Isolation of microglial cells and production of subcultures
Brains from mouse pups (post-natal day 1) were mechanically dissociated as described before [33] and cells in suspension were plated in PEI-coated T-75 culture flasks (Corning) containing 12 ml of DMEM supplemented with 10% FCS and a penicillin/streptomycin cocktail. Under these conditions, the purification of microglial cells occurs spontaneously after 14-18 days of culture. When required, purified microglial cells were maintained for up to one more week in cell culture flasks by adding 2-3 ml of DMEM supplemented with antibiotics and 1% FCS, only. To produce subcultures, purified microglial cells were trypsinized with 0.05% trypsin during 5 min. After trypsin neutralisation with 10% FCS, cells were plated onto uncoated Nunc 48 plates, at a density of 100,000 cells per well, using DMEM supplemented with antibiotics and 1% FCS as maintenance medium.