Isolation and culture of neural cells

Neural cells were isolated as previously reported (27 (link)). Briefly, newborn (~24 h old) SD rats were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight) followed by decapitation, and were sterilized in 75% alcohol for 5 min. The craniums were cut opened along the midline to isolate the brain tissue. Isolated tissue was washed with HBSS to remove blood, soft meninges and vascular network. The dentate gyrus was then isolated, cut into pieces of 1-2 mm in size after washing three times with HBSS, homogenized, filtered through a 100 mesh filter and digested with equal volume of 0.25% trypsin at 37˚C for 20 min. Digestion was stopped by adding two volumes of trypsin inhibitor. Digested cells were pelleted by centrifugation at 112 x g for 5 min at room temperature and cultured in DMEM medium with 10% FBS in 5% CO₂ at 37˚C for three passages.