Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Summary

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Abstract

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Introduction

Regulatory T (Treg) cells are critical for the homeostasis of immune system. Treg cells are defined by the expression of surface antigens CD4 and CD25, and the transcription factor forkhead box P3 (Foxp3)^1,2,3. Sakaguchi et al. first showed that CD25 is constitutively expressed on T suppressor cells in mice⁴, which provided a pioneering observation for the identification of human Treg cells. Treg cells play a central role in immune tolerance by exerting a suppressive ability via a variety of mechanisms, including cytolysis through the secretion of gran....

Protocol

The local animal ethics committee at Uppsala University approved the animal experiments.

1. Harvesting Organs from Animals

1. Sacrifice the mice by cervical dislocation.
2. Place thymic glands and spleens in 20 mL scintillation vials or 15 mL conical tubes filled with 5 mL of Hanks´ balanced salt solution (HBSS). Place PDLNs in 1.5 mL micro tubes filled with 1 mL of RPMI 1640. Use the whole thymus and spleen, and all the PDLNs.
   NOTE: Organs should be kept on ice throughout the procedure and the investigator should try to be fast while preparing single cell suspensions, especially when handling th....

Results

To investigate the expression of Nrp1 and Helios on tTreg and pTreg cells, we prepared single cells from thymic glands, PDLNs and spleens of normoglycemic NOD mice and stained them with the Treg cell markers CD4, CD25, Foxp3, Helios and Nrp1 for flow cytometric analysis. The results were analyzed as shown in the representative gating strategies (Figure 1). We found that the proportion of Helios⁺ cells among CD4⁺CD8^-CD25^+<.......

Discussion

In this study, we isolated single cells from thymic glands, PDLNs and spleens of NOD mice, and investigated the expression of Helios and Nrp1 in CD4⁺CD8^-CD25⁺Foxp3⁺ Treg cells using flow cytometry. In the present study, NOD mice were used, which is a murine model of type 1 diabetes. In a previous study, we have used the wild type mouse strains CD-1 and C57BL/6 mice to investigate whether Helios or Nrp1 is a better marker for distinguishing tTreg cells from pTreg cells. In that .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NOD mice			In-house breading
HBSS	Statens veterinärmedicinska anstalt	991750
RPMI-1640	Sigma-Aldrich	R0883-500ML
NH4Cl	EMD Millipore	1011450500
eBioscience Foxp3 / Transcription Factor Staining Buffer Set	ThermoFisher	00-5523-00	Contains Fixation/Permeabilization Concentrate , Fixation/Permeabilization Diluent and Permeabilization Buffer (10x)
eBioscience Flow Cytometry Staining Buffer	ThermoFisher	00-4222-26
CD4 Monoclonal Antibody (RM4-5), FITC, eBioscience	ThermoFisher	11-0042-85
CD25 Monoclonal Antibody (PC61.5), PE, eBioscience	ThermoFisher	12-0251-83
BD Pharmingen APC-H7 Rat anti-Mouse CD8a	BD Biosciences	560182
Mouse Neuropilin-1 APC-conjugated Antibody	R&D Systems	FAB5994A
FOXP3 Monoclonal Antibody (FJK-16s), PE-Cyanine7, eBioscience	ThermoFisher	25-5773-82
Pacific Blue anti-mouse/human Helios Antibody	BioLegend	137220
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	CORNING	352235	A 35 µm nylon mesh is incorporated into the dual-position snap cap
Tube 15 mL, 120 mm x17 mm, PP	Sarstedt	62.554.002
Micro tube 1.5 mL	Sarstedt	72.690.001
Disposable scintillation vials	WHEATON
Flow cytometry	BD
Flow cytometry analysis software	Inivai Technologies	Flowlogic