JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You have trial access to videos in this collection until May 31, 2014.

 JoVE Biology

Preparation of 2-dGuo-Treated Thymus Organ Cultures

1, 1, 1

1MRC Center for Immune Regulation, University of Birmingham

Article
    Downloads Comments Metrics
     

    Summary

    This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

    Date Published: 8/28/2008, Issue 18; doi: 10.3791/906

    Cite this Article

    Jenkinson, W., Jenkinson, E., Anderson, G. Preparation of 2-dGuo-Treated Thymus Organ Cultures. J. Vis. Exp. (18), e906, doi:10.3791/906 (2008).

    Abstract

    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).

    Protocol

    Please visit Springer Protocols for more information about the preparation of ex vivo thymus organ cultures.

    Disclosures

    The authors have nothing to disclose.

    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.

    Comments

    2 Comments

    This is interesting detail technique for dissecting, culturing thymus embryo to study the effect of ² d-Guo. The authors may be interesting to know about the effect of dGuo on islets. Please see those two abstracts of published articles.     1: Transplantation. 1991 May; 51(5):967-71. Links   Prolongation of allograft survival in diabetic rats treated with cyclosporine by deoxyguanosine pretreatment of pancreatic islets of Langerhans. al-Abdullah IH, Kumar AM, al-Adnani MS, Abouna GM. Department of Organ Transplantation and Pathology, Faculty of Medicine, Kuwait University, Safat. In vitro pretreatment of islets of Langerhans with deoxyguanosine (dGuo) has been shown to be effective for the prolongation of islet allograft survival in rats. [This study evaluates the effect of pretreatment of islets with dGuo transplanted into CsA-treated recipients.] Transplantation of dGuo-treated islets from Wistar rats into diabetic hooded (PVG) rats resulted in 36% graft survival without immunosuppression (dGuo-group) and 89% islet survival after a short course of cyclosporine was used in recipients (dGuo + CsA group). In contrast, transplantation of untreated islets into rats without immunosuppression (controls) and with CsA (CsA group) immunosuppression resulted in 0 and 56% survival, respectively. The differences in graft survival between dGuo versus control group (P less than 0.001), (dGuo + CsA) versus control group (P less than 0.0001), and CsA versus control group (P less than 0.00²) are statistically significant. Donor-strain skin-graft challenge failed to induce rejection of transplanted normoglycemic rats in (dGuo) and (dGuo + CsA) groups. The results indicate that a state of immunologic unresponsiveness may have been induced in the recipients of dGuo-treated islets, and further treatment with CsA synergistically prolongs islet survival in fully mismatched rats. PMID: ²031²80 [PubMed - indexed for MEDLINE]           1: Diabetes Res. 1991 Aug;17(4):181-7. Links   Improvement in allograft survival of islets of Langerhans by pretreatment with deoxyguanosine. al-Abdullah IH, Kumar MS, al-Adnani MS, Abouna GM. Department of Surgery, Hahnemann University, Philadelphia, Pennsylvania 1910². In vitro pretreatment of isolated islets of Langerhans prior to transplantation with deoxyguanosine (dGuo) was found to be effective in improving the survival in fully allogeneic diabetic rats (Wistar----PVG). Post transplant immunosuppression was not used. Islets pretreated with various concentrations of dGuo, 1, 1.35, 1.5 and ² microM dGuo per islet showed a graft survival of 9, 36, 9 and 14% respectively. PMID: 18²3639 [PubMed - indexed for MEDLINE]  
    Reply

    Posted by: AnonymousFebruary 9, 2009, 12:27 PM

    hello: it is wonderfull .and i is just doing some research using this method ! thank you very much!
    but i just have some techincal problem to ask you ,that is how to prepare the ²d-Guo stock solution ? because i am the first year in immunology research and i do not how to disvŒ the ²d-Guo in PBS ! is there and key prosedure to do it ?when i put the ²d-Guo to the PBS ,it dŒs not dissove at all ! i post this question for you , may you can reply me ,thank you very much ! my email is : xueliangxiu@hotmail.com
    Reply

    Posted by: xueliang x.June 3, 2009, 9:26 AM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter