Preparation of Single-cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

The thymus is an epithelial mesenchymal organ that promotes the development and repertoire selection of T lymphocytes. In the cortical and medullary microenvironments within the thymus, thymic epithelial cells (TECs) play a crucial role in directing T cell development and selection. It is well accepted that cortical TECs (cTECs) in the thymic cortex primarily contribute to the induction of early T cell development and positive selection of newly generated T-cell antigen-receptor (TCR)-expressing thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla are essential for the attraction of positively selected thymocytes from the cortex and the establishment of central self-tolerance by eliminating self-reactive T cells and by generating regulatory T cells^1,2. These functions of TECs in supporting T cell development and selection are mediated through a variety of molecules expressed in cTECs and mTECs.

These include Delta-like ligand 4 (DLL4), important for T-cell-lineage specification of lymphoid progenitors)^3,4, beta5t (also known as Psmb11, important for positive selection of CD8 T cells)^5,6, and Prss16 (also known as thymus-specific serine protease, or TSSP, important for positive selection of CD4 T cells)^7,8 expressed in cTECs, and Autoimmune regulator (Aire, important for promiscuous expression of self-genes)^9,10, Fez family zinc finger 2 (Fezf2, important for promiscuous expression of Aire-independent self-genes and the generation of thymic tuft cells)^11,12,13,14, and C-C motif chemokine ligand 21 (CCL21, important for medullary migration and accumulation of positively selected thymocytes)^14,15,16 expressed in mTECs. Flow cytometry provides a means for quantitatively detecting these functionally significant molecules in TECs, facilitating the assessment of both the development and function of cTECs and mTECs. Since many of the molecules in TECs are primarily expressed within the cells, such as in the cytoplasm (e.g., proteasome component beta5t) and the nucleus (e.g., transcriptional regulator Aire), it is advantageous to quantitatively analyze intracellular molecules in cTECs and mTECs using flow cytometry.

TECs are rare in the thymus. A female mouse at 5 weeks old has approximately 1 × 10⁶ cTECs and approximately 2.5 × 10⁶ mTECs, whereas the thymic cortex contains approximately 3 × 10⁸ CD4⁺CD8⁺ thymocytes and the thymic medulla contains approximately 5 × 10⁷ CD4⁺CD8^– and CD4^–CD8⁺ thymocytes¹⁷. Therefore, the vast majority (>99%) of cells in the thymus are hematopoietic cells, including thymocytes, rather than TECs, in young adult mice. Moreover, TECs are tightly associated with neighboring cells (e.g., thymocytes) and architectural extracellular components (e.g., collagen) within the thymus, so the liberation of TECs into cell suspension requires protease digestion of the thymus. Conventional methods for enzymatic digestion yield only a small fraction of TECs; approximately 1% (1 × 10⁴) and 10% (2 × 10⁵) for cTECs and mTECs, respectively, from 5-week-old mouse thymus^17,18,19. The low efficiency of TEC liberation from the thymus is likely due to the susceptibility of TECs to proteolytic enzymes and mechanical stress and the tight association of TECs with neighboring cells, particularly between cTECs and CD4⁺CD8⁺ thymocytes²⁰. In this regard, the results of single-cell analysis of mouse TECs, including flow cytometric analysis and single-cell RNA-sequencing analysis, based on current enzymatic digestion-mediated cell preparation methods are representative of only a small portion of TECs and therefore, are far from being comprehensive. Nevertheless, despite such limitations in the enzymatic retrieval of TECs, flow cytometric analysis of mouse TECs in single-cell suspension is still useful for the quantitative analysis of functionally relevant molecules in cTECs and mTECs. In cell suspension, TECs have been defined as cells positive for epithelial surface molecule EpCAM (CD326) and negative for hematopoietic surface molecule CD45. Within EpCAM⁺CD45^– TECs, cell-surface expression levels of Ly51 molecules (also known as BP-1 and CD249) and Ulex Europaeus agglutinin I (UEA1) binding capacity (primarily binding to glycoproteins and glycolipids containing α-linked fucose) have been used to phenotypically mark cTECs (Ly51^highUEA1^low) and mTECs (Ly51^lowUEA1^high). Here we describe our experimental protocols for the conventional preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis, focusing on the quantitative analysis of intracellular molecules beta5t, CCL21, and Aire in freshly prepared mouse TECs.