Measurement of ROS in the RVLM

The lucigenin-enhanced chemiluminescence assay was adopted to detect the ROS production in the RVLM (Chan et al., 2005). RVLM was homogenized in PBS (20 mM, pH 7.4) containing EDTA (0.01 mM). The homogenate was centrifuged at 1200 × g for 10 min at 4°C and the supernatant was collected for ROS assay. Background chemiluminescence in a 2-ml buffer mixed with lucigenin (5 μmol/L) was detected for 5 min. Adding 100 μl supernatant, the chemiluminescence was detected for 30 min at room temperature. The production of ROS was determined and expressed as average light units per minute per milligram of protein.