Dulbecco s modi ed eagle medium

Primary microglia cultures were isolated from 2-day-old Sprague-Dawley rat pups by shaking mixed glia cultures at 100 RPM for 1 hour at 37 °C yielding 90% purity as previously described (18). Cells were maintained at 37 °C and a humidi ed atmosphere augmented with 5% CO 2 in media containing Dulbecco's modi ed Eagle medium (Gibco), 1% l-glutamine (Gibco), 1% sodium pyruvate (Gibco), 10% FBS (Hyclone), and 1% penicillin and streptomycin (Thermo Fisher Scienti c). Media replacement occurred every 3-5 days.

The human OS SJSA1 cells of logarithmic growth phase were seeded in the 10 cm petri dish. When the cells acquired up to 70%~80% con uency, we added nutlin3a at the nal concentration of 5 µM. Two hours later, the media containing nutlin3a were discarded, then live cells were transferred into a new culture ask with the nutlin3a-free medium. When the cells reached up to 70% ~ 80% con uency, the above steps were repeated with 10 µM nutlin3a ( nal concentration) until cell mortality was less than 5%. The resulting resistant cell line was named NR-SJSA1.
Parent SJSA1 cells and nutlin3a-resistant NR-SJSA1 cells were grown in DMEM (Dulbecco's Modi ed Eagle Medium; Gibco, USA) and 10% FBS (fetal bovine serum; Gibco, USA) at 37°C and 5% CO 2 in a humidi ed incubator. In terms of the manufacturer's instructions, Lipofectamine 8000 (Beyotime, China) was used to transfect miR-206 inhibitor, miR-206 mimic, and siSLC7A11 (Genepharma, China). After constructing the pSEH-361-siSNHG14 retroviral vector, it was packaged by retrovirus and infected into NR-SJSA1 cells to obtain lncRNA SNHG14 knockdown cells, which were named siSNHG14 cells.

BV-2 microglial cells (Kunming Cell Bank, Chinese Academy of Sciences) were cultured in Dulbecco's modi ed eagle medium (Gibco, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scienti c) and 1% antibiotics (TransGen Biotech, Beijing, China) in a humidi ed incubator under 5% CO 2 at 37℃. Cells were split at 70%-80% con uence. OGD was induced to mimic ischemic conditions in vitro [16] . Brie y, cells were cultured in glucose-free Dulbecco's modi ed eagle medium, and ushed with 95% N2/5% CO2 gas mixture at a ow rate of 4 l/min for 10 minutes to create an anaerobic condition. A gas analyzer (Coy Laboratory, MI, USA) was used to monitor the anaerobic condition. Cells were then transferred to normal culture medium under 5% CO2 for reoxygenation. Our previous study showed that the regimen of OGD for 4 hours and reoxygenation for 24 hours was able to induce pivotal signaling events in cells without causing excessive death [16] . Control cells were treated without OGD.