Phenotype and function of CD25⁺ Treg cells freshly isolated from leukapheresis products. CD4⁺ T cells and CD25⁺ Treg cells were isolated from buffy coats and leukapheresis products (up to 1.5 × 10¹⁰ whole cells).⁸  (A) PBMCs and freshly isolated CD25⁺ Treg cells were stained with the indicated markers using fluorescence-labeled mAb and analyzed by flow cytometry. (B) Functional testing of CD25⁺ Treg cells. A total of 1 × 10⁵ freshly isolated human CD25⁺ Treg cells and CD4⁺ T cells were incubated with 3 × 10⁵ irradiated (5000 rad, 50 Gy) PBMCs in the presence of 0.5 μg/mL anti-CD3. Proliferation was determined by ³H-Tdr incorporation on day 3; measurements were performed in triplicates.