To measure the mitochondrial ROS production, freshly isolated mitochondria (25-?g protein) were suspended in 50 ?l of HMS medium (10 mM HEPES pH 7.4, 225 mM mannitol, 75 mM sucrose), and added in triplicate to 96-well, black flat-bottomed plates. The reaction was started with addition of 50 ?l of 2X reaction buffer (20 mM Tris-HCl at pH 7.4, 500 mM sucrose, 2 mM EDTA) containing 66-?M amplex red and 0.2U/ml horseradish peroxidase. Mitochondria were energized with complex I or complex II substrates, and amplex red oxidation by ROS to fluorescent resorufin was measured for three minutes at Ex563nm/Em587nm on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). Standard curve was prepared with H2O2 (50 nM–5 ?M) .
To measure the H2O2 levels, 50 ?l of tissue lysates (100 ?g) or cell lysates (1 x 104 cells) were added in triplicate to flat-bottom (dark-walled) 96-well plates. Then 100 ?l of reaction mixture containing 0.05 M sodium phosphate, pH 7.4, 33 ?M amplex Red, and 0.1 U/ml HRP was added. The plates were incubated for 30 min in dark, and ROS levels were recorded as above.
The level of 3-nitrotyrosine (3-NT) in heart tissue lysates was measured by using an ELISA kit (ab116691, Abcam, Cambridge MA). Protein carbonyls in tissue homogenates and plasma were measured by a colorimetric protein carbonyl assay (cat#10005020, Cayman Chemical, Ann Arbor MI). Malonyldialdehydes (MDA) provide a measure of lipid peroxidation products, and were measured in tissue lysates and plasma samples by a TBARS assay (10009055, Cayman Chemical). Concentration of lipid peroxides was calculated as an MDA equivalent using the extinction coefficient for the MDA–TBA complex of 1.56? 105 M?1 cm?1 at 532 nm.
Total antioxidant capacity was assessed by using lag time by antioxidants against the myoglobin-induced oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) with H2O2 (709001, Cayman Chemical). Briefly, 20 ?l of plasma samples (diluted 1:20, v/v) or heart homogenates (15 ?g) were added in triplicate to 96-well plates, and mixed with 90 ?l of 10 mM PBS (pH 7.2), 50 ?l of myoglobin solution, and 20 ?l of 3 mM ABTS. Reaction was initiated with H2O2 (20 ?l) and change in color monitored at 600 nm (standard curve: 2–25 ?M trolox).
To measure mitochondrial stress, cardiac myocytes (104/well) were incubated with Tc and/or PJ34, and then loaded for 30 min with 5 ?M MitoSOX red (detects mitochondrial ROS, Ex498nm/Em598nm) or 10 ?M JC-1 (5,5?,6,6?-tetrachloro-1,1?,3,3?-tetraethylbenzimidazolylcarbocyanine iodide). Cells were washed, and JC-1 red aggregates (Ex560nm/Em595nm) vs. green monomers (Ex485nm/Em/535nm) ratio recorded by using a Spectra MaxR M2 microplate reader. The fluorescent probes were purchased from Invitrogen/Molecular Probes.