Antioxidant effect of angiotensin (1-7) in the protection of pancreatic ß cell function

Zhang, Fen; Liu, Chang; Wang, Lei; Cao, Xi; Wang, Ying Ying; Yang, Jin Kui

ROS determination. INS-1 cells were grown in RPMI-1640 in 6-well plates for 48 h, followed by incubation with Ang (1-7) or A779 for 2 h, then a concentration of 250 µM H 2 O 2 was added in the last 15 min. Cells were loaded with 5 µmol/l dihydroethidium (DHE) ROS (Vigorous Biotechnology Co., Ltd.; Beijing, China) detection and suspended in PBS for 20 min at 37˚C in the dark. The cells were rinsed twice in PBS and collected with 0.05% trypsin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). Following centrifugation at 140 x g for 5 min, pellets were resuspended in 500 µl PBS. ROS was determined using intracellular ROS capture DHE with flow cytometry (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Briefly, an ex wavelength of 480-535 nm was used to determine em >590-610 nm. Cells were then divided into two subgroups: ROS-negative cells, which exhibit a very low fluorescence intensity and ROS-positive cells, which emit red fluorescence. Ten-thousand events per sample were collected.