Primary culture and identification of brain neurons in Chinese perch (Siniperca chuatsi)

The diversity of species makes the research at cell level of Chinese perch extremely limited. Establishing a simple and feasible method for primary culture of brain neurons from Chinese perch (Siniperca chuatsi) in vitro is beneficial to further study on fish nervous system. Our laboratory combined common methods of cell culture, Chinese perch brain neruons (born 3 months) were isolated by collagenase digestion and mechanical blow. L15+20%FBS suspended brain neruons were inoculated in the cell culture vessels, at 28 °C without CO₂ incubator. The medium was replaced after 3 days. Subculture was carried out after the cells covered the bottom of the cell culture vessels. The morphological changes of the neurons were observed under inverted phase-contrast microscope; Immunofluorescence staining for NeuN or β-tubulin was performed to identify the purity of neurons. The results showed that the cells began to adhere to the culture bottle and develop small neurites and form network gradually after primary culture for 2 days. On the 5^th day, many neurites extended to form dense network and Soma of neurons became well. Fluorescence staining with Neu-N or β-tubulin showed that the purity of neurons can reach above 95%. The present protocol is a simple and efficient method for culturing brain neurons of Chinese perch with high purity, which is of great significance for the further study of fish growth and development, expression regulation of various receptors and proteins, cell apoptosis and cell signal transduction.