YILIMART - MACSxpress WB Treg Isolation Kit, h

MACSxpress

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Whole Blood Treg Isolation Kit has been developed for the fast isolation of regulatory T cells (Tregs) 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 in a simple, fast two-step separation procedure.

Please note

: This kit cannot be combined with the MACSxpress Whole Blood Starting Kit (130-101-319)

Background information

Regulatory CD4

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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.

Detailed separation procedure

The isolation of CD4

⁺

CD25

⁺

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

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MACSxpress

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depletion cocktail and CD25 MicroBeads. During the first depletion step via the MACSxpress Separator non-CD4

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cells as well as and the majority of CD127

^hi

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

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cells, is instantly applied onto a MACS

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Column for enrichment of magnetically labeled CD25

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cells. This can be done manually via LS Column or automated with the autoMACS

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Pro Separator. The eluted cells represent CD4

⁺

CD25

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Tregs, which can be immediately used for further downstream analysis.

Applications

Isolation of CD4

⁺

CD25

⁺

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.

Figure 1

CD4

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CD25

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CD127

^dim/–

regulatory T cells were isolated from 8 mL of human EDTA-anticoagulated whole blood using the MACSxpress

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Whole Blood Treg Isolation Kit, an LS Column, an overhead rotator, a MidiMACS™ Separator, and a MACSxpress Separator. An aliquot of the original whole blood was taken and red blood cells were lysed. Cells taken before and after separation were fluorescently stained with CD45-VioBlue

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, CD4-FITC, CD25-PE, and CD127-APC and were analyzed by flow cytometry using the MACSQuant

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Analyzer 10. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

Before separation