brought to a single cell suspension by repeated pipeting. The suspension was filtered sequentially through a cell strainer 70 and 40 mm Oligomycin A meshes, dissociated cells were seeded on poly D lysine coated 100 mm Petri dishes and incubated at 37 8C in humidified 5% CO2/95% air. The medium was replenished 1 day after plating and changed every 4 6 days. After plating, the cells were cultured for 13 15 days until confluence. Analysis of the cultures has shown that 70 75% were PS-341 Bortezomib GFAP positive. About 25 30% cells in cultures reacted with Ricinus Communis Agglutinin 1. No neurons were detected, as confirmed by immunostaining method, using monoclonal antibody against MAP 2. Rat microglial cultures were received by shaking the primary mixed glial cultures with maximum yields between day 12 and 14.
The suspension of floating cells was filtered through 40 mM nylon mesh, centrifuged at 1200 rpm for 10 min, suspended in 200 ml culture medium, placed into 96 well tissue culture plates and incubated Sitagliptin MK-0431 at 37 8C for 15 min in humidified 5% CO2/95% air. Afterwards, the wells were washed 4 times with 200 ml of culture medium to remove nonadhering cells. Microglia cells, which firmly adhered to the bottom of the well, were incubated overnight before the experiment. Compound C was dissolved in DMSO at an initial concentration of 20 mM. Further dilutions were performed in the appropriate medium. Corresponding amounts of DMSO were added to the control cultures. The final DMSO concentration in medium did not exceed 0.05% and as previously checked, did not show any effect on microglia cell cultures.
After compound’s application the medium were harvested, centrifuged buy Riluzole and assayed. Each group of experiments was made up of 9 wells, and repeated in 4 independent experiments. The cells destined for Western blot analysis were sieved onto plastic dishes of 100 mm in diameter at the density of 15 106/ dish. Viability was determined using 0.1% trypan blue and MTT. More than 96% of cultured cells reacted with Ricinus Communis Agglutinin l and 3 4% were GFAP positive. Each group of culture plates was repeated in three independent experiments. On the day of experiment the culture medium was replaced with a fresh medium containing AICAR, compound C and LPS.Cells in 96 well tissue culture plates treated with AICAR, compound C and LPS in various concentrations were determined by reaction with Ricinus Communis Agglutinin 1, a lectin that binds to the surface glycoproteins on microglial cells.
Under 20objective, 9 fields of 0.135 mm2 were photographed and counted for lectin positive cells per well. Cytotoxic effects of treatments were determined by measuring the microglial structure cultures membrane integrity with 0.1% trypan blue exclusion test.Cell viability of microglia treated with studied compounds was evaluated with MTT conversion method. The cells ability to convert MTT indicates mitochondrial integrity andactivity, which might in turn indicate cell viability. The cleavage of tetrazoline ring in MTT takes place mainly with the participation of the mitochondrial succinate dehydrogenase and depends on the activity of the respiratory chain and the redox state of the mitochondria. MTT was added to the medium 3 h before the scheduled end of the experiment and then the cultures were incubated .