2.17. Cortical neuron cultures and oxygen–glucose deprivation

Oxygen–glucose deprivation (OGD) was used as an in vitro model of cerebral ischaemia. Cortices from embryonic day 14 brains of C57BL/6J mice were dissected and suspended in Krebs buffer containing trypsin (Sigma‐Aldrich). After 15‐min incubation at 37°C, fresh buffer with DNAse was added. The content was gently mixed and centrifuged at 230 x g for 3 min. The cell pellet was resuspended into neurobasal/B27/l‐glutamine/gentamicin medium. The viable cells were plated on culture plates pre‐coated with poly‐d‐lysine at 200,000 cells per well. Seven‐day‐old cortical neurons were used to induce hypoxia by incubation in a hypoxia chamber for 3 hr at oxygen concentration of 1% without glucose. Jurkat T Cells were transfected with control (20 nM) or agomir‐494 (20 nM). After 2‐day cultivation, cells were stimulated with PHA (20 μM) for 24 hr. Then neurons were exposed to conditioned medium from Jurkat T cells for 6 hr. After 24‐hr incubation of normal medium, the cell injury was assessed by immunofluorescence staining and high‐content screening assay for TUNEL and neurite outgrowth.