MACS Handbook

Regulatory T cells

1 Introduction

Regulatory T (Treg) cells are a subpopulation of CD4+ T cells. Treg cells can be identified by a combination of different surface markers and the intracellular transcription factor FoxP3. Their main function is the suppression and termination of proinflammatory immune responses. They are considered to play a crucial role in several human diseases and mouse models.

2 Treg cells from spleen and lymph nodes

2.1 Cell subsets, frequencies, and marker expression

Treg cells represent 1–4% of all lymphocytes in secondary lymphoid organs of wild type mice. By contrast, in most TCR-engineered mouse strains the frequency of Treg cells drops to <1% due to defective Treg cell development in the thymus. Unlike human Treg cells, mouse Treg cells represent a more homogenous population and are characterized by expression of CD4, CD25, and the intracellular transcription factor FoxP3. Apart from that, Treg cells isolated from secondary lymphoid organs express the spleen/lymph node homing receptors CCR7 and CD62L. Treg cell populations can also be distinguished according to their origin: natural Treg cells (nTreg cells) develop in the thymus whereas induced Treg cells (iTreg cells) develop from naive conventional T cells in the periphery. Both subsets have similar phenotypes and comparable suppressive function. However, they differ in the epigenetic modification of the FoxP3 locus, which correlates with the stability of the Treg cell phenotype. (PMID: 19109157, 17298177, 18493985).

2.2 Sample preparation of spleen and lymph nodes

Spleen and lymph nodes must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. Dissociation can be accomplished fully automatically using the gentleMACS™ Dissociator and specific Tissue Dissociation Kits (e.g. Spleen Dissociation Kit, mouse). Alternatively, tissues can be dissociated using a manual procedure. For details, see chapter Mouse Cell Sources.
MACS Handbook:

Sample preparation

2.3 Magnetic separation of Treg cells from spleen and lymph nodes

Miltenyi Biotec has developed numerous products for the magnetic separation of the various cell types and subsets that can be found in mouse spleen and lymph nodes.

 For details on MACS Cell Separation Technology, see the MACS Hadbook chapter Magnetic Cell Separation.
2.3.1 Isolation of Treg cells
At a glance: Kits and reagents for the separation of Treg cells from spleen and lymph nodes
Starting material Isolation strategy Comments Automation Product
Isolation of Treg cells
Single-cell suspension from spleen/lymph nodeCombination of depletion of non-target cells and subsequent positive selection of target cellsFinal positive (Treg cells) and negative (T cells) fractions can both be used for downstream assays/analysisYes*CD4+CD25+ Regulatory T Cell Isolation Kit, mouse
Pre-enrichment of Treg cells
Single-cell suspension from spleen/lymph node Positive selection of target cellsIsolation of CD25+ cellsYes*CD25 MicroBead Kit, mouse
Single-cell suspension from spleen/ lymph node Positive selection of target cellsIsolation of all CD4+ cellsYes*CD4 (L3T4) MicroBeads, mouse
Single-cell suspension from spleen/ lymph node Isolation via depletion of non-target cellsDepletion of all CD8+ and non-T cells.Yes*

CD4+ T Cell Isolation Kit, mouse

*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X

Treg cells can be isolated easily and fast from spleen and lymph node cell suspensions by using the Regulatory T Cell Isolation Kit, mouse.

View details

Isolation of Treg cells from mouse spleen. CD4+CD25+ regulatory T cells were isolated from a mouse spleen cell suspension by using the CD4+CD25+ Regulatory T Cell Isolation Kit, an LD and two MS Columns, a MidiMACS™ Separator and a MiniMACS™ Separator. The cells were fluorescently stained with CD25-PE and CD4‑FITC (A) or Anti-FoxP3-APC (B) and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from analysis based on scatter signals and propidium iodide fluorescence.

Treg cells from FoxP3 reporter mice, where FoxP3-positive cells co-express a fluorescent protein like GFP or RFP, are usually isolated by flow cytometry-based sorting. However, magnetic enrichment prior to flow sorting is useful to increase the target cell frequency. For this purpose, CD4 MicroBeads, CD4+ T Cell Isolation Kit, and CD25 MicroBead Kit are the best choice.

Related videos:

2.4 Characterization of Treg cells by flow cytometry

Mouse Treg cells can be distinguished from other T cells based on the surface markers CD4 and CD25 and more specifically by the transcription factor FoxP3. As there is no unique marker available for Treg cell identification, it is crucial to have a specific marker panel to avoid interference from other cells during flow cytometry analysis. 
Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ antibodies and flow analysis kits for comprehensive analysis.

2.4.1 Flow cytometry panels
Flow cytometry panels
Surface markersIntracellular markersCytokines
General markers:FoxP3IL-10
CD45HeliosTGF-b
CD3Ki-67IL-35
CD4
CD25
CD39
CD73
Tissue-specific markers:
CD62L (e.g., secondary lymphoid organs)
CCR7 (e.g., secondary lymphoid organs)

α4β7 (e.g., gut)

CCR5 (e.g., inflammatory sites, tumor)
CCR6
CCR3
CCR9  (e.g., gut and intestine)
CCR4 (e.g., skin)
CCR8
Activation-dependent markers:
GITR
GARP
Neuropilin
TIGIT
ICOS
CTLA-4
PD-1
Related PDFs:
Related documents:
Related products:
2.4.2 Analysis of cytokines and transcription factors
At a glance: Kits and reagents for the analysis of cytokines and transcription factors of Treg cells by flow cytometry
Use Comments Product
Intracellular staining of FoxP3Buffer set optimized to be used with Anti-FoxP3 antibodiesFoxP3 Staining Buffer Set
Detection of Treg cellsTreg Detection Kit (CD4/CD25/FoxP3), mouse
Detection of IL-10 secretion Enables cell enumerationMouse IL-10 Secretion Assay – Detection Kits
Detection and enrichment of IL-10 secreting cellsMouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE)

Since the transcription factor FoxP3 is one of the most reliable markers to identify Treg cells, intracellular staining is an important technique for flow cytometry analysis. Miltenyi Biotec offers a ready-to-use FoxP3 Staining Buffer Set which has been specifically developed to ensure effective staining with the various fluorochrome-conjugated Anti-FoxP3 antibodies

Dedicated mouse Treg Detection Kits (CD4/CD25/FoxP3) (PE) or (APC), which include fluorochrome-conjugated CD4, CD25, and Anti-FoxP3 antibodies and the FoxP3 Staining Buffer Set, allow the detection of Treg cells based on both surface (CD4 and CD25) and intracellular markers (FoxP3).

Under certain conditions, Treg cells secrete anti-inflammatory cytokines such as IL-10. These IL-10–secreting Treg cells can be detected at a single-cell level with the Mouse IL-10 Secretion Assay – Detection Kits (PE) or (APC). In addition, IL-10–secreting cells can also be enriched by using the Mouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE)

2.5 Cell culture of Treg cells

2.5.1 Expansion of Treg cells
At a glance: Kits and reagents for the expansion of Treg cells
Use Comments Product
Dedicated expansion kitProvides optimized stimulation conditions for in vitro Treg cell expansionTreg Expansion Kit, mouse
SupplementMouse IL-2, research grade
Treg cells are a small cell population. Therefore, cell expansion is necessary for downstream applications that require a larger number of cells. Miltenyi Biotec offers a harmonized combination of the cytokine IL-2 and the Treg Expansion Kit, mouse, providing optimized stimulation conditions for in vitro Treg cell expansion. Expanded Treg cells show stable FoxP3 expression and maintain the typical Treg cell phenotype.
View details

FoxP3 expression analysis after in vitro Treg cell expansion. Treg cells were isolated with the CD4+CD25+ Regulatory T Cell Isolation Kit, mouseIsolated cells were analyzed for FoxP3 expression directly after isolation (d0, left) and 7 (middle) and 10 days (right) after in vitro expansion with the Treg Expansion Kit, mouse.

MACS Handbook:

Cell culture