Du 600 uv vis spectrophotometer

Cells were incubated with 50 μM Amplex Red reagent (Thermo Fisher) at 37 °C for 30 min. Fluorescence was detected at ex/em 550/590 nm using a Gemini XPS Microplate Reader (Molecular Devices, San Jose, CA, USA). To account for H₂O₂-specific fluorescence, PEG-CAT was used as described above. Intracellular production of H₂O₂ was estimated using a sensitive assay based on the aminotriazole-mediated inactivation of CAT, as previously described by Wagner et al. [40 (link)]. Cells at 70–90% confluence in 100 mm tissue culture dish were treated with BET for 6 h, and 20 mM 3-aminotriazole (3-AT) was then added and incubated at 37 °C for 0, 5, 10, 15, 30, and 45 min. Cells were rinsed twice and harvested with ice-cold 50 mM phosphate-buffered saline (PBS), and pH 7.4 and pellets were collected. H₂O₂ concentration was calculated by kinetic analysis of the rate of decrease in CAT activity [41 (link)]. Spectrophotometric CAT activities were initiated by addition of 30 mM H₂O₂ and the loss of absorbance at 240 nm at 25 °C was monitored on a Beckman DU-600 UV–Vis spectrophotometer (Beckman–Coulter, Fullerton, CA, USA). Initial CAT activities were calculated by fitting experimental data to the first-order kinetics and expressed as catalase k mU/mg cell protein.

Twenty-four hours after transfection, the medium was removed and the wells were washed briefly with 200 μL of PBS. After removal of PBS, the cells were collected by adding 100 μL of 1× reporter lysis buffer (Promega, Fitchburg, WI, USA) to each well and the cell lysates were used for luciferase and protein assays. For the luciferase assay, 20 μL of cell lysate was transferred to a test tube and assessed directly by means of a Lumat LB 9507 luminometer (Berthold Detection Systems, Pforzheim, Germany), using a luciferase assay kit from Promega. The protein content was quantified using a bicinchoninic acid (BCA) assay (PIERCE, Rockford, IL, USA). The BCA assay was prepared as specified by the manufacturer. Forty microliters of cell lysate was mixed with 1 mL of BCA reagent in an acrylic cuvette, and the solution was incubated for 1 h at 37 °C. The light absorption of the solution was then read at 562 nm by means of a Beckman DU-600 UV-vis spectrophotometer (Palo Alto, CA, USA), and the protein content was estimated by comparison to bovine serum albumin standards. The luciferase activity was normalized by the protein content and expressed as relative luminescence units/μg of protein (RLU/μg protein).