Superoxide Generation

Freshly isolated retinas were incubated in 200 μL Krebs-Hepes buffer (20 mM HEPES in 0.12 mM CaCl₂ 0.08 mM MgSO₄), pH 7.2, with 5 or 25 mM glucose for 5 minutes at 37°C in 5% CO₂. Luminescence indicating the presence of superoxide was measured at 5 minutes after addition of 0.54 mM (final concentration) lucigenin, as published previously.^22–27 Luminescence intensity is stated in relative units per milligram protein. Superoxide data from opsin-deficient mice at 2 months of diabetes were reported previously,²⁸ and are reanalyzed differently here to emphasize superoxide generation in the experimental groups relative to nondiabetic C57Bl/6J controls.