Protocol for the Differentiation of Mouse Neural Stem Cells into Neurons
GUIDELINE
The generation of the different neural lines originates in adult neural stem cells that can self-renew or differentiate into astrocytes, oligodendrocytes, or neurons in response to specific stimuli. The abilities of stem cells to self-renew and form different mature cells expand the possibilities of applications in cell-based therapies such as tissue re-composition in regenerative medicine, drug screening, and treatment of neurodegenerative diseases.
METHODS
- Place a 12-mm cell culture slide in each well of a 4-well plate, add 100 μg/mL of PDL 500 μL to each well, and leave overnight at room temperature.
- The PDL is aspirated, and the slides are rinsed with sterile water and left to dry at room temperature. 10 μg/mL of laminin 500 μL is added to each well, and the slides are allowed to stand overnight at 37°C and 5% CO2.
- Mouse neural stem cells are digested with 0.05% trypsin for 2 min and terminated with an equal volume of 10% FBS. Centrifugation is performed at 1000 rpm for 5 min, the supernatant is discarded and the cells are resuspended with Neuron M. The cells are inoculated at a cell density of 1×104 cells/ml in a four-well plate in which laminin is discarded.
- Neuron M is replaced in half quantities every 2 days, and by day 21, differentiation is complete, and neuronal cells with neurofilaments can be observed microscopically.
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NOTES
Do not expose cells to air at any time after they have differentiated into neurons.