Astrocyte Culture and Senescence Induction

Primary astrocyte was cultured as described previously [27 (link)]. The neonatal midbrain (P0-3) was trypsinized with 0.25% trypase at 37 °C for 10 min and then centrifuged for 5 min at 1000g centrifugation. The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium containing 10% fetal bovine serum (FBS, GIBCO, Gaithersburg, MD, USA) and plated onto T-75 flasks at 50,000 cells/cm². After 10 d, confluent mixed glial cultures were shaken at 220 rpm for 6 h at 37 °C to remove unwanted cell types (microglia, oligodendrocytes, neurons, and fibroblasts). The purity of astrocytes was > 95% as determined with GFAP immunocytochemistry. To induce premature senescence model, astrocytes were pretreated with metformin at the indicated concentration for 1 h and then stimulated with MPP⁺ (200 μM, Sigma, St. Louis, MO, USA) for 24 h or α-synuclein aggregate (α-Syn PFF, 1 μg/mL, ab218819, abcam, USA) for 48 h. To induce naturally senescence model, astrocytes were cultured for 40 days and then treated with metformin for 10 days in vitro.