Method Article
Préparation de la 2-dGuo cultures traitées orgue Thymus
Dans cet article
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Résumé
Cette vidéo montre la dissection et l'enlèvement du thymus du fœtus ainsi la préparation des cultures ex vivo de la 2-dGuo traités thymus.
Résumé
Dans le thymus, les CD4 + thymocytes immatures 8 exprimant hasard réarrangés récepteur des cellules T α-et b-gènes de la chaîne subissent les épreuves de sélection positive et négative sur leur capacité à reconnaître complexe majeur d'histocompatibilité self-peptide/major (CMH) exprimées par thymique Les cellules stromales. Dans l'analyse in vivo du rôle des cellules stromales thymiques lors de la sélection intrathymique est rendue difficile par la complexité du microenvironnement cellulaire thymique dans le thymus adulte état stable, et par le manque de stratégies appropriées visant à manipuler l'expression des gènes, en particulier thymique compartiments stromal. Nous avons montré que le microenvironnement thymique peut être facilement manipulées in vitro par l'utilisation de cultures d'organes réagréger thymus, qui permettent la préparation de trois dimensions lobes de thymus de définir les cellules stromales et lymphoïdes. Bien que d'autres systèmes in vitro en charge certains aspects du développement des cellules T, réagréger culture d'organe thymus reste le seul système in vitro en mesure de soutenir efficacement CMH de classe I et II à médiation épreuves de sélection thymocytes, et peut donc être utilisé comme un outil efficace pour étudier la régulation cellulaire et moléculaire de la sélection positive et négative dans le thymus.
Protocole
S'il vous plaît visitez Protocoles Springer pour plus d'informations sur la préparation des cultures ex vivo d'orgue du thymus.
Déclarations de divulgation
The authors have nothing to disclose.
Erratum
Formal Correction: Erratum: Preparation of 2-dGuo-Treated Thymus Organ Cultures
Posted by JoVE Editors on 4/01/2012. Citeable Link.
A correction was made to: Preparation of 2-dGuo-Treated Thymus Organ Cultures. A revised abstract was republished due to a publisher error. The abstract was corrected to:
In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
from
In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor α- and b-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thymic stromal cells. In vivo analysis of the role of thymic stromal cells during intrathymic selection is made difficult by the cellular complexity of the thymic microenvironment in the steady-state adult thymus, and by the lack of appropriate targeting strategies to manipulate gene expression in particular thymic stromal compartments. We have shown that the thymic microenvironment can be readily manipulated in vitro through the use of reaggregate thymus organ cultures, which allow the preparation of three-dimensional thymus lobes from defined stromal and lymphoid cells. Although other in vitro systems support some aspects of T-cell development, reaggregate thymus organ culture remains the only in vitro system able to support efficient MHC class I and II-mediated thymocyte selection events, and so can be used as an effective tool to study the cellular and molecular regulation of positive and negative selection in the thymus.
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