Foxp3 staining buffer set

Flow cytometry analysis were performed as reported before (22 (link)) with slight modifications. For surface staining, single cells were incubated with fluorescein-labeled monoclonal antibodies for 30 min at 4°C after blockade of the Fc receptor, then washed twice and analyzed. For IL-13 staining, cells were stimulated with 50 ng/mL of PMA, 1 μg/mL of ionomycin and 10 μg/mL of monensin (all from Sigma–Aldrich) for 4 h at 37°C. After staining of cell-surface markers, cells were fixed and permeabilized with a Foxp3 Staining Buffer Set (BioLegend) according to the manufacturer's protocols and then stained with anti-mouse IL-13 or isotype control overnight at 4°C. Samples were analyzed by BD LSRII or LSRFortessa system (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA). Antibodies and isotype controls were purchased form BD Biosciences, BioLegend (San Diego, CA, USA), or eBioscience (San Diego, CA, USA) (Supplementary Table 1).

Fresh human peripheral blood mononuclear cells (PBMCs) were stained with anti-CD4 (PerCP-Cy 5.5 or APC-Cy7-conjugated from BD Bioscience), anti-CD25 (APC-Cy7 or APC-conjugated from BD Bioscience), and anti-CD45RA (FITC-conjugated from BD Bioscience). Intracellular detection of Foxp3 with anti-Foxp3 (PE-conjugated from BD Bioscience) was performed on fixed and permeabilized cells with the Foxp3 staining buffer set (Biolegend, USA) according to the manufacturer's instructions. The following fluorescence-conjugated antibodies were also used: CD39 (APC), Interferon-γ (IFN-γ) (PE-Cy7 or APC), and TGF-β (APC) obtained from BD Biosciences. PBMCs were stained according to the manufacturer's recommendations. The appropriate isotype-matched control antibodies were purchased from BD Bioscience. Cells were analyzed using a FACSCantoII flow cytometer (BD, USA) and Diva analysis software (BD, USA).