Biology

Beredning av 2-dGuo-behandlade Thymus Organ kulturer

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

Denna video visar dissekering och borttagning av fostrets bräss samt beredning av ex vivo kulturer av 2-dGuo behandlade bräss.

Abstract

I bräss, omogna CD4 8 + tymocyter uttrycka slumpmässigt ordnas T-cells receptor α-och b-kedjan gener genomgå positiva och negativa urval händelser baserat på deras förmåga att känna igen komplexa self-peptide/major histocompatibility (MHC) molekyler som uttrycks av thymic stromaceller. In vivo analys av den roll som thymic stromaceller under intrathymic val försvåras av det cellulära komplexitet thymic mikromiljön i steady-state vuxna bräss, och av bristen på lämpliga rikta strategier för att manipulera genuttryck i synnerhet thymic stromal fack. Vi har visat att thymic mikromiljön lätt kan manipuleras in vitro genom att använda reaggregate bräss orgel kulturer, som möjliggör framställning av tredimensionella bräss loberna från definierade stromaceller och lymfoida celler. Även om andra in vitro-system stöder vissa aspekter av T-cells utveckling, reaggregate bräss orgel kulturen är fortfarande den enda in vitro-system kunna stödja effektiva MHC klass I och II-medierad tymocyt händelser val, och så kan användas som ett effektivt verktyg för att studera den cellulära och molekylära regleringen av positiva och negativa val i bräss.

Protocol

Besök Springer protokoll för mer information om beredning av ex vivo bräss orgel kulturer.

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.