For antibody experiments, freshly plated C6 cells were incubated with neutraliz

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 For antibody experiments, freshly plated C6 cells were incubated with neutraliz Empty For antibody experiments, freshly plated C6 cells were incubated with neutraliz

Post  huwan123456 on Fri May 23, 2014 8:40 am

The products were separated on a 1. 2% agarose gel containing ethidium bromide, and were visualized under a gel imaging system. Western blotting analysis Cells were lysed in sample buffer containing ARQ 197 cell in vivo in vitro 60 mM Tris HCl, pH 6. 8, 5% glycerol and 2% SDS. Cell lysates were then boiled for five minutes and protein concentration was measured using a BCA kit purchased from Beyotime. Samples were subject to Western blot analysis as previously described. In brief, equal amount of proteins was loaded and separated on a 7 or 10% SDS PAGE gel and transferred to a PVDF membrane, which of Predictive Analytics Software 18. 0 for windows. Difference was considered significant when P 0. 05. Results Paroxetine reduces pro inflammatory cytokines in LPS stimulated BV2 cells Prior to study the impact of paroxetine on LPS induced microglial activation, we examined potential toxic effect of paroxetine on BV2 microglial cells.

The results showed that cell viability was not different from the control following the treatment of paroxetine at 0. 1, 0. 2, 1 or 5 uM. The dose of 10 uM led to a 15. 2% drop in cell viability compared with the control, which was then excluded in our following experiments. To evaluate the impact of paroxetine on cytokine production following LPS stimulation in BV2 cells, AZD1152-HQPA Aurora キナーゼ 阻害剤 we analyzed the release of two pro inflammatory cytokines, TNF and IL 1B, in the media. BV2 cells were treated with LPS for 24 hours in the presence or absence of paroxetine. Paroxetine alone did not elicit marked alteration in the release of TNF or IL 1B, whereas LPS stimulation significantly elevated the levels of these two cytokines.

Pretreatment with paroxetine led to a dose dependent inhibition on LPS induced production of TNF and IL 1B. In particular, paroxetine at 5 uM led to a significant purchase AMN-107 reduction by 68. 3% and 85. 3%, respectively, in TNF and IL 1B generation at 24 hours post LPS stimulation. In order to understand the mechanism underlying the inhibitory effect of paroxetine on LPS induced cytokine production, we analyzed the mRNA expression of TNF and IL 1B following LPS stimulation. Consistent with the cytokine release, LPS significantly up regulated mRNA expression of TNF and IL 1B at 24 hours, which was in turn suppressed by 21. 4% and 60. 7%, respectively, with 5 uM of paroxetine pretreatment.

Paroxetine alone also slightly decreased the basal mRNA level of TNF, whereas the basal IL 1B level seems undetectable using our current PCR program. Paroxetine suppresses LPS induced NO production in BV2 cells To assess whether paroxetine has an impact on NO release in microglial cells, we analyzed NO production following LPS stimulation. BV2 cells were treated with LPS for 24 hours in the presence or absence of paroxetine. As shown in Figure 3A, paroxetine alone did not lead to any change in NO production, whereas LPS significantly induced the generation of NO in BV2 cells. Pretreatment with paroxetine led to a dose dependent inhibition on LPS induced NO production by 15. 1% at 0. 1 uM, 19. 1% at 0. 2 uM, 36. 2% at 1 uM, and 59. 1% at 5 uM.

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