Purification of CD4⁺CD25⁺ cells from peripheral blood. Representative 2-color FACS plots of PBMCs and purified CD4⁺CD25⁺ and CD4⁺CD25^- cells. (A) Examination of peripheral blood reveals CD4⁺CD25⁺ cells constitute 1% to 3% of PBMCs. There are a variable number of non-CD4⁺ cells that express CD25, generally of lower intensity expression (mostly B cells). (B) Some donors evidence a more distinct CD4⁺CD25⁺ population. (C) CD4+CD25+ cells. Cells were purified by anti-CD25-FITC and anti-FITC cleavable microbeads and subsequently lineage depleted. Intensity of CD25-PE staining is slightly decreased by prior staining with anti-CD25-FITC. (D) CD4⁺CD25^- cells. Cells were purified by CD25 depletion of PBMCs, followed by CD4⁺ positive selection. Data are representative of 20 donor evaluations and 10 cell purification experiments.