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Treg cells represent approximately 1–4% of peripheral blood mononuclear cells (PBMCs). Several Treg cell subsets are described in the literature, all varying slightly from one another either by cell surface marker expression, secreted effector cytokines, or origin.
For example, Tregs that develop in the thymus are called natural Treg (nTreg) cells, whereas Treg cells that differentiate in the periphery from conventional naive T cells are called induced Treg (iTreg) cells. Both subsets have similar phenotypic characteristics and comparable suppressive function, but exhibit specific differences, including epigenetic modification of the FoxP3 gene and phenotype stability (PMID: 17694575).Treg cells also appear in blood in different developmental stages, such as naive and effector Treg cells.
|Naive Treg cells||0.1% of WBC; 30% of total Treg cells, which decreases with age (PMID: 16332974)||CD4+, CD25+, CD127dim/–, CD45RA+||Precursor Treg cells, develop into memory/activated Treg cells upon antigen encounter|
|Effector Treg cells||~3% of CD4+ T cells are CD49d– FoxP3+ (PMID: 18941119)||CD4+, CD25hi, CD49d–||Higher suppressive capacity than CD49d+ Treg cells (PMID: 25060807)|
|Natural Treg cells (nTreg cells)||1–4% of all WBCs||CD4+, CD25hi, CD127dim/– Helios+ (debated)||Suppress and terminate immune responses|
|Induced Treg cells (iTreg cells)||<3–30% of total circulating Treg cells||CD4+, CD25hi, CD127dim/–||Suppress and terminate immune responses|
Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of Treg cells and distinct Treg subsets. Treg cells can be isolated either straight from whole blood without density gradient centrifugation and erythrocyte lysis, or from PBMCs after density gradient centrifugation. All kits are based on a two-step isolation strategy: depletion of non-target cells followed by positive selection of target cells.For details on MACS® Cell Separation Technology, see the MACS handbook chapter Magnetic cell separation.
|Treg cell subset||Starting material||Isolation strategy||Comments||Automation||Product|
|CD4+CD25+ Treg cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Final positive (Treg cells) and negative (T cells) fractions can both be used for downstream analysis||Yes*||CD4+CD25+ Regulatory T Cell Isolation Kit, human|
|CD4+CD25+CD127dim/– Treg cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Isolation of Treg cell population with the highest FoxP3 expression||Yes*||CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II, human|
|CD25+CD49d– Treg cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Isolation of effector Treg cells based on absence of CD49d||Yes*||CD25+CD49d– Regulatory T Cell Isolation Kit, human|
|CD4+CD25+CD45RA+ Treg cells||PBMCs||Depletion of non-target cells and subsequent positive selection of target cells||Isolation of naive Treg cells based on CD45RA expression||Yes*||CD4+CD25+CD45RA+ Regulatory T Cell Isolation Kit, human|
|CD4+CD25+ Treg cells||Whole blood||Depletion of non-target cells and subsequent positive selection of target cells|
Isolation of CD4+CD25+ Treg cells directly from whole blood in less than 30 min.For up to 30 mL of whole blood
|Yes**||MACSxpress® Treg Isolation Kit, human|
** The second step can be performed on the autoMACS® Pro Separator
The MACSxpress Treg Isolation Kit, human enables fast isolation of Treg cells from up to 30 mL of freshly drawn anticoagulated whole blood without density gradient centrifugation. The isolation of CD4+CD25+ Treg cells is performed with only one labeling step and in a two-step separation procedure.
During the first isolation step, erythrocytes are aggregated and sedimented, while non-CD4+ and the majority of CD127hi cells are removed by immunomagnetic depletion with MACSxpress Beads. In a second enrichment step, CD25+ cells are magnetically sorted over a MACS Column. The eluted cell fraction represents the CD4+CD25+ Treg cells, which can be used immediately for further downstream analyses.
The CD4+CD25+ Regulatory T Cell Isolation Kit, human performs isolation in two steps. First, the non-CD4+ cells are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies as primary labeling reagent, and anti-biotin monoclonal antibodies conjugated to MicroBeads as secondary labeling reagent. The labeled cells are depleted by separation over a MACS Column.
In the second step, CD4+CD25+ Treg cells are directly labeled with CD25 MicroBeads and isolated by positive selection from the pre-enriched CD4+ T cell fraction. The magnetically retained CD4+CD25+ Treg cells are eluted as the positively selected cell fraction.
Isolation of CD4+CD25+CD127dim/– Treg cells with the CD4+CD25+CD127dim/– Regulatory T Cell Isolation Kit II, human is also performed in two steps. A cocktail of biotinylated antibodies and Anti-Biotin MicroBeads are used to deplete the non-CD4+ and CD127high cells in the first step. Then, the flow-through fraction of pre-enriched CD4+CD127dim/– T cells is labeled with CD25 MicroBeads for subsequent positive selection of CD4+CD25+CD127dim/– Treg cells.
The CD25+CD49d– Regulatory T Cell Isolation Kit, human first labels and magnetically depletes CD8+ and CD49d+ cells with a cocktail of CD8 and CD49d MicroBeads. In the second step, CD49d– Treg cells are labeled with CD25 MicroBeads and isolated by positive selection from the pre-enriched T cell fraction. The depletion of CD49d+ cells removes contaminating CD25+ effector T cells1, thus providing highly pure populations of Treg cells.
Finally, isolation of CD4+CD25+CD45RA+ Treg cells is performed in a two-step procedure using the CD4+CD25+CD45RA+ Regulatory T Cell Isolation Kit, human. First, non-CD4+ and memory T cells are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies and Anti-Biotin MicroBeads, and depleted over a MACS Column. In the second step, the flow-through fraction of pre-enriched CD4+CD45RA+ T cells is labeled with CD25 MicroBeads for subsequent positive selection of CD4+CD25+CD45RA+ Treg cells.
Treg cells can be discriminated from other CD4+ T cells via flow cytometry using a combination of different cell surface markers and, most specifically, the transcription factor FoxP3. There is no unique marker for Treg cell identification. Thus, a specific and reproducible flow cytometry staining strategy is critical to avoid contamination with other cell types.
The scientific poster below describes the use of antibody-fluorochrome conjugates for multiparameter flow cytometry analysis of T regs:
|General:||Activation-specific markers:||Tissue-specific markers:|
|CD45||CD137||E-selectin and P-selectin ligands|
|* Exclusion markers||CD103|
|Intracellular staining of FoxP3||Buffer set optimized to be used with Anti-FoxP3 antibodies||FoxP3 Staining Buffer Set|
|Detection of Treg cells||Treg Detection Kit (CD4/CD25/FoxP3), human|
|Detection of IL-10 secretion||Enables cell enumeration||IL-10 Secretion Assay – Detection Kit (PE), human|
|Detection and enrichment of IL-10 secreting cells||Uses an IL-10-specific catch reagent||IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE), human|
|Antigen-reactive T cell enrichment||CD137 MicroBead Kit, human|
Because the transcription factor FoxP3 is one of the most reliable markers to identify Treg cells, intracellular flow cytometry staining is essential for Treg cell research. The ready-to-use FoxP3 Staining Buffer Set was developed specifically for use in conjunction with Anti-FoxP3 antibodies to ensure the most reliable intracellular staining. The dedicated Treg Detection Kit (CD4/CD25/FoxP3), human includes CD4, CD25, and FoxP3 fluorescent antibodies as well as the FoxP3 Staining Buffer Set to enable detection of Treg cells based on the two cell surface markers (CD4 and CD25) and the FoxP3 intracellular marker.
Under some circumstances, Treg cells secrete anti-inflammatory cytokines. Treg cells secreting IL-10 can be detected at a single-cell level with the IL-10 Secretion Assay – Detection Kit (PE), human, and enriched with the IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE), human.
Antigen-reactive Treg cells can be identified, enriched, and analyzed via antigen-reactive T cell enrichment (ARTE) in combination with the CD137 MicroBead Kit, human. ARTE has been applied successfully to study antigen-specific Treg cells and is described in various publications (PMID: 23479226, 24301658, 27773482, 25172488).
|Reagents for in vitro stimulation of responder T cells||Optimized for use in suppression assays||Treg Suppression Inspector, human|
The suppression assay is one of the most common assays to test the functionality of Treg cells in vitro. Treg cells are cocultured with responder T cells (Tresp) and a proliferative stimulus (either polyclonal or antigen-specific). During coculture, functional Treg cells suppress the proliferation of Tresp cells. The Treg Suppression Inspector, human is a polyclonal stimulus optimized for T cell stimulation in a suppression assay. This cell culture tool stimulates Tresp cells in a way that Treg cells can suppress their proliferation reliably, and facilitates convenient handling and standardized conditions for reproducible results.