Primary cortical mixed culture
Xiqun Chen, Qing Ye
Abstract
To obtain cortical culture, pregnant mice were anesthetized, embryos were dissected, and cortex was collected in PBS. Tissues were incubated in 0.25% trypsin–EDTA at 37°C for 15 min. Trypsinized tissue was transferred into a high-glucose DMEM/F12 medium supplemented with 10% FBS. After centrifugation (1500rpm, 5 min), the pellet was resuspended. Cells were plated onto poly(L-lysine)-coated 24-well plates at 106cells per well and cultured in NB-A.
Steps
For primary cortical mixed culture - Use C57BL/6J mice at embryonic day 17
Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex.
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
Collect the cortices in PBS in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) On ice 5mL ) On ice
Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37°C for 0h 15m 0s Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
Centrifuge the dissociated cortices (1500rpm,0h 0m 0s, 0h 5m 0s) and resuspend the pellet in 10ml
medium supplemented with 10%
Seed the cells onto poly(L-lysine)-coated 24-well plates at a cell density of 1x106cells/well containing specialized
After 5 days of culture, these cells are ready for further experiment
