Endogenous accumulation of H₂O₂/ROSs results in down-regulation of IL-12 p40 production by macrophages. The RAW 264.7 macrophages were exposed to alloxan to allow endogenous accumulation of H₂O₂/ROSs. For measuring endogenous H₂O₂/ROS induction by alloxan, cells were loaded with 5 μM DCFH-DA in the absence or presence of 1 mM alloxan. After 15 minutes of incubation in the dark, the cells were further stimulated with LPS plus IFN-γ for another 15 minutes. The DCF fluorescence was measured by flow cytometer (A). The IL-12 p40 levels (mean ± SD) were quantified by EIA for LPS plus IFN-γ-activated RAW 264.7 macrophages treated with titrating doses of alloxan (B). This experiment is representative of 3 experiments performed with similar results.