CD25 is the low-affinity interleukin-2 receptor chain, which is expressed on activated T and B cells; therefore, it can be considered as a marker of cell activation. However, CD25 is also expressed on a subset of non-activated CD4+ T cells, which has a regulatory activity; these cells are known as regulatory T cells or Tregs. Although CD25 is expressed on activated human T cells as well as Tregs, it is still the best surface marker for identification and isolation of Tregs. In addition, depletion of CD25+ cells is a common method for investigating the inhibitory effect of Tregs on other cells.
CD25 Microbeads from Miltenyi Biotec are super-paramagnetic microbeads conjugated to monoclonal humanized CD25 antibody, and they are designed for positive selection or depletion of human CD25+ cells from peripheral blood mononuclear cells, lymphoid tissue or in vitro stimulated lymphocytes.
For the labeling protocol, washed cells are resuspended in MACS buffer (PBS containing 0.5% human AB serum and 2 mM EDTA) and incubated with CD25 Microbeads for 15 minutes at 6-12ºC. The same protocol is used for positive selection or depletion of CD25+ cells except that twice as many CD25 Microbeads are added when performing depletion. The magnetically labeled CD25+ cells can be positively selected or depleted using MACS columns or an autoMACS separator, and unlabeled non-CD25+ cells are collected as the negative fraction as they pass through the column. When using the autoMACS separator, the “Depletes” and “Posseld” functions are recommended for depletion and positive selection of CD25+ cells, respectively.
The purity of isolated CD25+ cells can be evaluated by flow cytometric analysis. The CD25 Microbeads recognize epitope A on the CD25 molecule; therefore, any antibody for control staining should bind to a different epitope (e.g. B) on the CD25 molecule. I have compared different antibodies and found that the anti-CD25-PE antibody from Miltenyi Biotec (Catalogue number 130-091-024, Clone 4E3) gave excellent staining without interfering with the antibody conjugated to Microbeads.
In summary, I normally use CD25 Microbeads for depletion of human CD25+ cells from peripheral blood mononuclear cells, and always find these Microbeads are able to efficiently remove CD25+ cells.
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Eyad Elkord, PhD
Post-Doctoral Fellow
Paterson Institute for Cancer Research
University of Manchester
United Kingdom
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