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.
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).
MAC manual:Spleen (rat) lymph node (mouse)
Investigation of regulatory T cells(User information)
2.2 Applications of Miltenyi Biotec for spleen and lymph node Treg cells
Miltenyi Biotec has created dedicated applications for working with and analyzing Treg cells.
- Multicolor flow cytometric analysis of regulatory T cells from mouse spleen
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.
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.
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 material||isolation strategy||Comments||Automation||products|
|Treg cell isolation|
|Single cell spleen/lymph node suspension||Combination of non-target cell depletion and subsequent positive target cell selection||Final positive (Treg cells) and negative (T cells) fractions can be used for further assays/analyses||Sim*||CD4+CD25+ regulatory T cell isolation kit, mouse|
|Pre-enrichment of Treg cells|
|Single cell spleen/lymph node suspension||Positive selection of target cells||CD25 isolation+cells||Sim*||CD25 Bead Kit, mouse|
|Single cell spleen/lymph node suspension||Positive selection of target cells||Isolation of all CD4+cells||Sim*||CD4 (L3T4) beads, mouse|
|Single cell spleen/lymph node suspension||Isolation by depletion of non-target cells||depletion 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.|
Treg cells can be easily and quickly isolated from spleen and lymph node cell suspensions using theRegulatory T cell isolation kit, mouse.
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
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.
MACS cell sorting: select the best(User information)
Investigation of regulatory T cells(User information)
- MACS cell sorting: select the best
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.
MACS Flow Cytometry - Antibodies
2.5.1 Flow cytometry panels
Flow cytometry panels
|surface markers||intracellular markers||cytokines|
|Fabric specific markers:|
|CD62L (e.g. secondary lymphoid organs)|
|CCR7 (e.g. secondary lymphoid organs)|
|α4β7 (e.g. intestine)|
|CCR5 (eg, inflammatory sites, tumor)|
|CCR9 (e.g. gut and gut)|
|CCR4 (e.g. skin)|
|Activation dependent markers:|
REAfinity Recombinant Antibodies(User information)
Antibody Panel Creator
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
|FoxP3 intracellular staining||Plug set optimized for use withAnticorpos anti-FoxP3||FoxP3 Staining Buffer Set|
|Treg cell detection||Kits de teste Treg, Anti-Mouse, REAfinity|
|Detection of IL-10 secretion||Enable cell enumeration||IL-10 secretion assay in mice: detection kits|
|Detection and enrichment of IL-10 secreting cells||IL-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.
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.
MACS Flow Cytometry - Support Kits and Reagents
2.6 Cell culture of Treg cells
2.6.1 Expansion of Treg cells
Summary: Treg Cell Reagents and Expansion Kits
|Dedicated Expansion Kit||Provides stimulation conditions optimized forin vitroTreg cell expansion||Treg Expansion Kit, Mouse|
|Supplement||IL-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.
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
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.
MAC manual:cell culture
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 .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).
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 studies13‐15 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.