Black 96 well plate

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).

Pyruvate dehydrogenase complex (PDH) activity was determined by detecting NADH formation from NAD⁺ catalyzed by the enzyme; additionally, LDH was inhibited in order to prevent its interference by NADH consumption. Inhibition was achieved by a catalytic site-targeting agent GNE-140. Sample preparation was performed as described in the methods for detection of OGDHC activity. Incubation buffer contained 1 g/L BSA, 0.1 mM coenzyme A, 0.3 mM DTT, 5 mM l-carnitine, 1 mM magnesium chloride, 2.5 mM NAD⁺, 50 mM potassium phosphate and 0.2 mM TPP; pH was adjusted to 7.5. GNE-140 stock was prepared in the concentration of 10 mM in DMSO (final concentration 0.09 mM); pyruvate stock was prepared in the concentration of 250 mM, with the final concentration being 11.5 mM. All reagents were obtained from Sigma-Aldrich, Germany. 200 μL incubation buffer, 2 μL GNE-140 stock and 5 μL sample were incubated at room temperature on a black 96-well plate (Sigma-Aldrich, Germany) for 20 min; 10 μL pyruvate stock were added directly before the measurement. NADH fluorescence was kinetically detected at 460 nm with the excitation wavelength of 340 nm; PDH activity was derived from the relative increase in fluorescence signal of the sample over time expressed in minutes, multiplied by the slope of the calibration curve obtained from the fluorescence values of a dilution series of NADH.