Mar 21, 2023
Differentiation NPCs to Dopaminergic/Midbrain Neurons
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi 2023. Differentiation NPCs to Dopaminergic/Midbrain Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn216qg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 20, 2023
Last Modified: March 21, 2023
Protocol Integer ID: 79099
Keywords: differentiation, dxn, NPC, Dopaminergic, Midbrain, Neurons, ASAPCRN,
Abstract
This protocol details methods for differentiation of NPCs to Dopaminergic/Midbrain Neurons.
Guidelines
Materials
Materials
Matrigel (Corning, #354230)
DMEM/F12 without HEPES (Gibco #11320033)
Accutase (Sigma Aldrich, #A6964-100 ml)
Propagation/NPC-Medium
ApoI (Selleckchem, #S1049)
Erhaltung/NPC-Medium
Differentiation Medium
Accumax (Gibco, #00-4666-56)
Maturation Medium
0.5µM PMA (Merck#540220-5MG)
Equipment
Centrifuge
37°C Incubator
Neubauer counting chamber
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Day -2: split NPCs
Day -2: split NPCs
35m
35m
Coat numbers of wells you need on a well-plate with Matrigel:
Note
Matrigel (Corning, #354230)
Dilute 4 °C aliquot 1/10 in DMEM/F12 without HEPES (Gibco, #11320033)
-> there is always one aliquot thawed in 4 °C . More aliquots are in -20 °C .
Keep Matrigel cool on ice!
Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES.
Note
1ml diluted Matrigel is enough for one complete plate. Only rinsing the wells!
Incubate 00:30:00 37 °C in incubator.
30m
Remove old medium and add 500 µL Accutase (Sigma Aldrich, #A6964-100 ml) into one well of a 6well-plate or 250 µL Accutase for well of a 12 well-plate.
Incubate 00:05:00 37 °C in incubator.
5m
Dilute and inactivate Accutase with 1 mL DMEM/F12 without HEPES (Gibco #11320033) and collect in 15ml Falcon containing 3 mL DMEM/F12 without HEPES . Centrifuge 1100 rpm, 00:05:00 .
Remove supernatant and resuspend pellet in 1 mL Propagation/NPC-Medium + ApoI (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 1.5 mL Propagation/NPC-Medium + ApoI (1/1000, Selleckchem, #S1049).
Day 0: Start Differentiation
Day 0: Start Differentiation
Remove old Erhaltung/NPC-Medium and add Differentiation-Medium.
Change Medium every 48:00:00 .
2d
Day 6
Day 6
5m
5m
Coat numbers of wells you need on a well-plate with Matrigel:
Note
Matrigel (Corning, #354230)
Dilute 4 °C aliquot 1/10 in DMEM/F12 without HEPES (Gibco, #11320033)
-> there is always one aliquot thawed in 4 °C . More aliquots are in -20 °C . Keep Matrigel cool on ice!
Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES.
Note
1ml diluted Matrigel is enough for one complete plate. Only rinsing the wells!
Incubate 00:30:00 37 °C in incubator.
Remove old medium and add 500 µL Accumax (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 00:05:00 37 °C in incubator.
5m
Dilute and inactivate Accumax with 1 mL DMEM/F12 without HEPES (Gibco #11320033) and collect in 15ml Falcon containing 3 mL DMEM/F12 without HEPES . Centrifuge 1100 rpm, 00:05:00 .
Remove supernatant and resuspend pellet in 1 mL Differentiation-Medium + ApoI (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 1.5 mL Differentiation-Medium + ApoI (1/1000, Selleckchem, #S1049).
Day 8: Start Maturation
Day 8: Start Maturation
Remove old Differentiation-Medium and add Maturation-Medium + 0.5uM PMA (1:2000, Merck #540220-5MG).
Day 10: Medium change
Day 10: Medium change
Change Medium to Maturation (without PMA!).
Change Medium every 48:00:00 .
2d
Day 14: Final Splitting
Day 14: Final Splitting
5m
5m
Coat numbers of wells you need on a well-plate with Matrigel:
Note
Matrigel (Corning, #354230)
Dilute 4 °C aliquot 1/10 in DMEM/F12 without HEPES (Gibco, #11320033)
-> there is always one aliquot thawed in 4 °C . More aliquots are in -20 °C . Keep Matrigel cool on ice!
Dilute thawed Matrigel out of 4 °C 1/10 in DMEM/F12 without HEPES.
Note
1ml diluted Matrigel is enough for one complete plate. Only rinsing the wells!
Incubate 00:30:00 37 °C in incubator.
Remove old medium and add 500 µL Accumax (Gibco, #00-4666-56) into one well of a 6well-plate. Incubate 00:05:00 37 °C in incubator.
5m
Dilute and inactivate Accumax with 1 mL DMEM/F12 without HEPES (Gibco #11320033) and collect in 15ml Falcon containing 3 mL DMEM/F12 without HEPES . Centrifuge 1100 rpm, 00:05:00 .
Remove supernatant and resuspend pellet in 1 mL Maturation-Medium + ApoI (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 1.5 mL Maturation-Medium + ApoI (1/1000, Selleckchem, #S1049).
Day >21:
Day >21:
After Day 21, cells are ready.