Isolation of Treg cells from total PBMC by single-step CD49d/CD127 depletion. Untouched Treg cells were isolated by CD49d/CD127 MACS depletion of total PBMC. (A) Depletion with α-CD49d and α-CD127 only. Depletion of human PBMC was carried out with α-CD49d and α-CD127 as described in Figure 4 except that total PBMC instead of purified CD4⁺ cells were used. Left panels show PBMC before depletion, right panel after depletion (CD49d⁻CD127⁻ PBMC). (B) CD49d/CD127 depletion combined with a CD4⁺ T-cell isolation kit. To increase the purity, the antibody mix of a commercial CD4⁺ T-cell isolation kit was added to the CD49d/CD127 depletion. The left panel shows PBMC before depletion, the right panel after depletion (CD49d⁻CD127⁻CD4⁺ cells). Numbers indicate the percentage of cells in each quadrant. (C) Statistical analysis of Treg enrichment. Treg cells were isolated in 25 independent experiments from PBMCs of healthy donors. CD49d/CD127 depletions were carried out as shown in Figure 4 (10 experiments) or panel B (15 experiments). The fractions of CD25⁺ effector T cells (Teff) and CD25⁺ Treg cells were determined by FACS with α-CD127 and α-CD25 (left panels). Analysis gates for CD25⁺ Teff (CD25⁺ CD127⁺) and CD25⁺ Treg cells (CD25⁺CD127⁻) are indicated. The percentages of CD25⁺ Treg (●) and of CD25⁺ Teff cells (○) of the total population of CD4⁺ cells before and after the CD49d/CD127 depletion are indicated in the dot plot (middle panel). Average percentages before depletion were 9.0% plus or minus 2.5% for CD25⁺ Treg and 32.1% plus or minus 9.8% for CD25⁺ Teff; after depletion they were 86.1% plus or minus 5.1% for CD25⁺ Treg and 0.9% plus or minus 1.2% for CD25⁺ Teff. (D) Percentage of Foxp3⁺ cells after depletion. Twelve Treg preparations were analyzed with α-CD25 and α-Foxp3. The dot plot indicates the corresponding values of CD25⁺Foxp3⁺ T cells and of total Foxp3⁺ T cells, including CD25⁻Foxp3⁺ T cells. Average values are indicated.