BV2 Cell Virus Transfection

293 T cells were cultured in DMEM basal culture medium with 10% FBS and 1% penicillin–streptomycin in an incubator at 37 °C and 5% CO₂. The 293 T cells were transfected with the phBL-CMV-MCS-3flag-EF1-PURo plasmid vector for lentivirus packaging. The supernatant was collected, and the virus particles were concentrated. BV2 microglia were incubated in 2 ml antibiotic-free DMEM overnight. Then 1 ml of the diluted viral mixture was added and the cells were incubated at 37 ℃ for 24 h. The medium was changed to FBS/DMEM 24 h later. The lowest cell lethal concentration was screened by adding the drug, G418. The drug concentration gradient was set at 200 μg/ml, 300 μg/ml, 400 μg/ml, and 500 μg/ml. Between the third day and the eighth day after adding G418, numerous cells began to die. The surviving cells formed monoclonal cultures, which were cultured further.