2.5 Primary astrocyte culture
The primary astrocytes were extracted according to our previous study with some modifications (Xu et al., 2022). In this study, wild-type (WT) mice and Astro-Lf mice were employed to obtain the primary astrocytes. Briefly, the newborn mice were sacrificed, and the cortexes were isolated and minced with forceps. Cortical tissues were further incubated with DMEM containing 0.25% trypsin-EDTA for 15 min at 37 °C. Dissociated cells were centrifuged at 500 g for 5 min and resuspended in DMEM containing 10% FBS and 1% penicillin/streptomycin. Cells were seeded into 75 cm2 culture flasks and grown at 37°C in a 5% CO2 incubator with a change of medium twice a week. After cell confluence, flasks were shaken in a rotator at 220 rpm for 6 h to purify astrocytes. Cells were treated with 10 μM cytosine arabinoside (Sigma, C1768) for 48 h to prevent the proliferation of other cell types, and the medium was replaced with DMEM containing 3% FBS and 0.15 mM dibutyryl cAMP (Sigma, D0267) to induce differentiation. Cells with GFAP-positive >95% were used for the following study. After the treatment of dibutyryl cAMP for 3 days, the medium was removed and changed to 1% FBS medium to further culture for 48 h. The medium was collected as the astrocytic conditional medium to treat the N2a-sw cells for 24 h or to enrich the medium proteins with acetone to detect the secreted Lf, and astrocytes were also collected for the western blot analysis.