Rat primary cortical astrocytes

Rat primary cortical astrocytes (Invitrogen, Poole, UK) were cultured routinely in DMEM supplemented with 20% (v/v) fetal bovine serum (Invitrogen), 1% penicillin/streptomycin and 1% glutamine in humidified 5% (v/v) CO₂ in air at 37°C. Primary human cortical astrocytes (Sciencell, Cat. No. 1800) were plated on 75 cm² culture flasks (Thermo Scientific Nunc Cell Culture Treated EasYFlasks) and cultured routinely in Astrocyte Medium (Sciencell; 1801) containing 1% fetal bovine serum (FBS, Sciencell Cat. No. 0010), 5 mL astrocyte growth supplement (AGS, Sciencell Cat. No. 1852) and 5 mL penicillin/streptomycin solution (P/S, Sciencell Cat. No. 0503). The cells were then incubated either in humidified 5% (v/v) CO₂ in air at 37°C for the normoxia work or in a controlled hypoxic atmosphere using a Coylab Hypoxia Chamber Glove Box, with a humidified airtight apparatus with inflow and outflow valves (into which a mixture of 90% N₂, 5% O₂ and 5% CO₂ was flushed) for the hypoxia-related experiments. HEK293 cells were cultured routinely in DMEM supplemented with 10% (v/v) fetal bovine serum (Invitrogen). HEK293 cells were transiently transfected when approximately 70% confluent, using 12 μg polyethyleneimine (branched, average M_r = 25,000, Sigma-Aldrich, 408727) and 2 μg plasmid DNA in 35 mm dishes.