Method Article
Preparación de 2-dGuo cultivos tratados con órganos timo
Erratum Notice
Resumen
Este video muestra la disección y remoción del timo fetal y la preparación de cultivos ex vivo de 2-dGuo tratados con el timo.
Resumen
En el timo CD4, inmaduros 8 + timocitos que expresan al azar reorganizado receptor de células T-α y la cadena de b-genes someterse a eventos de selección positiva y negativa sobre la base de su capacidad para reconocer complejos self-peptide/major de histocompatibilidad (MHC) expresadas por timo las células del estroma. En el análisis in vivo del papel de las células del estroma del timo durante la selección intratímica se hace difícil por la complejidad del microambiente celular del timo en el timo de adultos en estado estacionario, y por la falta de estrategias adecuadas de marketing para manipular la expresión génica, en particular, los compartimentos estromal tímica. Hemos demostrado que el microambiente del timo puede ser fácilmente manipulado in vitro mediante el uso de reaggregate culturas timo de órganos, que permiten la preparación de las tres dimensiones de los lóbulos del timo definido las células del estroma y linfoide. Aunque otros sistemas in vitro apoyar algunos aspectos de las células T de desarrollo, el cultivo de órganos reaggregate timo sigue siendo el único sistema in vitro capaces de soportar MHC de clase I y II eficiente mediada por eventos de selección de timocitos, y por lo tanto puede ser utilizado como una herramienta eficaz para estudiar la regulación celular y molecular de la selección positiva y negativa en el timo.
Protocolo
Por favor, visite Protocolos Springer para obtener más información acerca de la preparación de los ex cultivos de órganos vivo timo.
Divulgaciones
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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