Corti ces were harvested, while the meninges and blood ves sels were removed. Tissues were digested in 0. 25% trypsin containing the site 0. 1 M EDTA at 37 C for 15 min, and passed through a nylon sieve. The cells were seeded in Dulbeccos Inhibitors,Modulators,Libraries modified Eagles medium supplemented with heat inactivated 10% fetal calf serum, 50 ug ml penicillin, and 100 ug ml streptomycin. Cul tured cells were grown at 37 C in a humidified atmos phere with 5% CO2. After 10 days, the microglia and oligodendrocyte progenitors were depleted by shaking. The remaining astrocytes were then detached by trypsi nization and re plated at a density of approximately 1 �� 105 cells ml for future experiments. The purity of astro cytes was identified by immunohistochemical analysis with anti glial fibrillary acidic protein.
OGD reperfusion and 1400W treatment Inhibitors,Modulators,Libraries On the third day of subculture, astrocytes were sub jected to OGD with Earls balanced salt solution and incubated in a hypoxic in cubator filled with 1. 5% O2 and 5% CO2 for 8 h. The oxygen level in the OGD solution decreased to about 2% to 3% after 60 min in the hypoxic incubator. The cells were then provided with a normal amount of oxy gen and maintenance medium without glutamate Inhibitors,Modulators,Libraries to mimic in vivo reperfusion for up to 24 h. Normoxic con trol cells were incubated in 37 C with 5% CO2 and at mospheric air in a buffer almost identical to EBSS except containing 5. 5 mM glucose. iNOS inhibitor 1400W was prepared as a concentrated stock solution according to the manufacturers instructions. The final concentrations of 1400W in media applied to astrocytes were, 1, 10, and 50 uM.
1400W was added to culture medium 30 min prior to OGD exposure, and astrocytes were maintained in EBSS and Inhibitors,Modulators,Libraries maintenance medium dur ing the treatment. Measurement of NO level The concentration of NO in the culture medium was determined by the Griess reaction with minor changes. Inhibitors,Modulators,Libraries Briefly, 40 ul cell culture fluid, 10 ul NADPH, and 40 ul basal solution Rapamycin molecular weight were incubated in a 96 well microtiter plate for 45 min at room temperature. Next, 50 ul Griess reagent was added and the solution incu bated for 20 min in the dark at room temperature. Fi nally, the absorbance of the samples was measured at 540 nm. NO2 concentrations were calculated from a standard curve of sodium nitrite. Western blot Protein concentrations of cell lysates were determined by using the bicinchoninic acid method. Samples were loaded on 12% sodium dodecyl sulphate polyacrylamide gel for electrophoresis and then transferred to the PVDF mem brane. Membranes were blocked with 5% milk in TBS T buffer for 1 h and then incubated with pri mary antibodies for 16 h at 4 C, SOD1, iNOS, PDI. B actin was used as an internal control.