Journal: Journal of neurochemistry

Article Title: Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia.

doi: 10.1111/j.1471-4159.2011.07481.x

Figure Lengend Snippet: Fig. 5 S. aureus treatment induces ATP release from microglia. Pri- mary microglia isolated from C57BL/6 mice were exposed to live S. aureus strain USA300 for the indicated intervals, whereupon ATP release was determined using a luminescence-based assay. To evaluate whether P2X7R activation influenced ATP release, microglia were pre-treated with the P2X7R inhibitor AZ11645373 (AZ; 50 lM) for 30 min prior to S. aureus exposure. Significant differences in ATP release from microglia treated with S. aureus only versus S. aureus + AZ are denoted with asterisks (***p < 0.001; Student’s t-test). Results are reported as the mean + SD of three independent wells for each experimental treatment and were similar across two separate experi- ments.

Article Snippet: In select experiments, microglia were pre-treated with an inhibitory ODN (ODN 2088; Invivogen, San Diego, CA, USA) to block TLR9 signaling (Gurley et al. 2008) or various concentrations of the P2X7R inhibitor AZ11645373 (Tocris Biosciences, Ellisville, MO, USA), caspase 1 inhibitor Z-WEHD-FMK (R&D Systems, Minneapolis, MN, USA), or cathepsin B inhibitor CA-074 Me (Calbiochem, San Diego, CA, USA) prior to live S. aureus exposure.

Techniques: Isolation, Luminescence Assay, Activation Assay

Journal: Journal of neurochemistry

Article Title: Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia.

doi: 10.1111/j.1471-4159.2011.07481.x

Figure Lengend Snippet: Fig. 7 The majority of IL-1b processing in microglia is attributable to autocrine/paracrine actions of ATP mediated by P2X7R in addition to caspase 1. C57BL/6 WT microglia were pretreated with various con- centrations of the caspase 1-specific inhibitor Z-WEHD-FMK only (a) or Z-WEHD-FMK in combination with the P2X7R inhibitor AZ11645373 (AZ; b) for 30 min, whereupon microglia were exposed to live S. aureus USA300 for 6 h. IL-1b production was assessed by ELISA with concentrations normalized to the number of bacterial colony forming units (cfu) inoculated at time 0. Significant differences be- tween microglia treated with S. aureus only versus S. aureus + inhibitors are represented by asterisks (**p < 0.01; ***p < 0.001; Stu- dent’s t-test). Results are reported as the mean + SD of three inde- pendent wells for each experimental treatment and were similar across 3 separate experiments. N.D., not detected.

Article Snippet: In select experiments, microglia were pre-treated with an inhibitory ODN (ODN 2088; Invivogen, San Diego, CA, USA) to block TLR9 signaling (Gurley et al. 2008) or various concentrations of the P2X7R inhibitor AZ11645373 (Tocris Biosciences, Ellisville, MO, USA), caspase 1 inhibitor Z-WEHD-FMK (R&D Systems, Minneapolis, MN, USA), or cathepsin B inhibitor CA-074 Me (Calbiochem, San Diego, CA, USA) prior to live S. aureus exposure.

Techniques: Enzyme-linked Immunosorbent Assay