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.