Preparation of Single-cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis
Summary
We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.
Abstract
Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.
Introduction
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 cells1,2. 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 thymus17,18,19. 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 (Ly51highUEA1low) and mTECs (Ly51lowUEA1high). 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.
Protocol
Representative Results
Discussion
Collagenase, with or without Dispase, is widely used to digest mouse thymus to prepare TECs4,5,9,18,21. Liberase, which contains a blend of proteases including collagenase, is also used to digest the thymus to prepare TECs17,22. The use of Collagenase and Liberase gives essentially equivalent yields an…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Izumi Ohigashi for reading the manuscript. This work was supported by the Intramural Research Program ZIA BC 011806 of the National Institutes of Health, the National Cancer Institute, and the Center for Cancer Research.
Materials
| Collagenase/Dispase | Roche | 11097113001 | |
| Liberase TM | Roche | 5401127001 | |
| Foxp3 / Transcription Factor Staining Buffer Set | eBioscience | 00-5523-00 | |
| Ghost Dye Violet 510 | Tonbo | 13-0870-T500 | |
| BV421 anti-mouse CD45 monoclonal antibody, Clone 30-F11 | BD Horizon | 563890 | |
| Alexa Fluor 647 anti-mouse Ly51 monoclonal antibody, Clone 6C3 | BioLegend | 108312 | |
| PE/Cy7 anti-mouse EpCAM/CD326 monoclonal antibody, Clone G8.8 | BioLegend | 118216 | |
| DyLight 594 Ulex Europaeus Agglutinin I (UEA1) | Vector Laboratories | DL-1067-1 | |
| Alexa Fluor 488 anti-mouse Aire monoclonal antibody, Clone 5H12 | eBioscience | 53-5934-82 | |
| Anti-mouse Psmb11/beta5t rabbit monoclonal antibody, Clone CPTC-PSMB11(mouse)-1 | NIH NCI | CPTC-PSMB11(mouse)-1 | https://proteomics.cancer.gov/antibody-portal |
| Anti-mouse CCL21/exodus 2 rabbit polyclonal antibody | Bio-Rad | AAM27 | |
| Alexa Fluor 555 anti-rabbit IgG heavy chain goat recombinant antibody | Invitrogen | A27039 | |
| Anti-Mouse CD32/CD16 – Purified (Fc Block Antibody) | Leinco Technologies | C247 | |
| Nylon Mesh 60 Micron | Component Supply | W31436 | |
| Equipment | |||
| Thermomixer | Eppendorf | Heat block | |
| Cellometer Auto 2000 Cell Viability counter | Nexcelom | Viable cell counting | |
| LSRFortessa Cell Analyzer | BD Biosciences | Flow cytometer | Five lasers (355, 408, 488, 595, and 637 nm) |
| FACSDiva software version 9.0.1 | BD Biosciences | Flow cytometric analysis | |
| FlowJo 10.9.0 | BD Biosciences | Flow cytometric data analysis | |
| Antibody | Dilution rate (Working concentration) | Dilution Buffer | |
| Anti-Mouse CD32/CD16 – Purified (Fc Block Antibody) | 1:40 | FACS Buffer | |
| BV421 anti-mouse CD45 monoclonal antibody | 1:25 | FACS Buffer | |
| PE/Cy7 anti-mouse EpCAM/CD326 monoclonal antibody | 1:100 | FACS Buffer | |
| Alexa Fluor 647 anti-mouse Ly51 monoclonal antibody | 1:100 | FACS Buffer | |
| DyLight 594 Ulex Europaeus Agglutinin I (UEA1) | 1:400 | FACS Buffer | |
| Anti-mouse CCL21/exodus 2 rabbit polyclonal antibody | 1:200 | 1x Permeabilization Buffer | |
| Anti-mouse Psmb11/beta5t rabbit monoclonal antibody, Clone CPTC-PSMB11(mouse)-1 | 1:1000 | 1x Permeabilization Buffer | |
| Alexa Fluor 555 anti-rabbit IgG heavy chain goat recombinant antibody | 1:400 | 1x Permeabilization Buffer | |
| Buffer | Purpose | ||
| RPMI1640 containing 10 mM HEPES | For cell preparation | ||
| RPMI1640 containing 2% FBS | For cell preparation | ||
| FACS buffer (1x HBSS containing 1% BSA and 0.1% sodium azide) | For cell preparation and antibody staining | ||
| 1 mg/ml Collagenase/Dispase and 1U/ml DNase solution in RPMI1640 containing 2% FBS | For enzyme digestion | ||
| 2% PFA in PBS | For cell fixation | ||
| Fixation/Permeabilization Buffer (1 part of Fixation/Permeabilization Concentrate with 3 parts of Fixation/Permeabilization Diluent) | For cell fixation | ||
| 1x Permeabilization Buffer (1 part 10X concentrate with 9 parts distilled water) | For intercellular antibody staining |
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