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Curr Protoc Immunol. 2006 May;Chapter 7:Unit 7.4B. doi: 10.1002/0471142735.im0704bs72.

The purification and functional analysis of human CD4+CD25high regulatory T cells.

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Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.


Regulatory T cells were initially identified and isolated in the mouse, by virtue of their endogenous expression of CD25 (IL-2R alphachain) and shown to inhibit both the in vivo development of autoimmunity and the in vitro proliferation of nonregulatory, CD4+CD25- T cells. In contrast to mouse cells, human regulatory T cells are not purified by isolating all CD25-expressing CD4 T cells ex vivo. Such cells can be isolated by targeting only the small percentage of human CD4 T cells that express high levels of CD25. This is best achieved by FACS sorting using the level of CD25 expressed on CD4- T cells to place the gate for discriminating high expression of CD25. This unit provides two widely used methods to isolate (FACS) or to enrich (magnetic beads) human CD4+CD25+ regulatory T cells from blood, along with an in vitro coculture assay to measure the anergic and suppressive features of human CD4+CD25+ regulatory T cells.

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