HUVEC intracellular oxidant production in response to TNF. Live, unfixed HUVECs (original magnification, × 100) loaded with dichlorofluorescein (10 μg/mL DCF) were exposed to vehicle (A), or TNF (100 μ/mL) (B-D) for up to 15 minutes and were observed by confocal microscopy using a heated stage. Oxidant production, as evidenced by fluorescence (arrows), was discrete and localized to membrane sites. This observation was (B) present within 5 minutes of TNF exposure, (C) became maximal at 10 minutes, and (D) disappeared within 15 minutes. (E) Oxidant production at 5 minutes was also measured by flow cytometry, which showed that TNF significantly increased oxidant production (▪) compared with vehicle controls (□). This increase in oxidant production was prevented by DPI (▦).