Flow cytometric analysis of intracellular cytokine staining
Freshly isolated peripheral blood mononuclear cells (PBMCs) of KD patients were stimulated in duplicate with 50 ng/mL of phorbol myristate acetate (PMA) and 1.0 mg/mL of ionomycin (Sigma, St. Louis, MO, USA) in RPMI 1640 medium at room temperature in a humidified incubator with 95% air and 5% carbon dioxide for 2 hours and then cultured for another 4 hours in the presence of 0.5 mg/mL of brefeldin A (BFA, Sigma). The numbers of CD3+CD4+IL-22+Th22, CD3+CD4+IL-17+Th17 and CD3+CD4+IFN-γ+Th1 cells in individual samples were determined by flow cytometry following intracellular staining with anti-cytokine antibodies.