Quantifying Superoxide Production in Aorta

Superoxide production was measured using lucigenin-enhanced chemiluminescence (LgCL). The harvested aortic segments, stored on ice, were cut open to expose endothelial surface and equilibrated for 30 min in aerated Krebs-HEPES buffer at 37 °C. LgCL from intact vessels was measured in 2 ml buffer containing lowconcentration lucigenin (5 μM) using Berthold FB12 luminometer. Superoxide production was expressed as relative light units (RLU) per second per mg of dry weight of the vessel. An alternative, DHE-based method was used to quantify superoxide anion production in aorta homogenates of AngII-infused WT and Sphk1^−/− mice (n = 5 for both groups)55 (link). Fluorescence measurements were performed in 37 °C using Synergy H4 instrument. Protein content in homogenates was normalized using Bradford reagent.