The formazan crys tals in the cells were solubilized with DMSO. The absor bance at 550 nm was determined by a microplate reader Multiskan JX. Cell www.selleckchem.com/products/BAY-73-4506.html viability was expressed as a percentage of control. LDH assay Cells were treated with different concentrations of resveratrol with or without 0. 5 ug ml LPS for 8 h. The supernatant was collected and LDH release was detected using a cytotox 96 nonradioactive cytotoxicity assay kit according to manufactures instructions. Cell viability was expressed as a percentage of control. NO assay Production of NO was determined by measuring the accumulated level of nitrite in the supernatant after 24 h of LPS treatment with or without different concentrations of resveratrol using a colori metric reaction with Griess reagent.

Briefly, 100 uL of supernatant were mixed with 100 uL Griess reagent. After incubation at room temperature Inhibitors,Modulators,Libraries in the dark for 10 min, total nitrites were measured spectrophotometrically at 540 nm. The concentration of nitrite in the sample was determined from a NaNO2 standard curve. Proinflammatory cytokine measurement by ELISA Microglial cells or astrocytes were seeded into 48 well plates and cultured for 24 h. Cells were then washed twice with DPBS, and incubated in serum free DMEM with or without different concen trations of resveratrol for 30 min followed by 0. 5 ug mL LPS for an additional 24 h. The supernatants were col lected for measurement of TNFa, IL 6, and MCP 1, and cell lysate were made for detecting IL 1b, using ELISA as described by the manufacturer.

Western immunoblotting N9 cells or murine primary astrocytes were grown in 60 mm dishes until subconfluency and then were cul tured overnight in medium in the absence of FBS. The cells were pretreated with different concentrations of Inhibitors,Modulators,Libraries resveratrol for 1 h followed by LPS for different times, then were lysed with cold Inhibitors,Modulators,Libraries lysis buffer as described previously. Cell lysate proteins were electrophoresed on a 10% SDS PAGE gel, and transferred onto polyvinylidene Inhibitors,Modulators,Libraries difluoride membranes. The membranes were blocked with 5% nonfat milk, and then were incubated with anti phosphorylated ERK1 2, p38 or JNK antibody overnight at 4 C. After incubation with an HRP conjugated secondary antibody, the protein bands were detected with a Supersignal West Pico che miluminescenct substrate and X Omat BT film.

For detection of total ERK1 2, p38, Inhibitors,Modulators,Libraries or JNK, the membranes were stripped with Restore Western Blot Stripping Buffer, followed by incubation with specific antibodies. Immunoblot selleck kinase inhibitor results were quantified using Gel Pro Analyzer software. Transient transfection and NF B luciferase reporter assay One day before transfection, murine primary microglial cells or astrocytes were seeded into 24 well plates. Transient transfection of pNF B Luc plasmid and control vector was performed using Lipofectamin 2000 according to the man ufactures recommendations.