per l&#39;auto-tolleranza, o deviato verso linfociti intraepiteliali per scopi che non sono ancora chiare 3,7-10. Nel timo, cellule stromali del timo formano un microambiente unico che fornisce segnali per questi diversi destini di sviluppo delle cellule T 5,11,12. Cellule stromali del timo sono composti da cellule epiteliali timiche (TEC) - tra cui TEC corticali (CTEC) e TEC midollari (mTECs), cellule dendritiche, macrofagi, fibroblasti, cellule endoteliali, periciti derivati ​​dalla cresta neurale e di altre cellule mesenchimali 13-15. Tra questi, TEC sono cruciali nelle varie fasi di sviluppo delle cellule T 1,2,16,17. Tuttavia, la mancanza di un modo affidabile per isolare TEC ha impedito una comprensione globale delle loro funzioni 16. In particolare, CTEC, che formano una rete tridimensionale progenitori nella corteccia circostante, sono essenziali per la selezione positiva 13,18,19, per ragioni che non sono ancora chiare. Studi precedenti fornito indizi utili a capire l&#39;eterogeneità e il ruolo di TEC, per lo più basandosi su strumenti morfologici e istologici 13 </sup&gt;. Recentemente i ruoli unici di sottoinsiemi TEC sono state affrontate con approcci genetici in modelli murini 12,20. Un modo affidabile e riproducibile per isolare TEC è fondamentale per raggiungere la caratterizzazione imparziale di sottoinsiemi TEC, valutazione quantitativa e qualitativa delle funzioni TEC, e chiarimento dei meccanismi come CTEC supportano la selezione positiva. A causa della rarità della TEC in timo e le interazioni strette formano nell&#39;organo intatto, l&#39;isolamento di TEC è stato impegnativo. Il protocollo qui descritto è basato sulle scoperte precedenti, attualmente reagenti disponibili, le tecniche e la conoscenza della struttura del timo e della composizione stromale. Quasi due decenni fa, sono stati segnalati diverse procedure di disaggregare i tessuti timo 21-27, in cui i diversi enzimi sono stati usati durante la digestione, tra cui tripsina, Collagenase e dispasi. Grigio et al. comparano questi enzimi nella loro precedure 28, e ha registrato un miglioramento method una digestione più fasi di cocktail di enzimi 29 che si sono resi ampiamente utilizzato 20,30. Tuttavia, questo metodo richiede un lungo tempo di preparazione e mineralizzazione complessi e risultati in variabile finale numero di cellule e proporzioni di TEC anche nella stessa coorte murino 29,30. Diversi anni fa, Liberase grado di ricerca enzima, contenenti altamente purificato Collagenase e proteasi neutra cominciò ad essere utilizzato nel tessuto del timo dissociazione 30. Qui, descriviamo un protocollo basato digestione Liberase, con procedure di separazione meccanica ottimizzate, che produce un elevato numero di TEC vitali dai tessuti del mouse timo. . Per arricchire cellule stromali dissociate, studi precedenti hanno utilizzato sia gradienti di densità o di separazione tallone magnetica 21,29. Tuttavia, entrambi i metodi causano grave perdita di alcune popolazioni di cellule stromali del timo, in particolare la popolazione di CTEC 29. Eliminazione citotossici e tecniche di panning <sup&gt; 31,32 sono stati ampiamente utilizzati per esaurimento o separazione dei linfociti nel campo immunologica 12,31. Dopo il confronto di queste tecniche, abbiamo stabilito il protocollo panning attuale per l&#39;arricchimento della TEC. La condizione delicata durante la procedura di arricchimento porta a meno morte cellulare e imparziale e maggiore recupero TEC. La sospensione di cellule isolate timo descritto nella Sezione 3 può essere applicato direttamente alle analisi citofluorimetrica per l&#39;identificazione e la caratterizzazione dei sottoinsiemi TEC e cellule dendritiche. Sezione 4 descrive un modo semplice e utile per identificare sottoinsiemi TEC utilizzando il flusso multiparametrica citometro. Per gli esperimenti che cercano di ottenere CTEC purificate o mTECs, TEC arricchimento e cell sorting procedure può essere trovato nella sezione 5 e 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

Il timo è un organo vitale per lo sviluppo dei linfociti T. Di cellule stromali del timo, le cellule epiteliali timiche (TEC) sono particolarmente cruciali in più stadi di sviluppo delle cellule T: l&#39;impegno delle cellule T, selezione positiva e selezione negativa. Tuttavia, la funzione del TEC nel timo rimane completamente compresa. Nell&#39;articolo, mettiamo a disposizione un metodo per isolare sottoinsiemi TEC da topo fresco timo utilizzando una combinazione di perturbazione meccanica e digestione enzimatica. Il metodo permette di cellule stromali timiche e timociti per essere efficacemente rilasciato dalla cellula-cellula e connessioni cellula-matrice e per formare una cella singola sospensione. Usando le cellule isolate, citometria a flusso multiparametrica può essere applicato alla identificazione e caratterizzazione di TEC e cellule dendritiche. Perché TEC sono una rara popolazione di cellule nel timo, anche noi descriviamo un modo efficace per arricchire e purificare TEC mediante deplezione timociti, il tipo cellulare più abbondante nel timo. Folloala l&#39;arricchimento, cell sorting tempo può essere ridotto in modo che la perdita di vitalità cellulare può essere minimizzato durante la purificazione della TEC. Cellule purificate sono adatti per varie analisi a valle come Real Time-PCR, Western blot e profilo di espressione genica. Il protocollo promuoverà la ricerca della funzione TCE e così come lo sviluppo di ricostituzione in vitro di cellule T.

Usando questo protocollo, un organo del timo è stato rimosso da un topo adulto (sezione 2) e una sospensione di cellule del timo è stata preparata come descritto nella Sezione 3 La sospensione cellulare ottenuta consisteva di timociti, cellule stromali ematopoietiche derivate da cellule stromali e non-ematopoietiche. CD45 è una ematopoietiche pan-marcatore espresso su entrambi i timociti e cellule stromali ematopoietiche come i macrofagi e le cellule dendritiche. Al contrario, TEC non sono di origine emopoietica e no...

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

Qui si descrive un metodo efficace per l&#39;isolamento, l&#39;identificazione e la purificazione delle cellule epiteliali del timo topo (TEC). Il protocollo può essere utilizzato per gli studi di funzione del timo per il normale sviluppo delle cellule T, la disfunzione del timo, e la ricostituzione delle cellule T.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

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

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Research

JoVE Journal

Immunology and Infection

31.7K Views.  University of Minnesota.  Qui si descrive un metodo efficace per l'isolamento, l'identificazione e la purificazione delle cellule epiteliali del timo topo (TEC). Il protocollo può essere utilizzato per gli studi di funzione del timo per il normale sviluppo delle cellule T, la disfunzione del timo, e la ricostituzione delle cellule 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.

Applicazione di un mouse legatura Patch Peyer Assay intestinale Loop per valutare assorbimento da parte delle cellule batteriche M

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

Ricerca

Immunologia e infezioni

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.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

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.

Isolamento, elaborazione ed analisi dei murini gengivali

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.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

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.

Isolamento di cellule murine Linfonodo stromali

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.

Large-Scale Purificazione di suino o bovino fotorecettore Outer Segmenti per fagocitosi saggi su cellule retiniche dell&#39;epitelio pigmentato

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.

Isolamento e Trapianto di diversi invecchiato murini del timo innesti.

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.

La promozione 3-D di aggregazione delle cellule FACS purificata timica epiteliali con EAK 16-II / EAKIIH6 autoassemblanti idrogel

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.

Purificazione di DNA virale per l'identificazione delle proteine virali e cellulari associate

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.