Evaluation of intracellular reactive oxygen species

Levels of ROS in osteoblasts were determined with H2DCF-DA, a fluorescent dye, which could be rapidly oxidized into the highly fluorescent compound DCF in the presence of ROS (Ma et al., 2013). Following treatment with FAC, the osteoblasts were collected and washed with PBS, subsequently resuspended in serum-free media, and finally treated with 20 µM H2DCF-DA in dark at 37 °C for 20 min (Ding et al., 2012). After incubation, MC3T3-E1 cells were rinsed with serum-free a-MEM thrice, and subsequently the mean fluorescence intensity (MFI) was evaluated with a FACSCalibur flow cytometer (BD, Franklin Lakes, NJ, USA).