This work was supported by funding received from the National Institutes of Health (1R01AI083269).

Les études murins ont été approuvés par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) de l&#39;Université de Caroline du Nord à Chapel Hill et de tous les soins des animaux était en conformité avec les lignes directrices du IACUC. 1. Préparation du nouveau-né et de l&#39;adulte Thymi <ol><li> Préparer tous les réactifs et le matériel avant l&#39;euthanasie souris donneuses. </li><li> Stériliser les instruments chirurgicaux par autoclavage ou d&#39;autres méthodes appropriées. Toutes les interventions chirurgicales doivent être effectuées sous une hotte à flux laminaire afin de maintenir des conditions stériles et éviter la contamination. Assemblez les outils nécessaires pour l&#39;extraction de thymus de souris donneuses. </li><li> Remplir une boîte de 60 mm avec stérile 1x PBS (pH 7,4) et le placer sur la glace à l&#39;intérieur du capot. Il sera utilisé pour stocker brièvement thymus excisé de souris donneuses avant la transplantation. </li><li> Conformément aux lignes directrices éthiques, euthanasier souris donneuses nouveau-nés par des souris de décapitation et donneurs adultes par le CO 2 asphyxie suivie par dislocation cervicale. </li><li> Pour ra suppression du thymus: Posez la souris dans une position couchée dorsale sur une serviette de papier absorbant stérile et pulvériser avec 70% d&#39;éthanol avant de faire une incision. </li><li> Exposer la cavité abdominale et thoracique en faisant une incision de ligne médiane à travers la peau. Pliez la peau sur la poitrine et les pattes avant de révéler la cavité thoracique. </li><li> Faire deux incisions latérales à travers le diaphragme et la cage thoracique pour exposer le médiastin supérieur et antérieur cavité thoracique. Le thymus doit être visible sous forme de deux lobes blanc immédiatement au-dessus et à côté du coeur. </li><li> Démêler le tissu conjonctif qui entoure le thymus avec des pinces fines, afin d&#39;être certain de ne pas perturber la capsule. Tout en maintenant la cage thoracique arrière avec une pince, utiliser une deuxième paire de pinces pour extraire les deux lobes du thymus par le positionnement des pinces incurvées sous l&#39;organe de traction et verticalement. Cela peut être fait en utilisant un microscope à dissection pour extraire thymus de souris nouveau-nés. </li><li> Placez le thymus dans le 60 mm dish contient 1x PBS stérile (pH 7,4) sur de la glace et des lobes thymiques séparés en coupant à travers l&#39;isthme conjonctif. Enlevez les débris des lobes thymiques faisant certain de ne pas endommager la capsule, et couper le thymus dans le nombre approprié de sections pour la transplantation. </li><li> Ne pas manipuler le thymus obtenu à partir de souris nouveau-nés. NOTE: Utilisé pour un maximum de deux souris receveuses (1 lobe par bénéficiaire). </li><li> Transplanter le thymus obtenu à partir de souris adultes en un maximum de 4-6 souris receveuses. En utilisant une paire de pinces, saisir attentivement un adulte lobe thymique, comme le montre la figure 1, couper le thymus en trois parties égales à l&#39;aide de ciseaux chirurgicaux. </li><li> Répétez les étapes 1.3 à 1.4 pour chaque souris donneuse. Afin de limiter le temps de thymus sont exposés, préparer 1 donateurs thymus à la fois. </li></ol> 2. Thymus implantation sous la capsule du rein <ol><li> Assembler le matériel pré-stérilisé énumérés dans le tableau 1. Aseptique appropriéetechnique devrait être utilisée au cours de la procédure pour éviter d&#39;exposer transplantation bénéficiaires à des outils ou des réactifs contaminés. </li><li> Avant la transplantation, peser et marquer chaque souris receveuse. </li><li> Mettre en place le microscope à dissection et le circuit d&#39;anesthésie dans la hotte à flux laminaire. </li><li> Utiliser un rasoir électrique, de se raser le côté gauche de la souris receveuse et assurer pas de cheveux reste autour de la zone utilisée pour réaliser l&#39;incision. </li><li> Activer le vaporisateur isofluorane et anesthésier la souris en utilisant une dose comprise entre 1 à 2%. Déterminer anesthésie appropriée avant de commencer la procédure chirurgicale en vérifiant l&#39;absence de réflexe suivante pincement de l&#39;orteil. </li><li> Après la souris est correctement anesthésié, appliquer une pommade vétérinaire aux yeux de la souris pour prévenir la sécheresse et de préparer la souris pour la transplantation comme suit: </li><li> Placez la souris sous le microscope de dissection dans une position couchée sur le côté droit de sorte que le côté rasé est orientée vers le haut. Starting dans le centre de la zone chirurgicale, passer dans un mouvement circulaire en utilisant une pipette de transfert jetable éthanol à 70%, suivie par la povidone iodée (Bétadine). Répéter le traitement éthanol / bétadine 3 fois avant de faire une incision. </li><li> L&#39;aide de ciseaux de dissection font un flanc incision de 1-2 cm au-dessus du rein. </li><li> En utilisant des pinces moyennes saisir tissu conjonctif adjacent au rein et soulevez doucement le rein sorte qu&#39;il se trouve au sommet de la musculature. Pour garder le rein exposé et mis en place au cours de la musculature, insérez un bras de pinces moyennes sous le rein, en accordant une attention particulière à ne pas perturber l&#39;os iliaque rénale. </li><li> Afin d&#39;éviter la dessiccation du tissu, irriguer le rein avec PBS 1x stérile (pH 7,4) pendant chaque étape jusqu&#39;à ce que la manipulation de la capsule rénale du rein et est terminée. </li><li> En utilisant des pinces fines pincer et soulever la capsule à proximité du bord du rein la plus distale vers les glandes surrénales pour la séparer du rein. </li><li> Utilisez une jauge 18aiguille pour faire une incision assez grand pour insérer la pièce préparée de donateurs thymus (figure 2A). Assurez-vous de maintenir l&#39;incision capsulaire aussi faible que possible pour empêcher la dislocation de la greffe au cours du temps. </li><li> Tout en maintenant la capsule arrachée de rein, en utilisant une deuxième paire de pinces fines pour insérer la greffe en dessous de la capsule et appuyer sur le thymus le plus en avant à partir de l&#39;incision capsulaire que possible (figure 2B). </li><li> Retirer les pinces de dessous le rein, et retourner doucement le rein en place à travers l&#39;incision. </li><li> Fermez la musculature en suturant la paroi péritonéale et l&#39;application de la bétadine fois fermé. Après suture terminée, appliquer des agrafes pour fermer le derme et appliquer la bétadine à la zone environnante. </li><li> Retourner la souris dans une cage qui a été chauffé en utilisant une lampe chauffante. </li><li> Surveiller chaque souris post-opératoire jusqu&#39;à ce qu&#39;il retrouve la pleine conscience et la mobilité et ne placez pas la souris that ont pas récupéré après une anesthésie dans une cage avec d&#39;autres animaux. </li><li> Traiter les souris avec des médicaments de traitement de la douleur post-opératoire si nécessaire. Fournir des souris avec des analgésiques post-opératoires comme l&#39;acétaminophène dans l&#39;eau potable à une concentration de 1,6 mg / ml. </li><li> Répétez les étapes 2.3 à 2.9 pour chaque souris receveuse. </li><li> Veiller à ce que les souris retour à une activité normale dans les 1 h post-chirurgie. Surveiller le poids post-opératoire, de la motricité, ainsi que l&#39;activité de boisson et de l&#39;alimentation de chaque souris pour déterminer les complications découlant de la procédure de transplantation. REMARQUE: la perte de poids post-opératoire, par rapport à un poids pré-opératoire, ainsi que la mobilité altérée ou l&#39;échec de manger ou de boire peut indiquer complications. </li><li> Surveiller T reconstitution de cellules dans le sang périphérique par des souris saignements via queue nick suivie d&#39;une séparation des lymphocytes en utilisant Lympholyte médias de séparation des cellules selon les spécifications du fabricant. Accomplir T Établissement des caractéristiques de cellulestion par coloration des lymphocytes avec des anticorps conjugués à des marqueurs par fluorescence spécifiques de cellules T CD3, CD4, CD8 et, ainsi que d&#39;un discriminateur mort direct tel qu&#39;un ester de succinimidyle activé par fluorescence 8. Analyse comme représenté sur la figure 3 peut être réalisé par l&#39;intermédiaire de déclenchement sur ​​maillots de corps, des cellules vivantes et des cellules positives pour CD3. </li></ol>

<ol>
	<li>Kappler, J. W., Roehm, N., Marrack, P. <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+by+clonal+elimination+in+the+thymus.">T cell tolerance by clonal elimination in the thymus.</a> Cell. 49 (2), 273-280 (1987).</li><li>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=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat. Rev. Immunol. 6, 127-135 (2006).</li><li>Klein, L., Kyewski, B., Allen, P. M., 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+the+T+cell+repertoire:+what+thymocytes+see+(and+don&#8217;t+see).">Positive and negative selection of the T cell repertoire: what thymocytes see (and don&#8217;t see).</a> Nat. Rev. Immunol. 14 (6), 377-391 (2014).</li><li>Schwartz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+immunologic+self-tolerance.">Acquisition of immunologic self-tolerance.</a> Cell. 57 (7), 1073-1081 (1989).</li><li>Kishimoto, H., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+central+tolerance+in+NOD+mice.">A defect in central tolerance in NOD mice.</a> Nat. Immunol. 2 (11), 1025-1031 (2001).</li><li>Anderson, G., 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=Investigating+central+tolerance+with+reaggregate+thymus+organ+cultures.">Investigating central tolerance with reaggregate thymus organ cultures.</a> Methods. Mol. Biol. 380, 185-196 (2007).</li><li>He, Q., 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+development+of+autoreactive+T+cells+in+NOD+mice+is+regulated+in+an+age-dependent+manner.">Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.</a> J. Immunol. 191 (12), 5858-5866 (2013).</li><li>Stewart, C. C., Stewart, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.+Immunophenotyping.">Chapter 6. Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Rice, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+transplantation+for+complete+DiGeorge+syndrome:+medical+and+surgical+considerations.">Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations.</a> J. Pediatr. Surg. 39 (11), 1607-1615 (2004).</li><li>Markert, M. L., 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=Transplantation+of+thymus+tissue+in+complete+DiGeorge+Syndrome.">Transplantation of thymus tissue in complete DiGeorge Syndrome.</a> N. Engl. J. Med. 341, 1180-1189 (1999).</li><li>Market, M. L., Devlin, B. H., McCarthy, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation.">Thymus transplantation.</a> Clin. Immunol. 135 (2), 236-246 (2010).</li><li>Wells, A. D., Li, X., Strom, T. B., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+peripheral+T-cell+depletion+in+transplantation+tolerance.">The role of peripheral T-cell depletion in transplantation tolerance.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (1409), 617-623 (2001).</li><li>Albuquerque, A. 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=Human+FOXN1-Deficiency+is+associated+with+ab+double-negative+and+FoxP3++T-cell+expansions+that+are+distinctly+modulated+upon+thymic+transplantation.">Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.</a> PLoS One. 7 (5), e37042 (2012).</li><li>Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation+restores+the+repertoires+of+forkhead+box+protein+3+(FoxP3)++and+FoxP3-+T+cells+in+complete+DiGeorge+anomaly.">Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.</a> Clin Exp Immunol. 173 (1), 140-149 (2013).</li><li>Savino, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thymus+is+a+common+target+organ+in+infectious+diseases.">The thymus is a common target organ in infectious diseases.</a> PLoS Pathog. 2 (6), e62 (2006).</li></ol>

The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge syndrome, characterized by thymic hypoplasia or thymic aplasia9-10. Thymus transplantation, like most organ transplantation, is not free of complications, which are mostly due to transplant tolerance-related complications12 and development of autoimmune pathologies11. These issues, coupled with the need to better understand and characterize mechanisms involved in thymocyte tolerance induction led to the development of this method. The protocol described herein experimentally models thymic transplantation and demonstrates that efficient T cell reconstitution is obtained in recipients.
There are several key points to note in the protocol. It is necessary to be adept at surgery and aseptic technique prior to performing the transplantation surgery. Adherence to this will help minimize trauma, the time the mouse is anesthetized, and recovery time so that 100% post-surgery survival rate can be readily obtained. When preparing the donor thymus, it is imperative that the organ is kept on wet ice to minimize cell death. In addition the thymic lobes need to be cut to appropriate sized fragments to permit implantation without tearing the kidney capsule. Similarly, care is required to prevent any injury to the kidney upon making the incision of the renal capsule. Placement of the thymic graft underneath the kidney capsule is another important step in the process. Injury to the kidney or tearing of the renal capsule can be avoided with gentle handling, using appropriate fine-tipped surgical tools, and sufficient practice. One possibility to note is that successful T cell engraftment may also be achieved by implanting thymic lobes at sites other than under the kidney capsule, for instance at a subcutaneous site. The kidney however provides an ideal environment to receive thymic grafts due to a high level of vascularization, which promotes both graft survival as well as efficient T cell trafficking following thymic selection.
A distinct advantage of the above thymus transplant protocol is the technical ease in determining T cell engraftment. Using flow cytometry, success of the procedure is qualitatively and quantitatively assessed by measuring the frequency of T cells in peripheral blood. In addition to observing mature T cells in the blood, T cell engraftment is detected in various lymphoid organs. The latter permits further study of the expansion, activation, and differentiation of peripheral T cells in various host genetic environments under physiological conditions. One caveat that investigators may consider when interpreting results is that following thymic engraftment into an immunodeficient recipient, newly selected T cells will undergo lymphopenic expansion in the periphery. Although this rapid expansion will not alter thymic selection events, it may lead to preferential expansion of distinct T cell subsets in peripheral tissues. 
This protocol is widely applicable for studying mechanisms regulating T cell development and homeostasis in a variety of pathologies. For example, this approach can be used to study FoxP3 regulatory T cell generation and development7,13-14, as well as T cell renewal and export from the thymus in in vivo models of persistent viral, fungal, or bacterial infection15. In summary, this article describes a method of thymus transplantation that entails minimal manipulation of donor tissue, and a high success rate once the technique has been mastered.

Le thymus est un organe dans lequel les événements critiques dans le développement des cellules T se produisent 1. Thymocytes résidents, sur réarrangement du récepteur des cellules T (TCR) α et β gènes, sont soumis à une série d&#39;interactions avec des cellules de lymphostromal et présentant l&#39;antigène (APC) dans les régions corticales et médullaires du thymus 2. Sélection positive thymique est médiée par les cellules épithéliales thymiques corticales (TEC) pour produire thymocytes qui reconnaissent des peptides antigéniques dans le contexte de l&#39;hôte du complexe majeur d&#39;histocompatibilité (CMH) de classe I et II molécules 2-3. Après sélection négative thymique implique purge des thymocytes autoréactifs, entraînée par une interaction avec TEC médullaire ou des cellules dendritiques (DC) que les peptides présents dérivés de protéines du soi liés par le CMH de classe I et II 3 molécules. Le résultat de ces processus final est la création d&#39;un pool de cellules matures CD4 + et CD8 + T en mesure de répondre à un large éventaildes antigènes étrangers tout en présentant une réactivité minimale de quatre protéines du soi. L&#39;efficacité des événements de sélection thymiques est influencée par une multitude de facteurs, y compris la maturation thymique, la fréquence des médullaire et corticale TEC, la composition de sous-ensemble de thymique DC, et la source des précurseurs thymiques 3. Notamment, la sélection thymique aberrante peut entraîner des auto-immunes 5 ou immunodéficientes pathologies, qui découlent de la sélection négative ou positive avec facultés affaiblies, respectivement. Les événements moléculaires régulant la sélection thymique, cependant, sont mal comprises. Approches in vitro tels que des cultures d&#39;organes thymiques réagréger (RTOC) 6, se sont avérées utiles pour l&#39;analyse des événements de base associés à la sélection thymique, mais ne parviennent pas à récapituler pleinement la dynamique des cours dans les événements vivo. En conséquence, cette approche basée sur la transplantation-thymique a été créé afin de mieux les événements de sélection des thymocytes d&#39;étude in vivo 7 </sjusqu&#39;à&gt;. Ce protocole décrit la transplantation thymus de souris donneuses nouveau-nés et les adultes dans des souris immunodéficientes SCID bénéficiaires. Cette technique permet l&#39;étude des mécanismes qui régissent la sélection thymique positive et négative, ainsi que la sortie du thymus de différents sous-ensembles de cellules T au cours de l&#39;ontogenèse. Plus récemment, cette approche a été utilisée pour démontrer que l&#39;efficacité de la sélection thymique est limitée tôt après la naissance chez la souris conduit à un développement accru de lymphocytes T auto-réactifs, et un répertoire des cellules T réduite spécifiques pour des antigènes étrangers 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
</table>

isolation and transplantation of different aged murine thymic grafts.

Les mécanismes qui régulent l&#39;efficacité de la sélection thymique restent mal définis. La méthode présentée ici permet dans les analyses in vivo du développement et de la sélection des cellules T spécifiques de soi et des antigènes étrangers. L&#39;approche implique l&#39;implantation de greffes de thymus provenant de souris âgées dans divers destinataires SCID immunodéficientes. Au cours d&#39;une période relativement courte de temps les destinataires sont entièrement reconstituées avec des cellules T dérivées du thymus greffon implanté. Ensemencement Seulement thymocytes le thymus au moment de la sélection et de l&#39;isolement subissent développer en cellules T matures. En tant que tel, les changements dans la nature et la spécificité des cellules T greffées en fonction de l&#39;âge thymiques événements dépendants peuvent être évaluées. Bien que l&#39;expertise technique est nécessaire pour la transplantation thymique succès, cette méthode fournit une stratégie unique d&#39;étudier in vivo un large éventail de pathologies qui sont dues à un ou résultat de la fonction et / ou homeostas thymique aberranteest.

Le succès de ce procédé dépend de traumatisme chirurgical minimal, ainsi que le positionnement précis de la greffe sous la capsule du rein. Le greffon thymique doit être coupé afin d&#39;assurer convenablement dimensionné sections thymiques pour une transplantation subséquente comme représenté sur la Figure 1. En suivant le schéma de la figure 1, thymus de souris nouveau-nés ou les adultes peut être utilisé pour la réussite de la transplantation de sous-capsulaire avec les résultats de la prise de greffe des cellules T constants et reproductibles. Comme mentionné précédemment, le positionnement approprié de la greffe sous la capsule du rein est important dans le maintien de la survie à long terme, la fonction et la vascularisation du greffon. Comme on le voit sur ​​la figure 2, le greffon thymique doit être placé au-dessus du rein, le plus proche des glandes surrénales (antérieures de rein), sur l&#39;extrémité opposée de l&#39;incision capsulaire postérieur (de rein). Une greffe réussie thymique, lorsqu&#39;il est couplé avec un environnement hématopoïétique appropriée, peut rEmain productive au-delà de 30 semaines 7. À la suite de la transplantation, la prise de greffe est évalué par analyse cytométrique en flux de cellules T obtenues à partir de sang périphérique. La figure 3 et le tableau 2 montrent le niveau typique de la prise de greffe de cellules T observé dans les organes et le sang périphérique chez un receveur de greffe 6 semaines après transplantation. En utilisant cette technique, notre groupe a déjà montré la cinétique de CD4 + et CD8 + T reconstitution de cellules dans le sang périphérique au fil du temps chez les receveurs de greffe de thymus nouveau-né et l&#39;adulte. Brièvement, les cellules T circulantes peuvent être observées dans la périphérie en une semaine post-opératoire et le nombre de cellules CD4 + et CD8 + T continuent à augmenter jusqu&#39;à ce que le compartiment des cellules T périphérique est entièrement reconstitué à 5-6 semaines après la transplantation 7. <img alt="Figure 1" src="/files/ftp_upload/52709/52709fig1.jpg" /> <strong&gt; Figure 1:. Préparation de lobes et sections thymiques de souris nouveau-nés des bailleurs de fonds et donateurs thymus adultes sont excisées à partir de 1 jour-vieille (NB) et 4 semaines d&#39;âge (adulte). Les lignes en pointillés sont présentées comme un guide pour la préparation du nombre maximum de sections d&#39;un organe de donneur pour une transplantation subséquente. <img alt="Figure 2" src="/files/ftp_upload/52709/52709fig2.jpg" /> Figure 2:. Position d&#39;incision capsulaire et de placement d&#39;un greffon thymique section de taille appropriée préparée à partir du thymus de donneur est introduit dans l&#39;espace sous-capsulaire créé par une petite incision dans la capsule du rein à l&#39;extrémité postérieure du rein (A). Antérieur, le rein, le plus proche des glandes surrénales (non représenté) et à l&#39;extrémité opposée de l&#39;incision capsulaire, est la position optimale pour le greffage qui est inséré au-dessous de la capsule, comme illustré dans (B). <img alt = &quot;Figure 3&quot; src = &quot;/ files / ftp_upload / 52709 / 52709fig3.jpg&quot; /&gt; Figure 3. Détermination de la prise de greffe réussie via T reconstitution de cellules dans le sang et les organes lymphoïdes périphériques. Flux de données de cytométrie sont représentatifs de T reconstitution cellulaire observée 6 semaines après la transplantation chez un receveur d&#39;une greffe scid thymique NB. Des niveaux similaires de la prise de greffe de cellules T sont observées dans le sang périphérique, la rate, les ganglions lymphatiques pancréatiques (PLN), et les ganglions lymphatiques mésentériques (MLN) à la suite d&#39;une transplantation réussie greffon thymique. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Pinces fines de dissection </td></tr><tr><td> Forceps moyennes de dissection (2) </td></tr><tr><td> Forceps de dissection fine (de pointe recourbée) </td></tr><tr><td> Solution de Bétadine </td></tr><tr><td> 70% d&#39;éthanol </td></tr><tr><td> Aiguille de calibre 18 </td></tr><tr><td> Microscope de dissection w / source lumineuse </td></tr><tr><td> Dipipettes de transfert sposable (2) </td></tr><tr><td> Médicaments pour la gestion de la douleur: acétaminophène </td></tr><tr><td> Lingettes stériles </td></tr><tr><td> Lampe chauffante </td></tr><tr><td> Rasoir électrique </td></tr><tr><td> Isoflurane et de l&#39;isoflurane vaporisateur </td></tr><tr><td> Sutures (absorption rapide, intestin plaine) </td></tr><tr><td> Ciseaux de dissection </td></tr><tr><td> 9 clips mm en acier inoxydable plaies </td></tr><tr><td> Outil de suppression du clip plaies </td></tr><tr><td> PBS 1x stérile </td></tr><tr><td> 60 mm Lave- </td></tr></tbody></table> Tableau 1:. Matériel nécessaire lors de la transplantation chirurgicale thymique Liste des matériaux nécessaires utilisées pour implanter le thymus sous la capsule du rein de la souris receveuse. La quantité de matériaux spécifiés est listé dans la parenthèse. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Total Nombre de cellules T CD4 </td><td> Nombre total de cellules T CD8 </td></tr><tr><td> Sang </td><td> 1652 cellules / υl </td><td> 351 cellules / υl </td></tr><tr><td> Rate </td><td> 11546617 </td><td> 4203299 </td></tr><tr><td> PLN </td><td> 1241789 </td><td> 364918 </td></tr><tr><td> MLN </td><td> 2182266 </td><td> 532059 </td></tr></tbody></table> Tableau 2. Nombre total de lymphocytes T présents dans le sang et les organes lymphoïdes périphériques d&#39;un receveur de greffe du thymus. Le nombre total de cellules récupérées à partir des organes de CD4 + et CD8 + T ainsi que le sang périphérique d&#39;un receveur de greffe du thymus nouveau-né six semaines post- transplantation. Ces chiffres sont représentatifs de CD4 + et CD8 + T cellularité de la cellule qui est généralement observé chez les receveurs nouveau-nés et les adultes transplantation réussie thymiques.

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

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 ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.7K vues.  University of North Carolina at Chapel Hill. 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. 

Vidéo: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modèle murin de CD40-activation des cellules B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Recherche

Immunologie et infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Isolement des ARN Pseudomonas aeruginosa La colonisation du tractus gastro-intestinal murin

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

L&#39;examen de sélection positive et négative thymique par cytométrie en flux

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

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 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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolement et injection intraveineuse de moelle osseuse de souris dérivées monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

L&#39;isolement et la caractérisation par cytométrie en flux de l&#39;intestin grêle Lymphocytes murine

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Préparation et applications de cultures organotypiques de thymus Slice

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolement et activation de murins Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...