Collagen 1

A primary culture of mouse cerebellar astrocytes was prepared as previously described [51 (link),52 (link)] with slight modifications. Briefly, P1 mouse cerebella were dissected and digested with 2.5% trypsin (Wako, Osaka, Japan) in HBBS (Wako) for 30 min with continued shaking at 37 °C. The cells were resuspended in an astrocyte culture medium (DMEM high-glucose, 10% heat-inactivated FBS, 1% penicillin/streptomycin), and 10–15 million cells were plated on 10 cm dishes coated with Collagen-I (Iwaki, Tokyo, Japan). The cells were incubated at 37 °C in the CO₂ incubator. On DIV3, astrocyte culture medium was replaced with PBS. Dishes were then shaken by hand for 0.5–1 min until only the adherent monolayer of astrocytes was left. PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 with 0.25% trypsin 1 mmol/L Na.EDTA (Wako), and then plated on 12- or 24-well dishes. The quality of primary culture of cerebellar astrocyte was examined by immunocytochemistry with astrocyte marker including S100β and GFAP (Supplementary Figure S3). The cells were used for cell proliferation, invasion or F-actin activity assays.

The animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee, Gunma University (19-024, 17 December 2018), and all efforts were made to minimize animal suffering and the number of animals used.
A primary culture of mouse cerebral cortex astrocytes was prepared as previously described (29 (link), 30 (link)) with slight modifications. A 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 continued shaking at 37°C. Cells were resuspended in an astrocyte culture medium (high-glucose DMEM, 10% heat-inactivated FBS, 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), 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.

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.