Dopaminergic NSC
| Title | Dopaminergic Neuron Differentiation |
| Date Submitted | May 5, 2012 |
| Submitted by - | Efthymiou, Anastasia - anastasia.efthymiou@nih.gov |
| Adapted from - | Gibco Protocol |
| Contributors - | Efthymiou, Anastasia |
| Affiliation(s) - | NIH CRM - NIAMS – Laboratory of Stem Cell Biology |
Introduction:
Dopaminergic neurons expressing Tuj1 (green) and dopamineric neuron marker TH (red). - taken from ReproCELL, 2012.
Protocol:
- Coat the surface of the culture vessel (with or without cover slips) with poly-L-ornithine working solution at 20 ug/mL in distilled water (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish) and incubate the vessel overnight at room temperature.
- Wash the poly-L-ornithine-coated vessel 4 times with distilled water, and then coat it with laminin working solution at 10 ug/mL in D-PBS without calcium or magnesium (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish). Incubate the culture vessel for 3 hours at 37 C. Note: You may coat the culture vessels in advance, replace the laminin solution with D-PBS without calcium or magnesium, and store them wrapped tightly in Parafilm for up to 1 week. Make sure that the culture vessels do not dry out.
- After the neurospheres float in neural expansion medium for 6-8 days, transfer them into a 15-mL tube and centrifuge for 5 minutes at 200 × g.
- Aspirate the supernatant and incubate the neurospheres in pre-warmed StemPro Accutase Cell Dissociation Reagent for 10 minutes at 37 C.
- Gently pipet the cell clumps up and down to break the larger clumps into a single cell suspension.
- Centrifuge the cells for 5 minutes at 200 × g and aspirate the supernatant.
- Resuspend the cells in 10 mL of pre-warmed neural differentiation medium.
- Repeat steps 6 and 7.
- Aspirate the laminin from the coated culture vessels and plate the dissociated DA progenitors.
- Incubate the cells in a 37 C incubator with a humidified atmosphere of 5% CO2 and replace the spent medium with fresh neural differentiation medium every other day.
- You can evaluate DA neuron differentiation 3-4 weeks after plating.
Materials:
Poly-L-ornithine coated culture vessel
Distilled water
Laminin
D-PBS
Neural expansion medium
StemPro Accutase Cell Dissociation Reagent
Neural expansion medium
Neural differentiation medium
Neural Expansion Medium
| Component | Amount |
| D-MEM/F-12 | 96 mL |
| N-2 Supplement | 1 mL |
| B-27® Supplement | 2 mL |
| NEAA | 1 mL |
| Basic FGF Solution | 200 µL |
| Heparin Solution | 100 µL |
| Component | Amount |
| Neurobasal® Medium | 96 mL |
| L-Glutamine | 1 mL |
| B-27® Supplement | 2 mL |
| NEAA | 1 mL |
| GDNF Solution* | 100 µL |
| BDNF Solution* | 100 µL |
| Ascorbic Acid Solution* | 100 µL |
| dcAMP Solution* | 100 µM |
*Add GDNF, BDNF, ascorbic acid, and dcAMP at the time of medium change
