Regulatory T cells

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 CD127^hi 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⁺CD127^dim/– Treg cells with the CD4⁺CD25⁺CD127^dim/– 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 CD127^high cells in the first step. Then, the flow-through fraction of pre-enriched CD4⁺CD127^dim/– T cells is labeled with CD25 MicroBeads for subsequent positive selection of CD4⁺CD25⁺CD127^dim/– 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.

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 Phenotyping Kit, anti-human, REAfinity includes CD45, CD4, CD25, CD127, and FoxP3 fluorescent antibodies as well as the FoxP3 Staining Buffer Set to enable detection of Treg cells based on the four cell surface markers (CD45, CD4, CD25, 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).