Flow cytometry analysis of the numbers of circulating IL-10
B cells. PBMCs from individual subjects were plated in 24-well plates (106 cells/well) and stimulated in duplicate with 50 ng/mL of PMA, 1.0 μg/mL of ionomycin, and 50 ng/mL of LPS in complete RPMI-1640 medium for 2 h at 37°C in 5% CO2 followed exposing to Brefeldin A for another 4 h. The cells were then harvested, washed with ice-cold PBS, and stained with APC-anti-CD19, PerCP-anti-CD5, and PE-anti-CD1d. The immunostained cells were fixed and permeabilized with 0.5% saponin. After being washed, the cells were stained intracellularly with FITC-anti-IL-10 and analyzed by flow cytometry. The cells were first gated on living lymphocytes and then on CD19+ B cells for further analysis of total CD19+ B cells, CD5+CD19+, CD5+CD19+CD1dhigh B cells, CD5+CD19+CD1dhighIL-10+ Bregs. The isotype-matched antibodies served as controls. The levels of serum IL-10 in individual subjects were analyzed by ELISA. Data are representative FACS charts and expressed as the mean values of individual subjects and the difference between groups was analyzed by the Kruskal–Wallis test. (A) Flow cytometry analysis; (B) quantitative analysis of the numbers of total CD19+ B cells, CD5+CD19+, CD5+CD19+CD1dhigh B cells, CD5+CD19+CD1dhighIL-10+ Bregs and the levels of serum IL-10. The horizontal lines indicate the median values for different groups.