Fluoro hROS

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	The generation of other ROS by BITC treatment was measured using hROS detection kit (Cell Technology) according to user's manual with slight modification.

Protocol tips
The generation of other ROS by BITC treatment was measured using hROS detection kit (Cell Technology) according to user's manual with slight modification.

Publication protocol

The generation of other ROS by BITC treatment was measured using hROS detection kit (Cell Technology) according to user's manual with slight modification. This kit detects hROS [hydroxyl radical (·OH)], peroxynitrite (ONOO−) and hypochlorite (−OCl) production in the cells using a novel probe, aminophenyl fluorescein (APF). APF is a cell permeable non-fluorescent probe that oxidizes by hROS into strongly fluorescent APF. This probe has little reactivity toward other ROS such as singlet oxygen (O12), superoxide (O2−·), H2O2, nitric oxide (NO·) and alkyl peroxide (RO2·). 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. After that cells were rinsed with modified Hanks balanced salt solutions buffer supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM MgCl2, 2 mM CaCl2 and 2.7 mM glucose followed by incubation with 10 μM APF (diluted in modified Hanks balanced salt solutions buffer in 1:10 ratio) for 60 min at 37°C. After incubation with APF, 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 followed by measurement of fluorescence at excitation 488 nm and emission 515 nm in a fluorescent plate reader.

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