Sim a9

DRG cell lines (ND 7/23) and mouse microglial cells (SIM-A9) were obtained from Sigma-Aldrich (#92090903, USA) and Kerafast (END001, USA), respectively. The ND 7/23 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, #12100046; Gibco, USA) and the SIM-A9 cells were maintained in DMEM/F12 (#11320033; Gibco, USA) supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin-glutamate (Thermo Scientific, Utah, USA). Both cell lines were maintained in a 5% CO₂ incubator (humidified atmosphere) at 37 °C.

Murine BV-2 microglial cells and SIM-A9 (Kerafast, Boston, USA) were maintained and cultured referring to our previous study [15 (link)]. The BV2 cells were passaged in an uncoated 100 mm cell culture dish with DMEM/F-12 (Gibco, cat. #11320-033) containing 10% heat-inactivated FBS (Gibco, cat. #16000-044), 5% heat-inactivated horse serum (Invitrogen cat. #16050-122), and 1% penicillin/streptomycin (Gibco, cat. # 15140122). SIM-A9 cells were passaged in an uncoated 100 mm cell culture dish with DEME/F-12 (Gibco, cat. #11330-032) containing 10% heat-inactivated FBS (Gibco, cat, #16000-044), 5% heat-inactivated horse serum (Gibco, cat, # 16050-122), and 1% penicillin/streptomycin (Gibco, cat, #15140-122). Cells were cultured at 37 °C in an incubator with 5% CO₂.

NSCs were extracted from embryonic Sprague‒Dawley (SD) rats (E14). Telencephalons of each embryo were separated and cut into pieces under a microscope and then incubated with 0.25% trypsin solution (25200,072, Gibco, USA) at 37 °C for 5 min. Then, DMEM/F12 (11330,032, Gibco, USA) containing 10% FBS (044001A, Gibco, USA) was added to neutralize trypsin activity. The cell suspension was centrifuged and resuspended in proliferation medium (DMEM/F12 (11330032, Gibco, USA) containing 2% B27 (17504044, Gibco, USA), 1% N2 (17502001, Gibco, USA), 20 ng/ml bFGF (45033, PeproTech, USA), and 20 ng/mL EGF (31509, PeproTech, USA). Proliferation medium was applied to culture NSCs, and continuous passage culture was performed by incubating with Accutase (07920, STEMCELL, Canada) at 37 °C for 5 min to obtain single cells.
Murine microglial cells (SIM-A9) were purchased from the ATCC (CRL-3265, Manassas, VA, USA). SIM-A9 cells were cultured in DMEM/F12 medium (11330032, Gibco, USA) supplemented with 10% heat-inactivated foetal bovine serum (04-121-1A, BI, Israel) and 5% horse serum (04-004-1A, BI, Israel) under suitable conditions (37 °C, 5% CO₂). The cells were passaged every three days by incubating with PBS (containing 1 mM EDTA, 1 mM EGTA and 1 mg/mL glucose) for 12 min.