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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

All mouse experiments were performed under the approval of the Animal Care and Use Committee of the National Cancer Institute (ASP21-431, ASP21-432, and EIB-076-3).

1. Preparation of thymus cell suspension

NOTE: Mouse ontogeny affects the efficiency of enzymatic liberation of TECs from the thymus. Three methods depending on mouse age are described as follows.

  1. For postnatal mice more than 3 weeks old
    1. Harvest the thymus (two thymus lobes) from one mouse. Place the thymus in 5 mL of RPMI1640 containing 10 mM HEPES in a 60 mm plastic dish on ice.
    2. Remove blood, non-thymus tissues, and fat with curved fine-tip tweezers under a dissecting microscope. Cut the thymus into small pieces (approximately 1-2 mm in size) with dissecting scissors.
    3. Transfer the thymus pieces into a 15 mL conical tube containing 3 mL of 1 mg/mL Collagenase/Dispase, and 1 U/mL DNase solution in RPMI1640 containing 2% fetal bovine serum (FBS). Incubate in a water bath or a heat block at 37 °C for 20 min.
    4. Mix gently using a 3 mL plastic transfer pipet. Incubate for another 20 min in case the tissue fragments are still visible.
    5. Mix gently using a 3 mL plastic transfer pipet. Incubate for another 10 min in case the tissue fragments are still visible.
    6. Mix gently using a 3 mL plastic transfer pipet. Add 5 mL of 5 mM EDTA in 1x HBSS containing 1% FBS.
    7. Filter the cell suspension through a 60 µm nylon mesh and into a new 50 mL conical tube. Centrifuge the cell suspension at 350 × g at 4 °C for 5 min.
    8. Resuspend the cell pellet in 10 mL of FACS buffer (1x HBSS containing 1% bovine serum albumin [BSA] and 0.1% NaN3).
    9. Store the cells on ice and determine cell numbers using a cell counter.
  2. For neonatal mice between 1 and 2 weeks old
    1. Perform step 1.1.1.
    2. Remove blood and non-thymus tissues with dissecting tweezers under a dissecting microscope. Transfer the thymus into a 1.5 mL plastic tube containing 200 µL of RPMI1640 with 10 mM HEPES and cut the thymus into small pieces (approximately 1-2 mm in size) with dissecting scissors.
    3. Allow the thymus fragments to settle at the bottom of the tube, then gently remove the suspension with a 200 µL micropipette.
    4. Transfer the thymus pieces into a 5 mL polypropylene round-bottom tube containing 2 mL of 0.5 mg/mL Collagenase/Dispase and 1 U/mL DNase solution in RPMI1640 containing 2% FBS. Incubate in a water bath or a heat block at 37 °C for 10 min.
    5. Mix gently using a 3 mL plastic transfer pipet and then, with a 1 mL micropipette fitted with a plastic tip for further homogenization.
    6. Add 2 mL of 5 mM EDTA in 1x HBSS containing 1% FBS. Filter the cell suspension through a 60 µm nylon mesh and into a 50 mL conical tube. Add 16 mL of RPMI1640 containing 10 mM HEPES.
    7. Centrifuge the cell suspension 350 × g at 4 °C for 5 min. Resuspend the cell pellet in 10 mL of FACS buffer.
    8. Store cells on ice and determine cell numbers using a cell counter.
  3. For embryonic mice and neonatal mice up to 1 week old
    1. Harvest the thymus (two thymus lobes) from one mouse. Place the thymus in a 60 mm plastic dish and remove blood, non-thymus tissues, and fat with curved fine-tip tweezers under a dissecting microscope.
    2. Place the thymus in 0.2 mL of RPMI1640 containing 10 mM HEPES in a 1.5 mL microcentrifuge tube. Discard the liquid with a 200 µL micropipette.
    3. Add 0.5 mL of 0.5 mg/mL Collagenase/Dispase and 1 U/mL DNase solution in RPMI1640 containing 2% FBS. Incubate in a water bath or a heat block at 37 °C for 10 min. Mix gently using a 1 mL micropipette with a plastic tip.
    4. Add 0.5 mL of 5 mM EDTA in 1x HBSS containing 1% FBS. Filter the cell suspension through a 60 µm nylon mesh and into a new 15 mL conical tube. Add 9 mL of RPMI1640 containing 10 mM HEPES.
    5. Centrifuge the cell suspension at 350 × g at 4 °C for 5 min. Resuspend the cell pellet in 1-3 mL of FACS buffer.
    6. Store cells on ice and determine cell numbers using a cell counter.
  4. Alternative method: Liberase digestion
    1. Perform steps 1.1.1 and 1.1.2.
    2. Allow the thymus fragments to settle at the bottom of the tube; then gently remove the suspension with a 200 µL micropipette. Transfer the thymus pieces into a 5 mL tube containing 1 mL of 0.5 U/mL Liberase and 1 U/mL DNase in RPMI1640 containing 10 mM HEPES. Incubate in a water bath or a heat block at 37 °C for 15 min.
    3. Mix gently using a 1 mL micropipette with a plastic tip. Incubate for another 15 min in case the tissue fragments are still visible.
    4. Mix gently using a 1 mL micropipette with a plastic tip. Add 1 mL of 5 mM EDTA in 1x HBSS containing 1% FBS.
    5. Filter the cell suspension through a 60 µm nylon mesh and into a new 50 mL conical tube. Add 18 mL of RPMI1640 containing 10 mM HEPES.
    6. Centrifuge the cell suspension at 350 × g, 4 °C for 5 min. Resuspend the cells in 10 mL of FACS buffer (1x HBSS containing 1% BSA and 0.1% NaN3).
    7. Store cells on ice and determine cell numbers using a cell counter.

2. Antibody staining of thymus cells

  1. Place 16 × 106 cells in a 5 mL polystyrene round-bottom tube. Centrifuge the cells at 350 × g at 4 °C for 5 min. Discard the supernatant.
  2. Add 10 µL of working concentration of anti-Fc receptor monoclonal antibody (mAb, clone 2.4G2) in FACS buffer to block nonspecific binding of staining IgG to cell-surface Fc receptor. Incubate at 4 °C for 5 min.
  3. Add 40 µL each of working concentrations of anti-CD45 mAb (hematopoietic cell-specific; e.g., BV421-labeled), anti-EpCAM mAb (epithelial cell-specific; e.g., PE-Cy7-labeled), anti-Ly51 mAb (cTEC-detecting; e.g., AF647-labeled), and Ulex europaeus agglutinin I (UEA-1) (mTEC-detecting; e.g., AF594-labeled) in FACS buffer. In parallel, prepare tubes for single-color stained cells and unstained cells for setting up a flow cytometer.
  4. Incubate at 4 °C for 45 min. Mix well every 10 min. Add 2 mL of FACS buffer.
  5. Centrifuge cells 350 × g at 4 °C for 5 min. Discard the supernatant.
  6. Add 1 µL of Ghost Dye Violet 510 (to distinguish viable cells from dead cells in flow cytometric analysis of fixed and permeabilized cells). Use undiluted Ghost Dye reagent; mix well. Incubate at 4 °C for 30 min; mix well every 10 min.
  7. Add 2 mL of FACS buffer. Centrifuge cells 350 × g at 4 °C for 5 min. Discard the supernatant.
  8. Fix the cells by adding 1 mL of 2% paraformaldehyde (PFA) in PBS and incubating at room temperature for 10 min.
  9. Add 3.5 mL of PBS to the tube and centrifuge cells 700 × g at 4 °C for 8 min. Discard the supernatant.
  10. Permeabilize the cells by adding 1 mL of Intracellular Fixation/Permeabilization Buffer and incubating at 4 °C for 20 min.
  11. Centrifuge the cells 700 × g at 4 °C for 8 min. Discard the supernatant.
  12. Add 1 mL of 1x Permeabilization Buffer. Centrifuge the cells at 700 × g at 4 °C for 8 min. Discard the supernatant.
  13. Add 100 µL each of working concentrations of anti-beta5t antibody or anti-CCL21 antibody, or 40 µL of working concentrations of anti-Aire antibody (e.g., AF488-labeled), in 1x permeabilization buffer, mix well, and incubate for 60 min at room temperature.
  14. Add 1 mL of 1x Permeabilization Buffer. Centrifuge the cells at 700 × g at 4 °C for 8 min. Discard the supernatant.
  15. Repeat step 2.14.
  16. Add 100 µL of working concentrations of anti-rabbit IgG (e.g., AF555-labeled) and incubate for 30 min at room temperature.
  17. Add 1 mL of 1x Permeabilization Buffer. Centrifuge the cells at 700 × g at 4 °C for 8 min. Discard the supernatant.
  18. Resuspend the cells in 200 µL of FACS Buffer and mix well.
  19. Store the cells in the dark at 4 °C until flow cytometric analysis.
  20. Filter cells through 60 µm nylon mesh prior to flow cytometric analysis.

Results

The analysis requires a flow cytometer that allows the detection of at least six fluorescence channels (for CD45, EpCAM, Ly51, UEA1, Ghost Dye, and beta5t, CCL21, and/or Aire) as well as forward and side scatter parameters. Representative flow cytometric profiles of Collagenase/Dispase-digested thymus cells from 0-day-old mice and 2-week-old mice are shown in Figure 1 and Figure 2, respectively. In both figures, the dot plot in Panel A shows for...

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...

Disclosures

The authors have no conflicts of interest.

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

NameCompanyCatalog NumberComments
Collagenase/Dispase Roche11097113001
Liberase TMRoche5401127001
Foxp3 / Transcription Factor Staining Buffer SeteBioscience00-5523-00
Ghost Dye Violet 510 Tonbo13-0870-T500
BV421 anti-mouse CD45 monoclonal antibody, Clone 30-F11BD Horizon563890
Alexa Fluor 647 anti-mouse Ly51 monoclonal antibody, Clone 6C3BioLegend108312
PE/Cy7 anti-mouse EpCAM/CD326 monoclonal antibody, Clone G8.8BioLegend118216
DyLight 594 Ulex Europaeus Agglutinin I (UEA1)Vector LaboratoriesDL-1067-1
Alexa Fluor 488 anti-mouse Aire monoclonal antibody, Clone 5H12eBioscience53-5934-82
Anti-mouse Psmb11/beta5t rabbit monoclonal antibody, Clone CPTC-PSMB11(mouse)-1NIH NCICPTC-PSMB11(mouse)-1https://proteomics.cancer.gov/antibody-portal
Anti-mouse CCL21/exodus 2 rabbit polyclonal antibodyBio-RadAAM27
Alexa Fluor 555 anti-rabbit IgG heavy chain goat recombinant antibodyInvitrogenA27039
Anti-Mouse CD32/CD16 – Purified (Fc Block Antibody)Leinco TechnologiesC247
Nylon Mesh 60 MicronComponent SupplyW31436
Equipment
ThermomixerEppendorfHeat block
Cellometer Auto 2000 Cell Viability counterNexcelomViable cell counting
LSRFortessa Cell AnalyzerBD BiosciencesFlow cytometerFive lasers (355, 408, 488, 595, and 637 nm)
FACSDiva software version 9.0.1BD BiosciencesFlow cytometric analysis
FlowJo 10.9.0BD BiosciencesFlow cytometric data analysis
AntibodyDilution rate (Working concentration)Dilution Buffer
Anti-Mouse CD32/CD16 – Purified (Fc Block Antibody)1:40FACS Buffer
BV421 anti-mouse CD45 monoclonal antibody1:25FACS Buffer
PE/Cy7 anti-mouse EpCAM/CD326 monoclonal antibody1:100FACS Buffer
Alexa Fluor 647 anti-mouse Ly51 monoclonal antibody1:100FACS Buffer
DyLight 594 Ulex Europaeus Agglutinin I (UEA1)1:400FACS Buffer
Anti-mouse CCL21/exodus 2 rabbit polyclonal antibody1:2001x Permeabilization Buffer
Anti-mouse Psmb11/beta5t rabbit monoclonal antibody, Clone CPTC-PSMB11(mouse)-11:10001x Permeabilization Buffer
Alexa Fluor 555 anti-rabbit IgG heavy chain goat recombinant antibody1:4001x Permeabilization Buffer
BufferPurpose
RPMI1640 containing 10 mM HEPES For cell preparation
RPMI1640 containing 2% FBSFor 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% FBSFor enzyme digestion
2% PFA in PBSFor 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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Single cell SuspensionThymic Epithelial CellsTECsCortical TECsMedullary TECsT Cell DevelopmentFlow Cytometric AnalysisDLL4Beta5tAireCCL21Intracellular MoleculesMouse ThymusT Cell Selection

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