Penicillin streptomycin reagent

Human umbilical vein endothelial cells (HUVECs), the human glioblastoma cell lines U87 and U251, and the human microglial cell line HMC3 were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin reagent (Gibco) and incubated at 37°C in a humidified atmosphere with 5% CO
₂. The medium was refreshed every two days. U87, U251, and HMC3 cells with the same number were cultured in serum-free medium for 24 h. Then, the cell supernatant was collected as conditioned medium for the experiment of the human cytokine antibody array.

Primary astrocyte cultures were prepared from 1-day-old Sprague-Dawley rat brains (Animal Experiment Center, Southern Medical University, Guangzhou, China), as previously described (11 (link),12 (link)). Ethics approval was granted by the Medical Ethics Committee of Southern Medical University, Guangzhou, China. Briefly, a total of 12 rats (n=3/group) were deeply anesthetized with a mixture of ketamine and xylazine (70:6 mg/kg, intramuscular injection) and then decapitated, the striatums were dissected, and the meninges were removed. The cerebral hemisphere was freed of the meninges and cut into 1 mm³ cubes in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Fresno, CA, USA). The tissue was dissociated by vortex mixing for 60 sec, and the cell suspension was passed through 70 and 20 µm sterile mesh nylon filters. A volume of cell suspension containing approximately 4.5×10⁵ cells was seeded in a 35 mm² Falcon tissue culture dish (Corning, Albany, NY, USA). Primary astrocytes were cultured in DMEM containing 10% fetal bovine serum (FBS) and 0.15% penicillin-streptomycin reagent (both from Gibco Life Technologies) at 37°C with 5% CO₂. The medium was changed twice a week. Cultures of at least 4 weeks were used in the experiments.

MC3T3-E1 cells (subclone 14; American Tissue Culture Collection, Manassas, VA, USA) were cultured in α-minimal essential medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin reagent (Gibco). The cells were grown at 37°C in humidified air containing 5% CO₂ and fed with fresh medium every 3 days. MC3T3-E1 cells were seeded into six-well plates at a density of 1×10⁵ cells/well and grown to 95% confluence. MC3T3-E1 pre-osteoblasts were then induced by 10% fetal bovine serum osteogenic medium (α-minimal essential medium supplemented with 50 μmol/L ascorbic acid, 0.1 μmol/L dexamethasone, and 10 mmol/L β-glycerol phosphate). At the same time, we added BN (0.1 μM–10 μM) to the differentiation medium. The medium was changed every 3 days and the cells were maintained in differentiation medium for 14 days.