Mouse Brain Cell Culture Protocol

Whole brains from 3-d-old C57BL/6 mice were minced and mechanically disrupted using a nylon mesh. The cells obtained were seeded in culture flasks containing DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin and grown at 37°C in a 5% CO₂ atmosphere. The culture medium was changed initially after 5 d and then every 3 d. Cells were used after 14–21 d of culture. To check the cell-specific population, MGC were immunostained with anti-GFAP (mouse IgG, 1:1,000; BD Biosciences), anti-Iba-1 (rabbit IgG, 1:1,000; Wako) or anti-Olig2 (goat IgG, 1:500; R&D systems) antibodies for astrocytes, microglia, and oligodendrocytes, respectively. This was followed by incubation for 2 h at room temperature with the following fluorescence-conjugated secondary antibodies: FITC-conjugated anti-mouse (donkey IgG, 1:500; Jackson Immuno Research Laboratories), Cy3-conjugated anti-goat (donkey IgG, 1:500; Jackson Immuno Research Laboratories), or Cy5-conjugated anti-goat (donkey IgG, 1:500; Jackson Immuno Research Laboratories). DAPI was used for counterstaining (blue). Pure astrocyte cultures were prepared from MGCs by shaking overnight. Pure microglia cultures were obtained from MGCs by mild trypsinization (Saura et al, 2003 (link)). The BV-2 immortalized mouse microglial cell line was maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.