2.2 Cell culture and oxygen–glucose deprivation and reperfusion model
Mouse hippocampal HT22 cells were obtained from ATCC (USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin (Gibco, USA) and 10% fetal bovine serum(Sijiqing, China)at 37℃ and 5% CO2 in a Thermo Fisher incubator. The culture medium was refreshed every 48 hours, and subculturing was performed when cell confluence reached approximately 80-90%. Only logarithmically growing cells were used for subsequent experiments.
To initiate oxygen-glucose deprivation (OGD), the medium should be removed and rinsed three times with phosphate-buffered saline (PBS). Place the cultured cells in a dedicated chamber containing CO2/N2 (5%/95%) at 37 °C and DMEM was replaced by glucose-free DMEM, which was pre-gassed with N2/ CO2 (95%/5%) to remove other gas. According to experimental design, after a while, cells were removed from an anoxic chamber. After that, glucose-free DMEM was replaced by a neurobasal medium, which under normoxic conditions CO2/O2/N2 (5%/21%/74%) for 24h reoxygenation to generate the reperfusion injury. Control cells were cultured under normoxic conditions as part of routine culture procedures. At reoxygenation, 1.8mg/ml D-allose, which was soluble in PBS, was added to the DMEM medium.