The cortices of mice at postnatal 2–5 days (P2–P5) were isolated and sheared into pieces, and digested by 0.25% Trypsin in Ca2+Mg2+-free PBS for 30 min at 37 °C. Trypsinized tissues were triturated and passed through 70 μm mesh. Dissociated cells were collected and washed by DMEM medium once before plating into a poly-l-lysine-coated dish (Φ100 mm) with 10 ml DMEM supplemented with 10% FBS. Culturing media were replaced completely with fresh DMEM plus 10% FBS the next day to remove the dead cells. Remained cells were cultured until reaching 100% confluence with media changed every 2–3 days. Primarily cultured astrocytes could be passaged once at 1 to 3 ratios, and the next generation of astrocytes could be collected when reaching the 100% confluence and frozen into liquid nitrogen. Over 95% of cells in the cultures were positive for GFAP and Acsbg1. When using for measurement of cell growth rate, astrocytes were seeded into a 24-well plate at 5×103 cells per well. Astrocytes in one of the 24 wells were trypsinized and collected every 3 days to count the cell number by hemocytometer. Averaged numbers from three independent experiments were obtained for each time point.