PCH101 stains activated cells nonspecifically. FACS sorted human CD4⁺CD25⁻CD127⁺CD45RA⁺cells were stimulated for 5 days with plate-bound anti-CD3/CD28 and 100 IU/mL IL-2. (A) Gating on unactivated cells based on lower forward light scatter/side light scatter (FSC/SSC) and staining for FOXP3 with PCH101, 236A/E7, and 259D. (B) Gating on activated cells based on larger FSC/SSC and determining the cutoff for FOXP3 positivity from the unactivated cells. Numbers on plots are percentages of total cells.