Primary Culture of Mouse Cerebellar Astrocytes

Primary culture of mouse cerebellar astrocytes (25 (link), 26 (link)) was prepared using C57BL/6 pregnant mice (Japan SLC). Cerebella from postnatal day one of pups were dissected then digested in Hank’s balanced salt solution (Wako) containing 2.5% trypsin (Wako). Cerebellar tissue was incubated at 37°C for 30 min with continuous shaking. Mixed cerebellar tissue was centrifuge at 3500 rpm for 15 minutes at 4°C, the supernatant was discarded, and cells were resuspended in an astrocyte culture medium (high-glucose DMEM (Wako), 10% heat-inactivated FBS, and 1% penicillin/streptomycin). Cells were counted and 10–15 × 10⁶ cells were plated on Collagen I coated 10–cm dishes (Iwaki, Japan), then incubated at 37°C in a CO₂ incubator. The astrocyte culture medium was replaced with PBS on day 3 in vitro (DIV3). Oligodendrocyte precursor cells were removed by shaken the dishes for 2–5 minutes. Discard the supernatant then replaced with a fresh astrocyte culture medium. On DIV7, astrocytes were harvested and then plated on Collagen I coated 6 or 24 well dishes (Iwaki). Cells were then used for co-culture studies.