Best Aids Designed for PLX-4720

5 ml of 10% homogenate were oxidized to ubiquinones (CoQs) with 0.5 ml of 2% FeCl3 and 2.0 ml of ethanol. The total content of CoQs (CoQH2+ CoQ) was then determined according to Lang et al. (1986). The vitamin E content was determined using the HPLC procedure of Lang et al. (1986). Reduced glutathione (GSH) concentration was measured in homogenates as described by Griffith (1980). The rate of mitochondrial H2O2 release was measured at 30��C following the increase in fluorescence (excitation at 320 nm, emission at 400 nm) due to oxidation of p-hydroxyphenylacetate by H2O2 in the presence of horseradish peroxidase (Hyslop & Sklar, 1984) in a computer-controlled Jasko fluorometer equipped with a thermostatically controlled cell-holder. For measurement of H2O2 produced by the respiratory chain, the reaction mixture consisted of 0.1 check details mg ml?1 mitochondrial proteins, 6 U ml?1 horseradish peroxidase, 200 ��g ml?1?p-hydroxyphenylacetate, and 10 mm succinate added at the end to start the reaction in a medium containing 145 mm KCl, 30 mm Hepes, 5 mm KH2PO4, 3 mm MgCl2, 0.1 mm EGTA and 5 ��m rotenone, pH 7.4. Measurements in the presence of 500 ��m ADP or 10 ��m antimycin A were also performed. Capacity to remove H2O2 was determined by comparing the ability of mitochondrial samples to reduce H2O2-linked fluorescence emission with that of desferrioxamine solutions (Venditti et al. 2001b). Thus, the capacity of mitochondrial samples to remove H2O2 was expressed as equivalent desferrioxamine PLX 4720 concentration. Mitochondrial swelling was spectrophotometrically measured by determining the apparent absorbance at 540 nm in a medium containing 125 mm sucrose, 65 mm KCl, 10 mm Hepes (pH 7.2), 2 mm succinate, 4 ��m rotenone, 0.3 mg mitochondrial protein (ml reaction mixture)?1, 100 ��m Ca2+ and 50 mm EGTA or 100 ��m ciclosporin A where indicated. Mitochondrial membrane potential (����) was estimated through fluorescence changes of safranine (8 ��m) recorded on the Jasko fluorometer (excitation wavelength 495 nm, emission wavelength 586 nm) in a medium containing 125 mm sucrose, 65 mm KCl, 10 mm Hepes (pH 7.2), 2 mm succinate, 6 ��m rotenone, 0.3 mg mitochondrial protein (ml reaction mixture)?1 and 100 ��m Ca2+. The value of ���� was calculated according to ?ckerman & Wilkstr?m (1976), using a calibration curve obtained by incubating mitochondria in a medium containing Reelin 200 mm sucrose, 10 mm Hepes (pH 7.2), 6 ��m rotenone, 0.38 mm EDTA, 8 ��m safranine, 38.5 ng ml?1 valinomycin and KCl at concentrations ranging from 0 to 0.96 mm. The data obtained in eight different experiments are expressed as means �� SEM. Data were analysed with a one-way ANOVA method. When a significant F ratio was found, the Student�CNewman�CKeuls multiple range test was used to determine the statistical significance between means. Values of P