Previously, we have also shown that the debris resulting from Enterobacteriaceae lysed by trimethoprim could be imaged on PAO

g 10% FCS at 37uC for 24 h.Cells were incubated with an anti-BrdU monoclonal antibody, followed by a fluorescein-coupled goat anti-mouse Ig and Hoechst33324. To determine the percentages “9184477 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 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 A-83-01 manufacturer 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 chemiluminescent substrate . Several different exposure times were used for each blot to ensure linearity of band intensities. Immunoreactive bands were quantified using the ImageJ software. The relative expression of each protein was normalized to the intensity of b-actin. The expression level of each protein is reported as a Characterization of MeCP2-Deficient Astrocytes ratio relative to the level of normalized expression in a control sample. Glutamate Clearance Assay To measure extracellular glutamate concentrations, we used the Glutamate Assay Kit colorimetric assay . Assays were carried out in six independent trials. The clearance ratio of Glu was calculated from the Glu concentration in the medium sample ” of the drugtreated astroglial cells and the control non-drug treated glial cells. This is represented mathematically as follows: Glu clearance ratio = /. Threo-beta-benzyloxyaspartate, UCPH-101 -7–5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile), or dihydrokainate were applied to astroglial cells 60 min before Glu. treated with 0.011.0 mM Glu for 12 h, and subsequently analyzed for expression of GS and EAAT1 by western blot analysis. Statistic