Primary culture of neurons under low density conditions by immunomagnetic beads method combined with conditioned medium / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To establish a primary culture of neurons under low density conditions by immunomagnetic beads (1MB) based cell separation technology combined with astrocyte conditioned medium (A-CM). METHODS The cerebral hemispheres of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension for primary culture. After 10 d of culture, the microglia and other irrelevant cells were removed by constant temperature oscillation, while the purity of astrocytes was identified by immunofluorescence staining with anti-glial fibrillary acidic protein (GFAP) antibody. A-CM was prepared using cell cultures that met purity requirements. The cerebral cortex and hippocampus of neonatal KM mice within 24 h of birth were mechanically isolated and trypsin digested to prepare cell suspension. High-purity neurons were obtained by immunomagnetic beads based cell separation technology, and A-CM was applied to primary culture of neurons under low density conditions. Morphological parameters of neurons during culture were observed, and neurons were identified by immunofluorescence staining with anti-160 ku neurofilament antibody. RESULTS The purity of astrocytes was over 95% by cellular immunofluorescence staining identification, which met the cell purity requirements of subsequent experiments. Morphological observation and cellular immunofluorescence staining of the mouse cortex and hippocampal neurons showed that the neurons grew well under low density culture conditions with high purity, and the morphological characteristics were distinct at different developmental stages. CONCLUSION The primary culture of the mouse cerebral cortex and hippocampal neurons under low density conditions is successfully established.