Figure 2. Diphenyleneiodonium (DPI) inhibitor markedly attenuated the hydrogen peroxide (H₂O₂) generated signal in lens cells detected by peroxyfluor-6 acetoxymethyl ester (PF6-AM). A, top panel: Model indicating (red box) the region of E10 chick embryonic lens containing the equatorial zone (EQ) that is imaged below. A, bottom left panel: Lens placed in organ culture incubated with PF6-AM (green) showing H₂O₂ production in the EQ zone. A, bottom right panel: Lens placed in organ culture incubated with PF6-AM (green) before exposure to the inhibitor diphenyleneiodonium (DPI) attenuates PF6-AM fluorescence, suggesting the presence of an endogenous H₂O₂ signal. Both images are shown as an overlay with the differential interference contrast (DIC) image. B: Primary cultures of lens epithelial cells. (Top panels) Cultured lens epithelial cells incubated with PF6-AM (green, left panel) showed similar H₂O₂ production by cultured lens cells as occurs in the EQ region of the intact lens. Cells also shown imaged by DIC (middle panel) and presented as a PF6-AM/DIC overlay (right panel). (Bottom panels) Cultured lens epithelial cells incubated with the PF6-AM H₂O₂ probe (green, left panel) before exposure to DPI show a decrease in signal from PF6-AM. Cells also shown imaged by DIC (middle panel) and presented as a PF6-AM/DIC overlay (right panel). DPI results suggested that the PF6-AM signal is due to H₂O₂ production in cultured lens epithelial cells. Images were acquired live using confocal microscopy. Each image is a single optical slice of 1 μm; scale bar = 20 μm. Results are representative of three independent experiments.