An improved method for primary culture of rat cortical neuron and cell identification / 北京大学学报(医学版)

ABSTRACT

Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27.On the third day,10 ?mol/L cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.Half of the culture medium was changed every week.The morphological changes of neuron cells were observed by light microscope.Double immuno-staining of microtubule-associated protein 2(MAP2) and karyon were applied to assess the culture purity.Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre-and postsynaptic protein markers.Results:The improved method could remarkably increase the cell number and reduce neuronal damnification.The primary culture was characte-rized by high uniformity,purity,normal synapse formation and longtime livability.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.