一种高纯度原代神经元培养和高效率转染的方法.

BACKGROUND: Neuron culture and cell transfection technologies are important means to study the development and pathophysiologic mechanism of neurons in vitro, but the purity and transfection efficiency of primary cultured cortical neurons are poor. OBJECTIVE: To establish a simple, efficient and stable method of culture and transfection method of primary neurons in vitro. METHODS: Primary cortical neurons were harvested from neonatal 1-day rat brains under aseptic condition, which was digested with 0.25% trypsin prior to centrifugation and made into cell suspensions, followed by being seeded into Neurobasal-A medium (5×109 L-1 per pore). The morphological characteristics of neurons were observed by inverted microscope; two neuron-specific markers (MAP2 and Tuj1) were used for immunolabeling to identify the cultured cells; the transfection efficiency of neurons was tested by Lipofectamine 2000 and Block-iT Transfection Kit. RESULTS AND CONCLUSION: The neurons cultured in vitro exhibited interconnected networks after culture for 7 days. All the cultured neurons displayed MAP2 and Tuj1 immunoreactivity. The highly effective transfection was observed under fluorescence microscope using Lipofectamine 2000 transfected neurons. In summary, the culture method of primary cerebral cortex neurons can be adopted to obtain highly purified neurons. Besides, high transfection efficiency of primary neurons can be realized by artificial liposome. [ABSTRACT FROM AUTHOR]