Fluoro H202

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-0.5 × 10^6 cells were platted in 6 well plate. - Cells were pretread with BITC at different concentration and time intervals	- Cells were treated with 5 μM 6-carboxy-2′,7′-dichlorodihydrofluoroscein diacetate at 37°C for 15 min

Upstream tips
-0.5 × 10^6 cells were platted in 6 well plate. - Cells were pretread with BITC at different concentration and time intervals
Protocol tips
- Cells were treated with 5 μM 6-carboxy-2′,7′-dichlorodihydrofluoroscein diacetate at 37°C for 15 min

Publication protocol

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.

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Manufacturer protocol

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