Quantifying Cardiac Oxidative Stress

LV superoxide levels were detected via lucigenin-enhanced chemiluminescence. During tissue collection, fresh LV sections (4 mm³ × 1 mm³ per sample) were placed into individual wells of an opaque 96-well optiplate (Perkin Elmer) containing 100 μL of Krebs-HEPES buffer. β-NADPH was added to three of the four tissue-containing wells, assigning one as the non-substrate control. After a 1-h incubation at 37°C, lucigenin (5 μM) was added to every well, before being placed into an EnSpire Plate reader (Perkin Elmer) for superoxide detection via chemiluminescence (Tate et al., 2019 (link)). An Amplex Red assay kit (Invitrogen) was used to assess LV hydrogen peroxide content, as per manufacturer’s instructions. Hydrogen peroxide standards and LV protein samples were added to a black 96-well plate (Sigma-Aldrich). Standards and samples were then incubated for 30 min with Amplex Red/horseradish peroxidase (0.1 mM/0.2 U/ml, respectively) in the dark, prior to hydrogen peroxide detection using a fluorescence CLARIOstar plate reader (530 nm/590 nm excitation/emission).