Electron Paramagnetic Resonance (EPR) Spin Trapping of Superoxide Radical (O2.−)

To detect O₂^.− generation in HBMECs, measurements of supernatant O₂^.− levels were performed on cells treated with media and TL for 15 min. Cells were pretreated with scrambled control siRNA or CPT1A1 D1 siRNA for 48 h. Each treatment condition contained 0.5 mM 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) spin trap as previously described [15,49]. Spin traps are capable of rapidly trapping free radicals such as O₂^.− to form more persistent radicals (spin adducts) detectable by EPR. Conversion of the diamagnetic hydroxy-amine CP-H to the paramagnetic nitroxide was measured using a JEOL JES TE-100 EPR spectrometer. All spectra were obtained at room temperature by averaging two 2-min scans with a sweep width of 100 G at a microwave power of 3 mW and modulation amplitude optimized to the natural line width of the spin probe.