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.