Isolation of primary astrocytes from neonatal mice

To harvest astrocytes from neonates (under 96 hours old) resulting from homozygous wild-type or BNIP3 null crosses. Neonates are removed from the facility live in a clean cage with bedding material and a small heating pad warmed to body temperature and wrapped in green cloths. Neonates are brought to a flow hood which is designated for the isolation and maintenance of primary cell cultures exclusively. Neonates are sacrificed immediately by decapitation with scissors. Whole brains were immediately removed and placed in ice-cold serum-free astrocyte culture medium. The cortices were transferred to a fresh dish of ice-cold serum-free culture media. Cortices from each litter were combined and chopped into small pieces (<1mm3) and then transferred to a 50 mL conical centrifuge tube with 15 mL of serum-free culture media. The tissue was mechanically dissociated by vortexing to achieve a single-cell suspension. Cells were seeded in astrocyte culture media with 10% FBS at a density of 6x105 cells/mL. “Mature” astrocyte cultures were tested for purity by immunofluorescence staining for the astrocyte-specific marker protein GFAP (glial fibrillary acidic protein). Primary mouse astrocytes were cultured in custom astrocyte-selective media as described above. After isolation in serum-free media, astrocytes were supplemented with 10% FBS. Vigorous swirling was used to detach non-astrocytic cells at the time of passaging and/or changing media.