The supernatant medium from the 24 h-treated astrocytes and lysates from the brain cortices were used for cytokine determinations. Brain tissue was homogenized in cold lysis buffer (1% NP-40, 20 mM Tris-HCl, pH 8, 130 mM NaCl, 10 mM NaF, 10 μg/ml aprotinin, 10 mg/ml leupeptin, 10 μM DTT, 1 mM Na3VO4 and 1 mM PMSF; 250 mg tissue/0.5 ml). Lysis samples were kept on ice for 30 min and were centrifuged at maximum speed for 15 min, and the supernatant was collected for protein and cytokines determination. Protein was determined by the Bradford Assay (Bio-Rad, Hercules, CA, USA). The cytokine levels of IL-18 and IL-1β were measured using the enzyme-linked immunosorbent assay (ELISA) kits (Bender MedSystems GmbH, Austria) following the manufacturer’s instructions.
