Anti human foxp3 antibody

Prior to Th1, Th2, and Th17 analysis, cells were first stimulated for 5 h with a cell stimulation cocktail, which included protein transport inhibitors Phorbol 12-myristate 13-acetate (PMA), ionomycin, Brefeldin A, and monensin (eBioscience, San Diego, CA), according to the manufacturer’s protocol. Then, cells were extracellularly stained with anti-human CD4 antibody, consecutively fixed and permeabilized (BD Biosciences, San Jose, CA, USA), and intracellularly stained with anti-human interferon-γ, interleukin (IL)-4, and IL-17 antibody (eBioscience, San Diego, CA). For Tregs analysis, no stimulation with cell stimulation cocktail was needed. Monoclonal antibodies (mAbs) specific for anti-human CD4 and CD25 were used for surface staining, and anti-human FoxP3 antibody (eBioscience, San Diego, CA) was used for intracellular staining after fixation and permeabilization (eBioscience, San Diego, CA). Data were acquired using a FACS Calibur system (BD Biosciences) and analyzed by FlowJo software (Tree Star, Inc.).

Cells were washed with PBS and stained with a fixable viability dye. After washing, cells were labeled at 4℃ for 30 min with anti-CD3, -CD4, -CD8, and -CD25 antibodies conjugated with fluorescent dye (eBioscience, San Diego, CA, USA), anti-CD47 (eBioscience), and PerCP-Cy5.5 anti-rat secondary antibody. For intracellular labeling, cells were fixed and permeabilized with cytofix/cytoperm buffer (eBioscience) and labeled with anti-human FOXP3 antibody (eBioscience) conjugated with FITC. Labeled cells were quantified using a BD FACSVerse flow cytometer, and the data were analyzed using FlowJo Software (BD Bioscience, San Jose, CA, USA).