Immunology and Infection
Published: August 6th, 2016
We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.
Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.
T cells differentiate through a series of developmental intermediates in the thymus during which time they encounter several checkpoints that ensure the generation of a functional, self-tolerant T cell repertoire1-3. Positive selection promotes the survival of thymocytes with T cell receptors (TCR) capable of recognizing, with low to moderate affinity, peptide presented by major histocompatibility complex molecules (MHC) on cortical thymic epithelial cells (cTEC)2,3. Negative selection and regulatory T (Treg) cell development contribute to the establishment of self-tolerance via the elimination or diversion of thymocytes that respond s....
Protocols for all animal studies were approved by the Animal Care Committee at the Centre de recherche - Hôpital Maisonneuve-Rosemont.
1. Harvesting Mouse Thymus for Preparation of Thymic Slices and Single Cell Suspensions
Thymic slices support analysis of different aspects of T cell development such as positive and negative selection. For successful experiments, the quality of the thymic slice is paramount. Thus, thymic slices should be examined to ensure the integrity of the thymic tissue and that the agarose surrounding the thymic slice is intact (Figure 1A). Surface tension can be compromised when the agarose is damaged causing a significant decrease in the number of th.......
Here we describe a protocol for the preparation of thymic slices and representative results of efficient positive and negative selection of overlaid pre-selection MHC class I-restricted TCR transgenic thymocytes by flow cytometry. This system has been used with similar success to support positive selection of MHC class II-restricted CD4+ T cells from pre-selection DP thymocytes32, and, in the presence of agonist antigen, negative selection and thymic Treg development11,12,36,38,39,43.......
We would like to thank Marilaine Fournier for her comments on the manuscript and Josée Tessier for technical assistance. C57BL/6-Tg (OT-I)-RAG1<tmMom> #4175 were obtained through the NIAID Exchange Program, NIH. Support for this research is provided by a grant from the SickKids Foundation and CIHR-IHDCYN (NI15-002), an operating grant from the CIHR-III (MOP-142254), and start-up funds from the FRQS (Établissement de jeunes chercheurs) and Hôpital Maisonneuve-Rosemont Foundation to HJM. HJM is a junior 1 scholar of the FRQS, a CIHR New Investigator (MSH-141967), and a Cole Foundation Early Career Transition award recipient.....
|NuSieve GTG Agarose
|Low melting temperature agarose
|Embedding Mold (Truncated - T12)
|22mm x 22mm square, truncated to 12mm x 12mm
|Double Edge Prep Blades
|0.4 µm Cell Culture Inserts
|Of several brands tested, these maintained the cells atop the slices the best
|Dulbecco's Phosphate-Buffered Saline
|RPMI-1640 with L-glutamine
|Fetal Bovine Serum
|15 ml Tenbroeck Tissue Grinders
|Nylon Mesh Filter
|Microcentrifuge Tube Sample Pestle
|40 µm Nylon Cell Strainer
|Forceps Inox Tip
|Fine tip curved forceps, size .17 X .10mm
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