NADPH Oxidase Activity Assay With Lucigenin Chemiluminescence

Lucigenin chemiluminescence assay was used to measure membrane NADPH oxidase activity. Although lucigenin is known to undergo redox cycling at higher concentrations, lucigenin at 5 μM was validated for detecting superoxide production (Li et al., 1998; Skatchkov et al., 1999; Dikalov et al., 2007). Heart was homogenized (glass/glass) in a homogenization buffer containing 50 mM Tris•HCl pH 7.4, 2 mM dithiothreitol, and a protease inhibitor cocktail (Roche) and centrifuged at 2,000 g for 5 min at room temperature. The supernatant was removed, and the remaining lysate was centrifuged at 20,000 g for 20 min at 4°C. After the second centrifugation, the supernatant was removed and centrifuged at 100,000 g for 60 min at 4°C. Finally, the pellet was resuspended in homogenization buffer (without dithiothreitol) and further diluted in PBS to a final protein concentration of 0.2 mg/ml for the assay. Five micrometer lucigenin-derived chemiluminescence of the membrane suspensions was detected by a Lumat LB 9507 (Berthold) in the presence of 200 μM NADPH.