2.4. Isolation of neurons and glial cells
For primary mesencephalic neurons, refer to the previous method (Hanet al. , 2003). After the newborn C57BL/6 mice were anesthetized with isoflurane, the brain was taken out, and placed in cold DMEM culture medium, the midbrain was carefully cut off and the meninges and blood vessels were separated under stereological microscope, then digested into single cell suspension with 0.25% trypsin/PBS solution, precipitated and removed large impurities, and continued to be cultured in Neurobasal medium containing 2% B27. For primary microglia, after centrifuging the single cell suspension obtained by the above steps, use 10% FBS DMEM culture medium in the poly-L-lysine-coated culture plate, change the culture medium every 2 day, and continuously culture for 7-10d until the cells grow on the culture plate, until the microglia can be clearly seen attached to the surface of the underlying astrocytes under the microscope. Digest with 0.25% trypsin, observe that most microglia fall off under the microscope, terminate the digestion with culture medium, centrifuge the suspension containing microglia and culture it in DMEM medium containing 10% FBS for use. At the same time, identify the purity of microglia through Iba1 immunofluorescence to ensure that the proportion of Iba1+ cells exceed 95%. For primary astrocytes, GFAP immunofluorescence showed that the proportion of GFAP+astrocytes reached 90%.