And use of laboratory animals and were approved by the Italian Ministry of Health. Cortical glial cultures were obtained from the bark 1-3 day old Sprague-Dawley rats pr Parried. After isolation of the cortex and the removal of Hirnh Ute, the cells were caused by mechanical and enzymatic dissociation with trypsin-L Distributed solution in Hanks Balanced Salt Solution, pH 7.4. The cells were plated on 75 mm 2 flasks and cultured in DMEM erg Complements with 10% calf serum f Fetal K, Penicillin, streptomycin and 5% CO 2 for 14 and 37 DIV. Confluent cultures were shaken for 7 h at 37 to remove oligodendrocytes and microglia and a pure culture obtained from 90% astrocytes GFAP-F Judged staining. Astrocytes were used with a density of about 1 to 2105 and cells/cm2 when the corresponding plated reaching confluence. Cultures of pure cortical neurons from rat were obtained at embryonic day 15, prepared using a previously described method. In short, in Ca2/Mg2 cortices were free buffer, pH 7.4, dissected mechanically dissociated, and grown on ships or in multi-well-bo Your 35 mm with 0.1 mg / ml poly-D lysine coated. The cultures were grown in DMEM F12, erg complements with the following components: penicillin, streptomycin, BSA, glucose, insulin, transferrin, putrescine, glutamine, selenium, and progesterone. Cytoside arabinoside was added to reduce maintained for 18 h after plating on non-proliferation and the neuronal element for 72 h. Subsequent partial medium replacement was performed every 2 days.
After 7 DIV, cultures were processed for experiments. These conditions give a pure culture than by neuronal immunostaining Staining in 99% neuronal marker microtubule-associated protein 2 as previously stated specific analyzed by flow cytometry. Mixed cortical cultures containing both astrocytes and neurons were obtained from rats at embryonic day 17 and cultured on 0.1 mg / ml poly-D lysine Smad signaling pathway coated multiwell vessels. Cultures were maintained in minimal essential medium containing penicillin, streptomycin, glucose, 10% FCS, 10% horse serum, and glutamine. At 5 DIV FCS was removed from the middle and the cells were erg with 5 M cytoside arabinoside for 72 h Complements. Subsequent partial medium replacement was performed every 2 days. The culture were used for experiments at 14 DIV. Older plants contain about 40% of the neurons. Evaluation of neuronal death in cortical mixed cultures. A1 and A25 42 35 peptides were extracted from serum to mature mixed cortical cultures at 14 DIV applied. After 24 h, the neuronal toxicity T, which examined by light microscopy and quantitated by trypan blue-F Staining. Stained neurons were of three Feeder Llige fields / well gez Hlt. A variable number 80-300 dead neurons per field were hlt gez. All experiments were dinitroquinoxaline in the presence of glutamate receptor antagonists and dizocilpine maleate 2.3 6.7 dihydroxy to the toxicity t carried out to avoid of endogenous glutamate. Immunoblot analysis. Astrocytes and neurons were in lysis buffer Radioimmunpr Zipitation test with the addition of Triton X-100 and a mixture of protease and phosphatase inhibitor cocktail harvested. Transfected HEK293 cells were rinsed quickly in ice-cold PBS and solubilized in Triton X-100 lysis buffer. The proteins Were quantified by protein assay of Bradford.