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Miltenyi MACSxpress® Treg Isolation Kit, human

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Regulatory CD4+ T cells are suppressor cells that neutralize other immune cells by various mechanisms. Their characteristic marker is the transcription factor FoxP3. CD4+CD25+ regulatory T cells were originally discovered in mice, but a population with identical phenotype has also been identified in humans. CD25 is the interleukin-2–receptor α-chain which is not only expressed by regulatory T cells but also by activated effector T cells.

CD127, the α-chain of the IL-7 receptor, is expressed on most mature T cell and plays an important role in their proliferation and differentiation. However, on regulatory T cells CD127 is absent and its expression inversely correlates with FoxP3 expression. Thus, CD127 can be used as an additional marker to discriminate between human regulatory and activated T cells.

Detailed separation procedure

The isolation of CD4+CD25+CD127dim/– Treg cells is performed without density gradient centrifugation, only one labeling step and in a simple two-step separation procedure. The labeling cocktail contains both, the non-CD4+ MACSxpress depletion cocktail and CD25 MicroBeads. During the first depletion step via the MACSxpress Separator non-CD4+ cells are removed by immunomagnetic depletion with MACSxpress Beads, while erythrocytes are aggregated and sedimented. Due to its small nature, the CD25 MicroBeads are not affected by the magnetic field of the MACSxpress Separator. After incubation the supernatant, containing CD4+ cells, is instantly applied onto a MACS Column for enrichment of magnetically labeled CD25+ cells. This can be done manually via LS Column or automated with the autoMACS Pro Separator. The eluted cells represent CD4+CD25+CD127dim/– Tregs, which can be immediately used for further downstream analysis.

Applications

Isolation of CD4+CD25+CD127dim/– regulatory T cells from whole blood samples without density gradient centrifugation for further phenotypical or functional characterization.

Columns

For the second magnetic separation (positive selection): LS or autoMACS Columns.