Primary Culture of Mouse Astrocytes

Primary culture of mouse cerebral cortex astrocytes was prepared as previously described^{44 (link),57} . Pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1, mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank's balanced salt solution (Wako) for 30 min with continuous shaking at 37 °C. Cells were resuspended in an astrocyte culture medium (high-glucose Dulbecco's Modified Eagle Medium, 10% heat-inactivated fetal bovine serum, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37 °C in a CO2 incubator. On day 3 in vitro (DIV3), the astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako) and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.