Differentiation NPCs to Dopaminergic/Midbrain Neurons
michela.deleidi
Abstract
This protocol details methods for differentiation of NPCs to Dopaminergic/Midbrain Neurons.
Steps
Day -2: split NPCs
Coat numbers of wells you need on a well-plate with Matrigel:
Dilute thawed Matrigel out of 4°C 1/10 in DMEM/F12 without HEPES.
Incubate 0h 30m 0s 37°C in incubator.
Remove old medium and add 500µL (Sigma Aldrich, #A6964-100 ml) into one well of a 6well-plate or 250µL for well of a 12 well-plate.
Incubate 0h 5m 0s 37°C in incubator.
Dilute and inactivate Accutase with 1mL (Gibco #11320033) and collect in 15ml Falcon containing 3mL. Centrifuge 1100rpm.
Remove supernatant and resuspend pellet in 1mL (1/1000, Selleckchem, #S1049).
Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5mL (1/1000, Selleckchem, #S1049).
Day 0: Start Differentiation
Remove old Erhaltung/NPC-Medium and add Differentiation-Medium .
Change Medium every 48h 0m 0s.
Day 6
Coat numbers of wells you need on a well-plate with Matrigel:
Dilute thawed Matrigel out of 4°C 1/10 in DMEM/F12 without HEPES.
Incubate 0h 30m 0s 37°C in incubator.
Remove old medium and add 500µL (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 0h 5m 0s 37°C in incubator.
Dilute and inactivate Accumax with 1mL (Gibco #11320033) and collect in 15ml Falcon containing 3mL. Centrifuge 1100rpm.
Remove supernatant and resuspend pellet in 1mL (1/1000, Selleckchem, #S1049).
Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5mL (1/1000, Selleckchem, #S1049).
Day 8: Start Maturation
Remove old Differentiation-Medium and add Maturation-Medium + 0.5uM PMA (1:2000, Merck #540220-5MG).
Day 10: Medium change
Change Medium to Maturation (without PMA!) .
Change Medium every 48h 0m 0s.
Day 14: Final Splitting
Coat numbers of wells you need on a well-plate with Matrigel:
Dilute thawed Matrigel out of 4°C 1/10 in DMEM/F12 without HEPES.
Incubate 0h 30m 0s 37°C in incubator.
Remove old medium and add 500µL (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 0h 5m 0s 37°C in incubator.
Dilute and inactivate Accumax with 1mL (Gibco #11320033) and collect in 15ml Falcon containing 3mL. Centrifuge 1100rpm.
Remove supernatant and resuspend pellet in 1mL (1/1000, Selleckchem, #S1049).
Count cells with Neubauer counting chamber (4big squares) and seed 1.000.000 cells in one matrigel-coated well of a 6well-plate. Use 1.5mL (1/1000, Selleckchem, #S1049).
Day >21:
After Day 21, cells are ready.
