Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (2023)

1. Introduction

Regulatory T cells (Tregs) 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. Its main function is the suppression and termination of pro-inflammatory immune responses. They are considered to play a crucial role in several human diseases and mouse models.

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (1)MAC manual:
CD4+ T cells (mouse) CD8+ T cells (mouse) Pan T cells (mouse)

2 Treg cells of the spleen and lymph nodes

2.1 Cellular subsets, frequencies and expression of markers

Treg cells represent 1 to 4% of all lymphocytes in the secondary lymphoid organs of wild-type mice. In contrast, in most TCR-engineered strains of mice, the frequency of Treg cells drops to <1% due to defective development of Treg cells in the thymus. Unlike human Treg cells, mouse Treg cells represent a more homogeneous population and are characterized by the expression of CD4, CD25 and the intracellular transcription factor FoxP3. Furthermore, Treg cells isolated from secondary lymphoid organs express the spleen/lymph node receptors CCR7 and CD62L. Treg cell populations can also be distinguished based on their origin: wild-type Treg cells (nTreg cells) develop in the thymus, while induced Treg cells (iTreg cells) develop from naive conventional T cells in the periphery. . Both subsets have similar phenotypes and comparable suppressor 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).

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (2)MAC manual:
Spleen (rat) lymph node (mouse)
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Investigation of regulatory T cells(User information)

2.2 Applications of Miltenyi Biotec for spleen and lymph node Treg cells

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (4)

Miltenyi Biotec has created dedicated applications for working with and analyzing Treg cells.

  • Multicolor flow cytometric analysis of regulatory T cells from mouse spleen
(Video) Comprehensive solutions for your Treg mouse and human research [Webinar]

2.3 Preparation of spleen and lymph node samples

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. Decoupling can be achieved fully automatically using theDissociador gentleMACS™and tissue-specific dissociation kits (eg.Spleen dissociation kit, mouse). Alternatively, tissues can be dissociated by a manual procedure. For more details, see chaptermouse cell sources.

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (5)MAC manual:
sample preparation

2.4 Magnetic separation of Treg cells from the spleen and lymph nodes

Miltenyi Biotec has developed several products for the magnetic separation of the various types and subsets of cells that can be found in the spleen and lymph nodes of mice.

For details on MACS cell sorting technology, refer to the MACS Handbook chapterMagnetic cell separation.

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (6)MAC manual:
Magnetic cell separation

2.4.1 Isolation of Treg cells

Overview: kits and reagents for spleen and lymph node Treg cell separation

starting materialisolation strategyCommentsAutomationproducts
Treg cell isolation
Single cell spleen/lymph node suspensionCombination of non-target cell depletion and subsequent positive target cell selectionFinal positive (Treg cells) and negative (T cells) fractions can be used for further assays/analysesSim*CD4+CD25+ regulatory T cell isolation kit, mouse
Pre-enrichment of Treg cells
Single cell spleen/lymph node suspensionPositive selection of target cellsCD25 isolation+cellsSim*CD25 Bead Kit, mouse
Single cell spleen/lymph node suspensionPositive selection of target cellsIsolation of all CD4+cellsSim*CD4 (L3T4) beads, mouse
Single cell spleen/lymph node suspensionIsolation by depletion of non-target cellsdepletion of all CD8+and non-T cells.Sim*CD4+ T cell isolation kit, mouse
*Automation options range from fully automated benchtop solutions such as theautoMACS® Pro Separatorfor high-performance platforms such asSeparador Plus MultiMACS™ Cell24oMultiMACS X.
(Video) Comprehensive solutions for your Treg mouse and human research

Treg cells can be easily and quickly isolated from spleen and lymph node cell suspensions using theRegulatory T cell isolation kit, mouse.

see details

Isolation of mouse spleen Treg cells.CD4+CD25+Treg cells were isolated from a suspension of mouse spleen cells using CD4+CD25+Regulatory T cell isolation kit, one LD column and two MS columns, one MidiMACS™ Sorter and one MiniMACS Sorter. Cells were fluorescently stained with CD25-PE and CD4-FITC (A) or Anti-FoxP3-APC (B) and analyzed by flow cytometry using a MACSQuant® analyzer. Cell debris and dead cells were excluded from analysis based on propidium iodide scatter and fluorescence signals.

Isolation of mouse spleen Treg cells using CD4+CD25+Regulatory T cell isolation kit, mouse

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (9)

CD4+CD25+Treg cells were isolated from a suspension of mouse spleen cells using CD4+CD25+Regulatory T cell isolation kit, one LD column and two MS columns, one MidiMACS™ Sorter and one MiniMACS Sorter. Cells were fluorescently stained with CD25-PE and CD4-FITC (A) or Anti-FoxP3-APC (B) and analyzed by flow cytometry using a MACSQuant® analyzer. Cell debris and dead cells were excluded from analysis based on propidium iodide scatter and fluorescence signals.

Treg cells from FoxP3 reporter mice, where the FoxP3-positive cells co-express a fluorescent protein such as GFP or RFP, are usually isolated by cell sorting based on flow cytometry. However, magnetic enrichment before cell sorting is useful to increase the target cell frequency. For this purpose,CD4 microspheres, CD4+ T cell isolation kit, youKit of beads CD25they are the best option.

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2.5 Characterization of Treg cells by flow cytometry

Mouse Treg cells can be distinguished from other T cells on the basis of CD4 and CD25 surface markers and more specifically by the FoxP3 transcription factor. As there is no single marker available for identifying Treg cells, it is critical to have a dedicated panel of markers to avoid interference from other cells during flow cytometry analysis.
Miltenyi Biotec offers a broad portfolio of conventional and recombinant productsAnticorpos REAfinity™and flow analysis kits for complete analysis.

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MACS Flow Cytometry - Antibodies

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2.5.1 Flow cytometry panels

Flow cytometry panels

surface markersintracellular markerscytokines
General tags:ZorroP3IL-10
CD45héliosTGF-β
CD3Ki-67IL-35
CD4
CD25
CD39
CD73
Fabric specific markers:
CD62L (e.g. secondary lymphoid organs)
CCR7 (e.g. secondary lymphoid organs)
α4β7 (e.g. intestine)
CCR5 (eg, inflammatory sites, tumor)
CCR6
CCR3
CCR9 (e.g. gut and gut)
CCR4 (e.g. skin)
CCR8
Activation dependent markers:
GITR
GARP
neuropilina
TIGITUS
ICOS
CTLA-4
PD-1
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Antibody Panel Creator

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primary antibodies

2.5.2 Analysis of cytokines and transcription factors

Abstract: Kits and Reagents for Analysis of Treg Cell Cytokines and Transcription Factors by Flow Cytometry

To useCommentsproducts
FoxP3 intracellular stainingPlug set optimized for use withAnticorpos anti-FoxP3FoxP3 Staining Buffer Set
Treg cell detectionKits de teste Treg, Anti-Mouse, REAfinity
Detection of IL-10 secretionEnable cell enumerationIL-10 secretion assay in mice: detection kits
Detection and enrichment of IL-10 secreting cellsIL-10 Secretion Assay in Mice: Cell Detection and Enrichment Kit (PE)

Since the FoxP3 transcription factor is one of the most reliable markers for identifying Treg cells, intracellular staining is an important technique for analysis by flow cytometry. Miltenyi Biotec offers a turnkey solutionFoxP3 Staining Buffer Setthat was specifically developed to ensure efficient staining with the various conjugated fluorochromesAnticorpos anti-FoxP3.

(Video) T cell culture fine-tuned to your cells’ needs [WEBINAR]

dedicated mouseKits de teste Treg, Anti-Mouse, REAfinity, which include fluorochrome-conjugated anti-FoxP3, CD4, and CD25 antibodies and the FoxP3 Staining Buffer Set, allow detection of Treg cells based on surface (CD4 and CD25) and intracellular (FoxP3) markers.

Under certain conditions, Treg cells secrete anti-inflammatory cytokines such as IL-10. These IL-10 secreting Treg cells can be detected at the single cell level with theIL-10 Secretion Assay in Mice: Detection Kits (PE) or (APC).Furthermore, IL-10 secreting cells can also be enriched using theIL-10 Secretion Assay in Mice: Cell Detection and Enrichment (PE) Kit.

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2.6 Cell culture of Treg cells

2.6.1 Expansion of Treg cells

Summary: Treg Cell Reagents and Expansion Kits

To use Comments products
Dedicated Expansion KitProvides stimulation conditions optimized forin vitroTreg cell expansionTreg Expansion Kit, Mouse
SupplementIL-2 Mouse, Research Grade

Treg cells are a small population of cells. Therefore, cell expansion is necessary for downstream applications that require a larger number of cells. Miltenyi Biotec offers a harmonized combination ofIL-2 cytocinit's himTreg Expansion Kit, Mouse, providing stimulation conditions optimized forin vitroTreg cell expansion. The expanded Treg cells show stable FoxP3 expression and maintain the typical Treg cell phenotype.

see details

Analysis of the FoxP3 expression afterin vitroTreg cell expansion. Treg cells were isolated with CD4+CD25+Regulatory T cell isolation kit, mouse. Isolated cells were analyzed for FoxP3 expression directly after isolation (d0, left) and 7 (middle) and 10 (right) days later.in vitroexpansion with the Treg Expansion Kit, mouse.

Analysis of the FoxP3 expression afterin vitroTreg cell expansion

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (19)

Treg cells were isolated with CD4+CD25+Regulatory T cell isolation kit, mouse. Isolated cells were analyzed for FoxP3 expression directly after isolation (d0, left) and 7 (middle) and 10 (right) days later.in vitroexpansion with the Treg Expansion Kit, mouse.

Mouse regulatory T cells | Treg cells and markers | Manual | Miltenyi Biotec (20)MAC manual:
cell culture

FAQs

What is CD127 a marker for? ›

CD127 is a useful marker for identifying memory and effector T cells. Its expression is down-modulated on Treg cells. The ligation of IL-7 with its receptor is important for stimulation of mature and immature T cells as well as immature B cell proliferation and development.

How do you identify Tregs? ›

The transcription factor FoxP3 has been shown to identify Tregs. However, the detection of FoxP3 requires cell permeabilization, thereby preventing isolation of viable Tregs. Subsequently, the extracellular marker CD127 was established for the identification of Tregs.

Which marker is used to identify T regulatory cells? ›

Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells.

What are the activation markers for mouse T cells? ›

Mouse T cells are characterized by CD3 expression and are subdivided into CD4+ helper and CD8+ cytotoxic groups. T cell activation increases expression of CD69 and CD25, which are frequently used as markers of activation.

What is CD45RA a marker for? ›

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

What is CD45RO a marker for? ›

CD45RO is a marker of memory T-cells [1–3]. The first monoclonal antibody (mAb) identifying human CD45RO was UCHL1 [4].

Are Tregs CD4 or CD8? ›

Natural Treg are characterised as expressing both the CD4 T cell co-receptor and CD25, which is a component of the IL-2 receptor. Treg are thus CD4+ CD25+.

Do Tregs express CD3? ›

Tregs express CD3, CD4, CD25, and FOXP3 but lack CD127. CD4 and CD3 identify helper T lymphocytes, of which Tregs are a subset. CD25 is IL-2Rα, an essential activation marker that is expressed in high levels on Tregs.

Are regulatory T cells CD4 or CD8? ›

Regulatory T cells come in many forms with the most well-understood being those that express CD4, CD25, and FOXP3 (CD4+CD25+ regulatory T cells).

What is the best activation marker for T cells? ›

CD25 is the alpha chain of the trimeric IL-2 receptor and considered to be the most prominent cellular activation marker. It is expressed constitutively on the surface of several subsets of peripheral blood lymphocytes, such as regulatory and resting memory T cells.

What are biomarkers of T cells? ›

Biomarkers for T Cell Differentiation Level

These distinct subsets can be distinguished using polychromatic flow cytometry based on the presence of different surface markers (differentiation markers such as CD45RA, CD45RO, CD62L, CCR7, CD27, CD28, and activation markers such as CD25, CD127, CD57, and CD137) (35).

What are CD4 and CD8 markers? ›

CD4 and CD8 are glycoproteins found on the surface of T lymphocytes and other immune cells. Researchers have long been able to detect these T cell subset markers in formalin-fixed, paraffin-embedded human tissues.

What is CD27 a marker for? ›

CD27+ B cells are larger cells with abundant cytoplasm carrying somatic hypermutation, and have an ability to produce immunoglobulin, indicating that CD27 is a memory marker of B cells. The ligation of CD27 yields crucial signals that positively control the entry of B cells into the pathway to plasma cells.

Do T cells express CD127? ›

Certain studies1315 report that the expression of CD127, known as the interleukin-7 receptor (IL-7R) α chain, on the surface of T cells is involved in the regulatory mechanism of IL-7–induced promotion to maturation of thymocytes and the survival and function of T cells, maintaining homeostatic expansion in T-cell ...

What do CD markers indicate? ›

CD is an abbreviation “for cluster of differentiation”. CD molecules are cell surface markers which are very useful for the identification and characterization of leukocytes and the different subpopulations of leukocytes.

Do NK cells express CD127? ›

Human NK cells can also express the pan-ILC markers CD127 and CD161 (18–20), but NK cells are typically distinguished by their surface expression of CD16 (FcγRIIIA), CD94/NKG2 heterodimers, killer immunoglobulin-like receptors (KIRs), NKG2D, and NKp80 (21).

What is CD22 a marker for? ›

CD22 has been previously characterized as an activation marker of mature B lymphocytes. However, we found that CD22 is expressed early during the ontogeny of B cells in the bone marrow and spleen, and was found on B cells isolated from all the different lymphoid compartments.

What is CD45RO vs CD45RA? ›

CD45RA is the long isoform of CD45 and is expressed on naive T cells. CD45RO is the shorter isoform, and is expressed on T cells that have encountered antigens.

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