Figure 3
An NG-to-HG shift increases the production of TNF-α, NO, peroxides, and ROS in cultured BV-2 microglia. (a,b) At an assigned time point of 0 h, the culture media used to maintain NG- and HG-cultured cells was replaced by fresh media with or without a shift in glucose concentration, as indicated. To avoid any influence of different cell proliferation rates on the assays, ARA-C (2 µg/ml) was added to all treatment groups. Cell media was collected at the indicated time points for the measurement of TNF-α using ELISA (a) or NO using an assay of the Griess reaction to determine nitrite accumulation (b). Data are represented as mean ± SEM from five independent experiments performed in triplicate. *p < 0.05 versus constant NG group at the same time point; #p < 0.05 versus constant HG group at the same time points. (c,d) Culture media was replaced by fresh media at the assigned time point of 0 h with or without an NG-to-HG shift. ARA-C was added to all treatment groups to eliminate the influence of different cell proliferation rates on the detected parameters. At different time points within 0−24 h, cell-permeable fluorogenic probes, DHR-123 (c) and H2DCFDA (d), were employed individually. DHR-123 monitors the production of ROS, such as peroxide and peroxynitrite. H2DCFDA serves as a tool to detect intracellular ROS, reflecting the degree of overall oxidative stress. Data are represented as mean ± SEM from four independent experiments performed in triplicate. *p < 0.05 versus constant NG group at the same time point. RFU: relative fluorescence unit.
