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With rose bengal maleimide; sodium hydroxide; at 20.0℃; for 0.5h;
Production of ROS was detected using 20,70-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Wako). H2DCF-DA (10 mM) wasfirst treated with NaOH (10 mM) for 30 min at room temperature to generate H2DCF, which is a ROS indicator that can be rapidly oxidized to generate highly fluorescent 2',7'-dichlorodihydrofluorescein. H2DCF (10 lM) and rose bengal (10 lM) were mixed in100 lL T buffer and loaded on a MicroWell 96-well optical bottom plate (Nunc, Rochester, NY, USA) or on a glass (12-mm)-bottomed dish (IWAKI). The solution was irradiated with US (1 MHz, dutycycle 30%, 0.3 W/cm2) using a Sonitron2000V equipped with anUS probe (/ 6-mm). After irradiation, fluorescence spectra were measured at an excitation wavelength of 492 nm using an FP-6600 spectrofluorometer (Jasco).