Fitc conjugated anti human cd4

Mononuclear cells were isolated from the decidual samples by disaggregating the tissue, filtering it through a sterile net (100 μm; BD Biosciences, Heidelberg, Germany), and lysing the erythrocytes with RBC lysis buffer. The lymphocytes were then harvested by a discontinuous density gradient method. Peripheral blood mononuclear cells (PBMCs) were obtained following removal of the erythrocytes with RBC lysis buffer. Thereafter, the cells were washed, incubated with cell-surface staining monoclonal antibodies (FITC-conjugated antihuman CD4; BD Biosciences, San Jose, CA, USA; PEconjugated antihuman CCR6, eBioscience, San Diego, CA, USA) for 30 min at 4°C in darkness, and then fixed in 1% paraformaldehyde for 40 min at 4°C in darkness. Staining for intracellular FOXP3 protein (BD Biosciences) was performed using a cell-fixation/ cell-permeabilization kit (eBioscience), according to the manufacturer instructions. Isotype controls were used to exclude false-positive cells. Depending on the experiment, approximately 10 5 cells were analyzed using fluorescence-activated cell sorting (FACS-Calibur, BD Biosciences).