Time course of intracellular IL-12 expression by mature, activated dendritic cells.

DCs prepared and cryopreserved as described previously were thawed and plated in AIM V medium alone or with the combination of CD40L (1 μg/mL) and IFN-γ (1000 U/mL) for the indicated period, and brefeldin A was added for the last 4 hours. Cells were harvested, fixed, permeabilized, and then stained with α-CD14 PerCP, α-CD11c-APC, α-CD83-FITC, and α-IL-12(p40/p70)-PE and were analyzed with a FACSCalibur multiparameter flow cytometer. Approximately 10 000 CD11c⁺ large cells were analyzed. The percentage of CD83-expressing DCs (●, left y-axis) and the associated percentage of IL-12–producing DCs (▪, righty-axis) are plotted versus the duration of treatment with CD40L and IFN-γ (x-axis).