Primary cultured astrocytes of mice cortices (WT or TLR4-KO) from newborn pups were prepared and characterized as previously described (Minana et al., 2000). Cells were plated on 60-mm diameter plates in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Invitrogen Corporation, Barcelona) containing 20% FBS, supplemented with L-glutamine (1%), glucose (1%), fungizone (1%), and antibiotics (1%). Cultures were grown in a humidified atmosphere of 5% CO2/95% air at 37°C. After 1 week of culture, FBS was reduced to 10%, and the medium was changed twice a week. Cells were grown to confluence and were used after 12 days in culture. The purity of astrocytes was assessed by immunofluorescence using: anti-glial fibrillary acidic protein (anti-GFAP, astrocyte marker, Sigma-Aldrich, Madrid, Spain), anti-CD11b (microglial marker, Serotec, Bionova, Madrid, Spain), anti-myelin basic protein (MBP, olygodendroglial marker, Sigma–Aldrich, Madrid, Spain) and anti-microtubule-associated protein 2 (MAP-2, neuronal marker, Sigma–Aldrich, Madrid, Spain). Astrocyte cultures were found to be at least 98% GFAP-positive and 2% CD11b-positive.
