Biology

2-dGuo Boyalı Thymus Organ Kültürleri hazırlanması

Published: August 28, 2008 doi: 10.3791/906

William Jenkinson¹, Eric Jenkinson¹, Graham Anderson¹

¹MRC Center for Immune Regulation, University of Birmingham

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

Summary

Bu video, diseksiyon ve fetal timus çıkarılması de 2-dGuo ile tedavi edilen timus ex vivo kültürlerin hazırlanmasında gösteriyor.

Abstract

Timus, olgunlaşmamış CD4 8 + T-hücre reseptör rastgele yeniden düzenlenmiştir ifade timositleri α-ve b-zincir genleri self-peptide/major histokompatibilite kompleksi (MHC) timik tarafından ifade molekülleri tanıma yeteneklerine göre pozitif ve negatif seçim olaylar tabi stromal hücreler. Kararlı durum yetişkin timus timik mikroçevresinin hücresel karmaşıklığı zor intrathymic seçimi sırasında timik stromal hücrelerin rolü in vivo analizi yapılır ve uygun hedefleme stratejilerinin eksikliği, özellikle de timik stromal bölümlerinde gen ekspresyonu işlemek için. Biz timik mikroçevresinin in vitro olarak tanımlanan stromal ve lenfoid hücrelerin üç boyutlu timus lobları hazırlanmasına olanak timus organ kültürleri, reaggregate kullanımı yoluyla kolayca manipüle edilebilir olduğunu göstermiştir. In vitro sistemleri diğer T-hücre gelişiminin bazı yönlerini desteklemesine rağmen, timus organ kültürü, I ve II-aracılı timosit seçim olaylar, ve böylece eğitim için etkili bir araç olarak kullanılabilir sadece in vitro sistemde verimli MHC sınıf desteği mümkün kalır reaggregate timus pozitif ve negatif seçim hücresel ve moleküler düzenlenmesi.

Protocol

Ziyaret ediniz Springer Protokolleri , ex vivo timus organ kültürlerinin hazırlanması ile ilgili daha fazla bilgi için .

Erratum

Formal Correction: Erratum: Preparation of 2-dGuo-Treated Thymus Organ Cultures
Posted by JoVE Editors on 04/01/2012. 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.