FIG. 5.

hUCB-MSCs show low antioxidant activity. (A) Cells incubated with 20 μM of 2′,7′-dichlorodihydrofluorescein diacetate for 30 min were washed, and then incubated with fresh media containing the indicated concentration of hydrogen peroxide for 30 min. Intracellular ROS levels were detected using the FACS Calibur instrument (BD Bioscience). Mean fluorescence intensity of DCF was used as measure of ROS, and values were normalized with the basal intensity of each cell line. (B) Total antioxidant activity in each cell lysate was measured using Trolox E as described in the Materials and Methods section. Values per 1 μg of cell lysate are shown. (C) Activities of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured as described in the Materials and Methods section. Enzyme activity per 1 μg of cell lysate is indicated. (D) 40 μg of total protein from each cell line was immunoblotted with the appropriate antibodies (see also Supplementary Fig. S6A). Relative band intensities normalized to the β-actin band were calculated using Multi Gauge 3.0 software (Fujifilm). SOD1, superoxide dismutase 1; SOD2, superoxide dismutase 2; GPx1, glutathione peroxidase 1. (E) 1 μg of mRNA from each cell line was reverse transcribed using specific primers (Materials and Methods). Relative band intensities from gel electrophoresis following RT-PCR (Supplementary Fig. S6B) were measured using Multi Gauge 3.0 software. Quantitative data for each RT-PCR product were normalized against the GAPDH level. The P values were 0.036 (A); 0.017 (B); 0.0081, catalase activity in (C); 0.0076, SOD activity in (C); and <0.0001, GPx activity in (C). DCF, 2′,7′-dichlorodihydrofluorescein; ROS, reactive oxygen species.