Clone 259D is a less sensitive detector of total FOXP3 expression. Human CD4⁺CD25⁻ T cells were stained with CFSE and cultured in the presence of no antigen (top row), irradiated, allogeneic T-cell–depleted peripheral blood mononuclear cells (PBMC, middle row) or irradiated, autologous T-cell–depleted PBMC with anti-CD3. Cells were harvested and stained at various time points for CD4, CD25, and FOXP3 and analyzed on a BD LSR II (BD Biosciences, San Jose, CA), as described before² ; this is similar to the authors' protocols for permeabilization (eBioscience reagents). The data represent day 6 of in vitro stimulation and demonstrate CFSE on the x-axis and phycoerythrin (PE) staining on the y-axis, with the indicated PE-conjugated antibodies (FMO indicates “fluorescence minus one”; ie, no stain in the PE channel). Thus, clones PCH101 and 236A/E7 showed similar results, with FOXP3 expression robustly detected in virtually all activated, dividing cells (panels I, K, O, Q). Nondividing cells at this late time point showed very few FOXP3⁺ cells. In contrast, clone 259D could not robustly detect total FOXP3 expression (panels L,R). PCH101 (used here in titrated amounts) did not show appreciable high background staining of unstimulated cells (compare panels A-C). However, clone 236A/E7 showed high background with unstimulated cells (panel E; represents cutoff with isotypic control from panel D). Thus, panel E (not D) provided the correct cutoff for the analysis of panels K and Q. Numbers on plots are percentages of total CD4⁺ T cells.