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 ....

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 th.......

Discussion

Collagenase, with or without Dispase, is widely used to digest mouse thymus to prepare TECs^4,5,9,18,21. Liberase, which contains a blend of proteases including collagenase, is also used to digest the thymus to prepare TECs^17,22. The use of Collagenase and Liberase gives essentially equivalent yields an.......

Materials

Name	Company	Catalog Number	Comments
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