Wallac victor 3 multilabel counter

Lipid peroxidation was determined using the Lipid Peroxidation Assay Kit (Sigma–Aldrich) following manufacturer instructions. Malondialdehyde levels were quantified using the Victor 3 Multilabel Counter (Wallac, Perkin Elmer, Waltham, MA, USA) at 532 nm for excitation and 553 nm for emission. Finally, results were normalized on cell count.

ROS were determined by incubating H460^siRNA and H460^siFHC with the redox-sensitive probe 2’-7’-DCF (CM-H₂CFDA; Molecular Probes). In detail, 1 × 10⁶ H460 cells were plated in 96-well plates and incubated with Hanks balanced saline solution (HBSS), 10 mm glucose and 20μm DCF for 15 min at 37°C. After two cyclewashes, cells were maintained in HBSS supplemented with 10 mm glucose. Fluorescence was revealed, after 60 min incubation, using the Victor3 Multilabel Counter (Wallac, Perkin Elmer) at 485 nm and 535 nm for excitation and emission, respectively. Results were normalized on protein concentration evaluated by the bicinchoninic acid (BCA) method (Thermo Fisher Scientific).

ROS level in the liver was determined using the fluoresenct probe dichlorofluorescein (DCF) as previously described [25 (link)]. The stock solution of 2’,7’-Dichlorofluorescin diacetate (DCFDA, D6883, Sigma-Aldrich, St. Louis, MO) was prepared by dissolving DCFDA in 12.5 mM of ethanol and stored at -80°C until used. Approximately 20 mg of liver was homogenized in 0.5 ml HEPES buffered saline (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose), centrifuged at 1000 × g for 10 min. Homogenate containing 100 μg protein was pipetted into a black 96-well plate. The DCFDA was diluted to 125 μM immediately before use and pipetted into each well to a final concentration of 25 μM. The plate was placed on a shaker for 2 min and incubated at 37°C in the dark for 30 min. The fluorescence was read on a Wallac Victor 3 Multilabel Counter (PerkinElmer Inc., Waltham, MA) at excitation 485 nm/emission 530 nm at 0 and 50 min. The ROS level was expressed as the increased absorbance value between 0 and 50 min. Mean of fluorescence intensity was calculated from five mice per group.