S-Mtb induces intracellular ROS generation and MAPK (ERK1/2 and p38) activation in murine microglial BV-2 cells and in primary cultures of mixed glial cells. A) BV-2 cells were incubated with DCFH-DA (H2O2 detection, left panel) or DHE (superoxide detection, right panel) in the presence or absence of 1% s-Mtb or 100 ng/ml LPS for 30 min. Live cells were washed with serum-free medium and imaged using a confocal microscope. Enhanced H2O2 or superoxide was abolished by pre-treatment with 20 mM NAC or 20 μM DPI, respectively. Images are representative of three independent experiments. B) Primary cultures of mixed glial cells were analyzed for H2O2 or superoxide production in response to s-Mtb or LPS treatment. The experimental conditions were identical to those outlined in A. Quantitative data are the mean ± SD of values from three random fields and are representative of three independent experiments. C) NADPH oxidase activity was quantified by measuring the production of ROS using a lucigenin-derived chemiluminescence assay. The effect of the NADPH oxidase inhibitor DPI (20 μM) was examined. M; mock. D) BV-2 cells (Upper panel) or primary mixed glial cells (Lower panel) were stimulated with 1% s-Mtb or 1 μg/ml LPS for the indicated times (0–480 min). The cells were then harvested and subjected to Western blot analysis to detect phosphorylated and total forms of p38 and ERK1/2. The same blots were washed and blotted for α-actin as a loading control. Data are representative of three independent experiments.