Intracellular ROS Measurement by DCFH-DA

The determination of intracellular reactive oxygen species (ROS) production was followed by the previous report [13 (link)] using 2′,7′-Dichlorofluorescin diacetate (DCFH—DA, Sigma, St. Louis, MO, USA). Briefly, cells were cultured in 96 well plates with DMEM. The culture medium was removed and replaced with 2 µM DCFH-DA in serum-free DMEM in a dark room for 24 h at 37 °C. After H/R protocol, the medium was removed and replaced with a completed DMEM with DCFH-DA and incubated at 37 °C for 1 h. The fluorescence intensity was measured by using an EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.