Objective: To improve the efficiency of primary culture of hippocampal neurons and obtain highly purified neurons with good in vitro growth and minimal risk of contamination.
Methods: The hippocampal neurons of neonatal Wistar rats were isolated and the single cell suspension was prepared by mechanical trituration and sedimentation in stead of trypsin digestion and filteration. Twenty-four hours after the cell plating, the culture medium was removed and replaced by serum-free DMEM/F12 with B27 supplementation. Half of the culture medium was changed 2-3 times every week. The morphological changes of the neurons were observed under inverted phase-contrast microscope. Immunofluorescence staining for NSE was performed to identify the neurons, and the purity of neurons was calculated. The hippocampal neurons were stained with calcium-sensitive fluorescent dye to monitor the effect of KCl on neuronal excitability by a calcium imaging system.
Results And Conclusion: This simplified method is time-saving and cost-effective for primary culture of hippocampal neurons with reduced risk of contamination, and the neurons obtained showed high uniformity, purity and long-term viability.
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