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.