*** <0.0001 and ** <0.001. Comparison of anti-Foxp3 antibodies used to stain frozen PBMCs from 10 individuals In a third set of experiments, we used all clones of anti-Foxp3 antibodies in combination with the eBioscience buffer to stain PBMCs from 10 different individuals (Fig. statistically higher levels of Foxp3 MGC116786 cells than 150D and 3G3 antibodies (mean=6.9, 5.1, 4.7, and 3.7% compared to 1.7, and 0.3% of CD25+Foxp3+ events within CD4+ cells, respectively). Importantly, the non-specificity of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on non-Tregs. Better separation of Foxp3+ and Desacetyl asperulosidic acid Foxp3- populations was observed using the PCH101 clone coupled to Alexa647 compared to FITC, or the 259D/C7 clone coupled to PE compared to Alexa488 fluorochrome. Conclusions Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid including non-Tregs in the Foxp3+ gate. The experiments presented here will aid in optimization of flow cytometry staining panels to quantify Treg frequencies in humans. Keywords: Foxp3 staining, Anti-human Foxp3 antibodies, PCH101, 259D/C7, 236A/E7, 3G3, 206D, 150D, Regulatory CD4+ T cells INTRODUCTION CD4+ regulatory T cells (Treg cells) suppress effector T cells and prevent or limit reactivity to self-antigens (1) and pathogens (2), blunt inflammation, and maintain antigen-specific T cell homeostasis (3). Multiple Treg cell populations have been reported (3), with natural Treg cells the best characterized (4). Natural Treg cells constitutively express CD25 (IL-2-receptor -chain) (5), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 or CD152) (6), and the forkhead/winged helix transcription factor Foxp3, which is a key control gene in their development and function (7). While no single marker uniquely identifies Treg cell populations, panels of markers allow identification of CD4+ T cells with suppressive activity. Treg cells are often defined as CD3+CD4+CD25+Foxp3+ cells (5-9). The expression of intracellular CTLA-4 and Foxp3 is positively correlated with surface CD25 expression (10). Higher levels of intracellular Foxp3 and CTLA-4 and lower levels of surface CD127 consistently distinguish CD25hi cells from CD25int/low and CD25? cells (11). More recent studies demonstrated that Treg cells also Desacetyl asperulosidic acid express low levels of CD127 marker (IL-7 receptor) (11,12). Our laboratory focuses on the role of T cell responses in controlling viral infections (13-15) and the ability of the immune system to reject donor white blood cells during blood transfusion (16). Regulatory T cells likely play an important role in both these processes. The materials used in many immunology studies reside in repositories of frozen peripheral blood mononuclear cells (PBMCs) derived from study subjects, so we Desacetyl asperulosidic acid aimed to optimize Treg cell identification in frozen blood samples. After blood collection, PBMCs were isolated Desacetyl asperulosidic acid on Ficoll-Paque and frozen to be further used in batches for flow cytometry studies. We present here the results of optimization studies on Foxp3 staining that were performed on healthy normal controls to ensure the reproducibility of the staining, acquisition, and analysis before quantifying Treg cells in frozen PBMCs from infected study subjects. In early studies, Desacetyl asperulosidic acid few clones of anti-human Foxp3 antibodies were available, with the PCH101 clone from eBioscience (San Diego, CA) the most frequently used in published studies (17-20). Prior publications noted concerns about the specificity of Foxp3 staining using clones such as the PCH101 antibody from eBioscience (21,22), and other antibodies have been recommended (23). In our system, we noted several levels of variability in the results obtained using different clones of Foxp3 antibodies coupled to different fluorochromes and even using antibodies paired with different fixation/permeabilization buffers to.