Intracellular peroxide levels were detected by flow cytometric analysis using an oxidation-sensitive fluorescein labeled dye, carboxylated dichlorodi-hydrofluorescein diacetate (carboxy-H2DCFDA, Molecular Probes, Carlsbad, CA, USA) (15 (link)). Upon oxidation by intracellular ROS, the non-fluorescent dye is converted into its fluorescent form. INS-1 cells were labeled with 100 M carboxy-H2DCFDA for 1 hour at 37°C. Following cell loading of the dye, the cells were washed twice with PBS and then put back into culture conditions for 2 hours. INS-1 cells were then harvested, washed twice with PBS, and re-suspended in trypsin–EDTA (0.25% trypsin, 2 mM Na4-EDTA, Invitrogen) for 5 minutes at 37°C. In order to disperse the cells into a single cell suspension, INS-1 cells were gently passed 20 times in and out of a 200–1,000 lL tip. The cells were then washed twice with ice-cold PBS. Analysis of cells was performed using a 488 nm argon laser EPICS XL-MCL flow cytometer controlled by EXPO 32-ADC software (Beckman Coulter, Fullerton, CA, USA). ROS values were analyzed based on fluorescence intensity.