Isolation and Culture of Astrocytes from APOE Mice

Primary astrocytes were isolated from postnatal day 0–4 pups of mice homozygous for E3 or Ε4. The brain was surgically excised and meninges were removed from cortical tissue in cold DMEM. Tissue from pups of the same genotype was pooled and coarsely chopped to encourage suspension. Tissue homogenates were incubated in serum free DMEM with 0.25% trypsin and DNAse for 30 min with gentle shaking. Cell suspension was then filtered through 40 μm strainer and spun for 5 min at 1100 x g. Suspended primary cells were then plated in a poly-lysine coated plate and allowed to grow to confluence in Advanced DMEM (Gibco) with 10% FBS. Immortalized astrocytes were derived from targeted replacement mice expressing human APOE alleles (kind gift from Dr. David Holtzman). These immortalized cell lines secrete human ApoE in HDL-like particles at equivalent levels to primary astrocytes from targeted replacement APOE knock-in mice and have been relied upon for studies of APOE’s role in astrocyte metabolism by several groups [39 (link)–41 (link)]. Cells were maintained in Advanced DMEM (Gibco) supplemented with 1 mM sodium pyruvate, 1X Geneticin, and 10% fetal bovine serum unless otherwise noted.