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The optical density (OD) was measured at 540 nm. Viability of untreated control cells was set to 100%, and the relative viability of the experimental group was calculated as follows: cell viability = (OD of experimental group/OD of control group) �� 100%. Apoptosis of microglia was scored by TUNEL assay using a commercially available kit, according to the manufacturer's protocol (In Situ Cell Death Detection Kit; peroxidase; Roche Applied Science) as previously described (Jung et al.,2005). The percentage of TUNEL-positive cells was quantitated by counting cells in 10 random microscopic fields. Matrix metalloproteinase (MMP) activity in the culture media was determined by substrate gel electrophoresis as previously described (Birkedal-Hansen and Taylor,1982). MMP secretion was CCI-779 research buy also assessed by Western blot analysis as previously BVD523 described (Lee et al.,2004). All data are presented as mean �� SD of three or more independent experiments. Statistical comparison of different treatments was performed with either Student's t-test or one-way ANOVA with Dunnett's multiple-comparisons test in GraphPad Prism (GraphPad Software Inc., San Diego, CA). Differences with a P value OPHN1 LPS/IFN-�� stimulation as demonstrated by quantitative RT-PCR (bottom panels in Fig. 2). In the end-point analysis of GITR expression, as shown in the upper panels of Figure 2, the expression level appeared to be saturated, making it impossible to evaluate the effect of LPS/IFN-��. RAW 264.7 cells were again used for comparison. These results indicate that GITR/GITRL are expressed in brain microglia cells and that the level of expression is increased by inflammatory stimuli. The mRNAs of GITR and GITRL were expressed in the C6 astrocytoma cell line (data not shown), suggesting that the GITR/GITRL system also participates in the regulation of brain astrocytes. To determine whether the stimulation of GITR or GITRL induces proinflammatory activation of microglia cells, we examined NO production in BV-2 mouse microglia cells after stimulation with agonistic monoclonal antibodies (mAb) against GITR or GITRL (Fig. 3A).