TITLE: Differentiation	NPCs	to Dopaminergic/Midbrain Neurons
AUTHORS: michela.deleidi

[DESCRIPTION]
This protocol details methods for differentiation of NPCs to Dopaminergic/Midbrain Neurons. 

[STEPS]
SECTION: Day	-2: split NPCs
1. Coat numbers of wells you need on	a well-plate with Matrigel: 
 

SECTION: Day	-2: split NPCs
1.1. Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES. 
 
	

SECTION: Day	-2: split NPCs
1.2. Incubate 30 min 37 °C in incubator.

SECTION: Day	-2: split NPCs
2. Remove	old medium and add 500 µL (Sigma Aldrich, #A6964-100 ml) into one well of a 6well-plate or 250 µL for well of a 12 well-plate. 

SECTION: Day	-2: split NPCs
4. Dilute and inactivate Accutase with 1 mL (Gibco #11320033) and collect in 15ml Falcon containing 3 mL. Centrifuge 1100 rpm, 5 min.

SECTION: Day	-2: split NPCs
5. Remove supernatant and resuspend pellet in 1 mL (1/1000, Selleckchem, #S1049).

SECTION: Day	-2: split NPCs
6. Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5 mL (1/1000, Selleckchem, #S1049).
  
SECTION: Day	0: Start Differentiation
7. Remove old Erhaltung/NPC-Medium and add Differentiation-Medium.
SECTION: Day	0: Start Differentiation
8. Change Medium every 2880 min.

SECTION: Day 6
9. Coat numbers of wells you need on	a well-plate with Matrigel: 
 

SECTION: Day 6
9.1. Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES. 
 
	

SECTION: Day 6
9.2. Incubate 30 min 37 °C in incubator.

SECTION: Day 6
10. Remove old medium and add 500 µL (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 5 min 37 °C in incubator.

SECTION: Day 6
11. Dilute and inactivate Accumax with 1 mL (Gibco #11320033) and collect in 15ml Falcon containing 3 mL. Centrifuge 1100 rpm, 5 min.

SECTION: Day 6
12. Remove supernatant and resuspend pellet in 1 mL (1/1000, Selleckchem, #S1049).

SECTION: Day 6
13. Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5 mL (1/1000, Selleckchem, #S1049).
  
SECTION: Day 8: Start Maturation
14. Remove old Differentiation-Medium and add Maturation-Medium + 0.5uM PMA (1:2000, Merck #540220-5MG).
SECTION: Day 10: Medium change
15. Change Medium to Maturation (without PMA!). 

SECTION: Day 14: Final Splitting
17. Coat numbers of wells you need on	a well-plate with Matrigel: 
 

SECTION: Day 14: Final Splitting
17.1. Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES. 
 
	

SECTION: Day 14: Final Splitting
17.2. Incubate 30 min 37 °C in incubator.

SECTION: Day 14: Final Splitting
18. Remove old medium and add 500 µL (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 5 min 37 °C in incubator.

SECTION: Day 14: Final Splitting
19. Dilute and inactivate Accumax with 1 mL (Gibco #11320033) and collect in 15ml Falcon containing 3 mL. Centrifuge 1100 rpm, 5 min.

SECTION: Day 14: Final Splitting
20. Remove supernatant and resuspend pellet in 1 mL (1/1000, Selleckchem, #S1049).
SECTION: Day 14: Final Splitting
21. Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5 mL (1/1000, Selleckchem, #S1049).
  
SECTION: Day &gt;21:
22. After Day 21, cells are ready.
SECTION: Day	-2: split NPCs
3. Incubate 5 min 37 °C	in incubator.
SECTION: Day 10: Medium change
16. Change Medium every 2880 min.