Superoxide dismutase sod

Superoxide dismutase (SOD) was purchased from MP Biomedicals, USA. High purity (> 99%) 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) was obtained from Enzo Life Sciences. Diethylenetriaminepentaacetic acid (DTPA; > 99%), a metal chelator, was purchased from Sigma-Aldrich, USA.

To test for involvement of reactive oxygen species, supernates were treated with catalase (MP Biomedicals, Santa Ana, CA), superoxide dismutase (SOD, MP Biomedicals, Santa Ana, CA), carboxy-PTIO (Sigma-Aldrich, St. Louis, MO), uric acid (UA, Pointe Scientific, Canton, MI), or MnTBAP (Enzo Life Sciences, Farmingdale, NY) at indicated concentrations to neutralize/scavenge hydrogen peroxide, superoxide, nitric oxide, or peroxynitrite, respectively. Concentrations depicted represent the final concentration after transfer. Supernate assays were performed as described above (with wells receiving antioxidant-treated supernate from responder cells cultured alone used as a control to ensure lack of effect of antioxidants on baseline proliferation), and percent blockade of suppression was calculated by comparing supernates treated with the antioxidants to untreated supernate controls, as described above. As a positive control for catalase activity, the ability to break down hydrogen peroxide was measured spectrophotometrically.