4.6. Lucigenin Assay

To assess the activity of NOX in NRCMs, a lucigenin assay was performed as described previously [32]. Total cell lysates were harvested by lysis buffer (Nacalai tesque). Phosphate buffer (200 µL: 50 mM NaH₂PO₄ and NaHPO₄, 1 mM EGTA and 150 mM sucrose at pH 7.0) containing lucigenin (10 µM), NADPH (1 mM) and cell lysate (20 µg) was poured into assay wells (96-well plates). Then, the chemiluminescence was continuously measured for 30 min at 37 °C by a TriStar LB941 luminometer (Berthold, Bad, Wildbad, Germany). The chemiluminescence of relative light units per second (RLU/s) was obtained every 10 s, and the results were calculated as area under the curve.