The generation of ROS was evaluated by measuring the levels of hydrogen peroxide produced in the cells using Fluorescent Hydrogen Peroxide/Peroxidase Detection Kit (Cell Technology) according to user's manual with slight modification. The fluoro H2O2 detection kit utilizes a non-fluorescent detection reagent to detect H2O2. H2O2 oxidizes the detection reagent in a 1:1 stoichiometry to produce a fluorescent product resorufin which is catalyzed by peroxidase in a homogeneous no-wash assay system. Briefly, 40 000 Capan-2, MiaPaCa-2 or HPDE-6 cells were plated in 96 well plates in 100 μl medium and allowed to attach overnight. To evaluate the generation of ROS in a time-dependent manner, cells were treated with 10 μM BITC for 0.5, 1, 2, 4 and 6 h. In a separate experiment, cells were treated with 0, 2.5, 5, 10 and 20 μM BITC for 2 h. After the completion of BITC treatment for indicated time points, cells were washed twice with their respective plain mediums (without 10% FBS) followed by incubation with 100 μl reaction cocktail (10 mM detection reagent, 10 U/ml horseradish peroxidase in plain medium) for 10 min at room temperature in dark. After incubation, the fluorescence was measured at excitation 540 nm and emission 595 nm in a fluorescent plate reader. ROS generation was also evaluated by measuring the levels of hydrogen peroxide produced in the cells by flow cytometry. Levels of hydrogen peroxide in control and BITC-treated Capan-2 cells were determined by staining the cells with 6-carboxy-2′,7′-dichlorodihydrofluoroscein diacetate as described by us previously (19,20). 6-Carboxy-2′,7′-dichlorodihydrofluoroscein diacetate is a cell permeable probe and is cleaved by non-specific esterases and oxidized by peroxides produced in the cells to form fluorescent 2′,7′-dichlorofluoroscein. The intensity of 2′,7′-dichlorofluoroscein fluorescence is proportional to the amount of peroxide produced in the cells. Briefly, 0.5 × 106 cells were plated in 25 cm2 flasks and allowed to attach overnight. After treatment with BITC, cells were further incubated with 5 μM 6-carboxy-2′,7′-dichlorodihydrofluoroscein diacetate at 37°C for 15 min. Subsequently, cells were washed and re-suspended in phosphate-buffered saline and analyzed for 2′,7′-dichlorofluoroscein fluorescence by using Coulter XL flow cytometer. Approximately 20 000 cells were evaluated for each experiment. In all determinations, cell debris and clumps were excluded from the analysis. Full paper
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