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 pro-inflammatory immune responses. They are considered to play a crucial role in several human diseases and mouse models.
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 homogeneous 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).
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Regulatory T cell research (brochure)
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
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 Handbook chapter Magnetic cell separation.
MACS Handbook:
Magnetic cell separation
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 node | Combination of 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 assays/analysis | Yes* | CD4+CD25+ Regulatory T Cell Isolation Kit, mouse |
| Pre-enrichment of Treg cells | ||||
| Single-cell suspension from spleen/lymph node | Positive selection of target cells | Isolation of CD25+ cells | Yes* | CD25 MicroBead Kit, mouse |
| Single-cell suspension from spleen/lymph node | Positive selection of target cells | Isolation of all CD4+ cells | Yes* | CD4 (L3T4) MicroBeads, mouse |
| Single-cell suspension from spleen/lymph node | Isolation via depletion of non-target cells | Depletion 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.
Treg cells from FoxP3 reporter mice, where FoxP3-positive cells co-express a fluorescent protein such as GFP or RFP, are usually isolated by flow cytometry–based cell sorting. However, magnetic enrichment prior to cell 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.
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MACS Cell Separation - Select the best (brochure)
Regulatory T cell research (brochure)
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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.
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| Surface markers | Intracellular markers | Cytokines |
|---|---|---|
| General markers: | FoxP3 | IL-10 |
| CD45 | Helios | TGF-β |
| CD3 | Ki-67 | IL-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 |
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 FoxP3 | Buffer set optimized to be used with Anti-FoxP3 antibodies | FoxP3 Staining Buffer Set |
| Detection of Treg cells | Treg Analysis Kits, anti-mouse, REAfinity | |
| Detection of IL-10 secretion | Enables cell enumeration | Mouse IL-10 Secretion Assay – Detection Kits |
| Detection and enrichment of IL-10 secreting cells | Mouse 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 Analysis Kits, anti-mouse, REAfinity, 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).
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At a glance: Kits and reagents for the expansion of Treg cells
| Use | Comments | Product |
| Dedicated expansion kit | Provides optimized stimulation conditions for in vitro Treg cell expansion | Treg Expansion Kit, mouse |
| Supplement | Mouse IL-2, research grade |
MACS Handbook:
Cell culture
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