4.7. Measurement of Intracellular Reactive Oxygen Species (ROS)

A previously described method [45] was conducted to measure the intracellular ROS generation induced by t-BHP. In brief, an adequate (5 × 10⁵/mL) number of cells (RAW 264.7 cells and BV2 cells) were first cultured in 96-well plates for 24 h. The cells were pretreated with indicated concentrations of NNFE. After 1 h, cells were stimulated with t-BHP (100 μM) and incubated for 24 h followed by washing with phosphate buffer saline (PBS) twice and then 25 μM 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was added and incubated at 37 °C for 30 min. The fluorescence intensity was measured at an excitation wavelength of 485 nm and emission wavelength of 528 nm using a fluorescence microplate reader (Victor3, PerkinElmer, Waltham, MA, USA).