z-VAD-fmk inhibits TNFα-stimulated intra- and extracellular ROS accumulation. Neutrophils were cultured for 30 minutes in the presence or absence of 0.03-300 μM z-VAD-fmk before the addition of TNFα (200 U/mL) or PBS for a further 30 minutes. (A) Lucigenin (0.25 mM) or (B) luminol was then added and steady-state ROS generation assessed using a luminometer. For reference, these values are approximately 100 times lower than the values obtained under TNFα-primed, 100 nM fMLP-stimulated conditions. (C) The inclusion of a ROS scavenger, DPI (10 μM), did not effect z-VAD-fmk (300 μM) augmentation of TNFα-stimulated cell death, (D) but was sufficient to inhibit TNFα-primed (30 minutes)/fMLP stimulated (48 seconds) ROS generation. ^*P < .05 (significant inhibition of TNFα-stimulated ROS generation analyzed by ANOVA). Data represent the mean ± SEM of n = 3 separate experiments each performed in triplicate. AnV, annexin V; RLU, relative light units.