To further confirm this study, BrdU incorporation assay was performed using Sema 3A treated SK-Mel-28 cells

ontaining 15% fetal calf serum by trituration though 10-ml plastic pipettes. Cells were pelleted at 1006g for 5 min, resuspended in Dulbecco’s modified Eagle’s “7901789 medium containing 15% FCS, and seeded into 100-mm culture dishes previously coated with poly-D-lysine. Upon reaching confluency, cells were trypsinized and replated. Cells were used after the third passage in all experiments, and were seeded at 36104 cells/cm2 in 6-well plate dishes or 35-mm culture dishes. MedChemExpress SB-590885 cultures were assayed by immunochemical analysis using antibodies against GFAP, MAP2, and CD11b in order to determine the degree of enrichment; the astrocyte cultures were nearly pure without contamination of microglia and neurons. Cell growth and bromo-29-deoxyuridine uptake assay To determine growth rate, cells were plated at 26105 cells/dish in 35-mm dishes. At each passage, three dishes per cell line were harvested by trypsinization, and cell numbers were determined using a hemocytometer. Growth rate was expressed as the number of harvested cells divided by the number of seeded cells. BrdU incorporation during DNA synthesis was determined using the 5-Bromo-29-deoxy-uridine Labeling and Detection Kit I. Cells were incubated with an anti-BrdU monoclonal antibody, followed by a fluorescein-coupled goat anti-mouse Ig and Hoechst33324. To determine the percentages of BrdU-positive cells, fluorescent images were obtained by a Biorevo BZ-9000 fluorescence microscope; images were analyzed using the BZ-II application. BrdU-positive cells and total cells were counted in random 3 fields per well. Results were obtained from four independent experiments. each gene were carefully determined by several preliminary experiments. The number of cycles for GFAP, S100b, EAAT1, EAAT2, and GS was 25, 32, 35, 32, and 25, respectively. The amplified cDNA was electrophoresed on 2% agarose gels containing ethidium bromide, and quantities were analyzed by densitometry using ImageJ software . The relative expression of each gene was normalized to the intensity of a housekeeping gene, hypoxantine-phosphoribosyltransferase. The expression level of each gene is reported as a ratio relative to the level of normalized expression in a control sample. Cell Viability Analysis Cell were seeded at 16104 cells per well in 96-well plates and incubated in D-MEM containing 15% FCS at 37uC for 24 h. In injury models of drug and oxidative stress, cells were incubated with 0.0110 mM glutamate for 24 h, 12.5200 mM NH4Cl for 4 h, or 0.1251.0 mM H2O2 for 1 h as previously described. After 24 h of drug treatment, cell viability was determined using the WST-8 assay . Immunocytochemistry Cultures were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.05% Triton-X 100 for 5 min. After blocking of nonspecific binding sites with 10% nonfat dry milk in PBS for 1 h, cultures were immunocytochemically stained using antibodies against MeCP2, b-tubulin type III, or glial fibrillary acidic protein , followed by secondary fluorescent antibodies as described previously. Cultures were “9357531 additionally stained with Hoechst33342 and examined using an Olympus IX-70 microscope. Photomicrographs were captured using an Olympus DP70 digital camera. PCR analysis Immunoblotting Cell extracts were prepared from astroglial cultures as described previously. Western blot analysis was performed using antiglutamine synthetase, anti-excitatory amino acid transporter 1, horseradish peroxidase-conjugated anti-rabbit IgG, and chemiluminescen