4.7. Lucigenin-Enhanced Chemiluminescence

NADPH-dependent superoxide production was assessed using lucigenin-enhanced chemiluminescence [1,5]. Briefly, cells or freshly-dissected LV (approximately 30 mg) were lysed in Buffer B (50 mmol/L monobasic potassium phosphate, 1 mmol/L EGTA, 150 mmol/L sucrose) with protease inhibitor cocktail (Roche, Basel, Switzerland) prior to sonication for 3 × 10 s on ice. Following centrifugation, the supernatant was removed and pellets re-suspended in Buffer B with protease inhibitor cocktail, before quantification using the Bradford assay. Protein (50 μg) was then loaded in to white 96-well plates before the superoxide reaction was initiated by adding 25 μL NADPH (1 mg/mL; Sigma-Aldrich) to each sample and incubating for 10 min at 37 °C. An initial fluorescence reading was taken before 25 μL lucigenin (10 μmol/L; Sigma-Aldrich) was added to each well and serial measurements taken every 60 s for 30 min (Berthold Tristar). Superoxide production was quantified as area under the curve, calculated by subtracting the mean background reading from each mean sample value. All experiments were performed in triplicate with mean superoxide levels presented in relative light units.