Method Article
La preparazione di 2-dGuo-trattati Organ Culture Timo
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Riepilogo
Questo video dimostra la dissezione e la rimozione del timo del feto e la preparazione delle colture ex vivo di 2-dGuo trattati con timo.
Abstract
Nel timo, immaturi CD4 +8 + timociti che esprimono in modo casuale riarrangiati cellule T recettori α-e b-catena di geni sottoposti a selezione di eventi positivi e negativi in base alla loro capacità di riconoscere self-peptide/major complessi di istocompatibilità (MHC) molecole espressa dal timo cellule stromali. L'analisi in vivo del ruolo di cellule stromali del timo durante la selezione intratimica è resa difficile dalla complessità cellulare del microambiente timico in steady-state timo adulto, e dalla mancanza di adeguate strategie di targeting per manipolare l'espressione genica, in particolare, comparti timica stromale. Abbiamo dimostrato che il microambiente timica può essere facilmente manipolate in vitro tramite l'uso di reaggregate culture organo del timo, che permettono la preparazione di lobi timo tridimensionale definito da cellule stromali e linfoidi. Anche se gli altri sistemi in vitro supportano alcuni aspetti di cellule T sviluppo, reaggregate coltura d'organo thymus rimane l'unico sistema in vitro in grado di supportare efficienti MHC di classe I e II-mediata eventi di selezione timocita, e quindi può essere utilizzato come uno strumento efficace per lo studio la regolazione cellulare e molecolare di selezione positiva e negativa nel timo.
Protocollo
Si prega di visitare Protocolli Springer per ulteriori informazioni sulla preparazione di ex vivo culture organo timo.
Divulgazioni
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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