2.18. EPR spin trapping of Superoxide

Spin trapping measurements of superoxide and superoxide-derived radicals were performed using 50 mM DMPO with 1 μM acetylcholine in perfusate immediately prepared and infused by a Harvard pump at 0.5 mL/min. The DMPO-infused samples were collected into tubes containing methyl-β-cyclodextrin (final concentration of 100 mM) to enhance spin adduct stability. EPR measurements were performed in a quartz flat cell and spectra obtained at room temperature at a microwave frequency of 9.77 GHz using 20 mW of microwave power and 1.0 G modulation amplitude. L-NAME was administered for 10 minutes prior to DMPO infusion at a concentration of 1 mM to block the production of NOS-dependent superoxide.