10,000.00 cells were counted per samples. Relative fluorescence intensities were monitored by BD FAXSCANTO™ flow cytometer (BD Bioscience, CA, USA) and analyzed by the software Modfit and Cell-Quest (BD Biosciences, Franklin Lakes, NJ) with settings of FL1 (green)

at 530 nm and FL2 (red) at 585 nm (Liu et al., 2007). Cellular ATP was determined by means of the firefly luciferin–luciferase assay system. Cells (1 × 105) were incubated as for the viability assay and suspension was centrifuged at 50 × g for 5 min at 4 °C. The pellet was treated with 1 ml of ice-cold 1 M HClO4. After centrifugation at 2000 × g for 10 min at 4 °C, aliquots (100 μl) of the supernatants were neutralized with

70 μl buy Bleomycin of 2 M KOH, suspended in 100 mM Tris-(hydroxymethyl) aminomethane (Tris)–HCl, pH 7.8 (1 ml final volume), and centrifuged again. Bioluminescence was measured in the supernatant with a Sigma-Aldrich assay kit according Doramapimod manufacturer to the manufacturer’s instructions, using an AutoLumat LB953 Luminescence photometer (Perkin-Elmer Life Sciences, Wilbad, Germany). Intracellular oxidation of dichlorodihydrofluorescein diacetate (H2DCFDA) to 2,7-dichlorofluorescein (DCF) by ROS was monitored through fluorescence increase. HepG2 cells were seeded in a 12-well plate at a density of 1 × 105 cells/well and incubated as for the cell viability assay. After incubations, the well plates were washed with PBS and then 100 μl/well pentoxifylline of 10 μmol/l H2DCFDA was added to each well, remaining incubated at 37 °C for 45 min in a 5% CO2 incubator. Fluorescence was measured in a model F-4500 Hitachi fluorescence spectrophotometer (Tokyo, Japan) at the 503/529 nm excitation/emission wavelength pair (slits 5/10 nm) (Halliwell and Whiteman, 2004). Mitochondria were isolated by standard differential centrifugation (Pedersen et al., 1978). Male Wistar rats weighing approximately 200 g were euthanized by decapitation; livers (10–15 g) were immediately removed, sliced in medium (50 ml)

consisting of 250 mM sucrose, 1 mM ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and 10 mM HEPES-KOH, pH 7.2, and homogenized three times for 15 s at 1 min intervals using a Potter-Elvehjem homogenizer. Homogenates were centrifuged (580 × g, 5 min) and the resulting supernatant further centrifuged (10300 × g, 10 min). Pellets were then suspended in medium (10 ml) consisting of 250 mM sucrose, 0.3 mM EGTA and 10 mM HEPES-KOH, pH 7.2, and centrifuged (3400 × g, 15 min). The final mitochondrial pellet was suspended in medium (1 ml) consisting of 250 mM sucrose and 10 mM HEPES-KOH, pH 7.2, and used within 3 h. Mitochondrial protein contents were determined by the Biuret reaction. Mitochondria were energized with 5 mM potassium succinate (plus 2.