Mar 19, 2024
Differentiation of WTC11 and KOLF2.1 iPSCs to dopaminergic neurons
- Nisha Mohd Rafiq1,
- Pietro De Camilli2,3,4,5
- 1University of Tuebingen;
- 2Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 3Department of Cell biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 4Program in Cellular Neuroscience, Neurodegeneration and Repair;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Protocol Citation: Nisha Mohd Rafiq, Pietro De Camilli 2024. Differentiation of WTC11 and KOLF2.1 iPSCs to dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp39m8vzp/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: September 18, 2023
Last Modified: March 19, 2024
Protocol Integer ID: 87937
Keywords: iPSC, Dopaminergic neurons, Protocol, Differentiation
Funders Acknowledgement:
ASAP
Abstract
This protocol describes the differentiation of iPSCs (WTC11 and KOLF2.1) to dopaminergic neurons according to Bressan et al 2021.
Materials
1 Reagent list
2-MercaptoethanolGibco - Thermo FischerCatalog #21985023
Accutase cell dissociation reagentGibco - Thermo FischerCatalog #A1110501
B27 supplement minus vitamin AGibco - Thermo FischerCatalog #12587010
BDNF (Brain-Derived Neurotrophic Factor)peprotechCatalog #450-02
CHIR99021R&D SystemsCatalog #4423
DAPTCayman Chemical CompanyCatalog #Cay13197
Db-cAMP (dibutyryl-cyclic AMP)Sigma AldrichCatalog #D0627
DMEM/F-12, HEPESGibco - Thermo FischerCatalog #31330095
DMSO (dimethyl sulfoxide)Sigma AldrichCatalog #D2650
DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190169
Essential 8 Flex complete medium (E8)Gibco - Thermo FischerCatalog #A2858501
Fibronectin (Fibro)CorningCatalog #356008
FGF-8b (Recombinant human/murine Fibroblast Growth Factor-8b)peprotechCatalog #100-25
GDNF (Glial cell line-Derived Neurotrophic Factor)peprotechCatalog #450-10
GlutaMAXGibco - Thermo FischerCatalog #35050038
Knockout DMEM/F-12Gibco - Thermo FischerCatalog #12660012
Knockout Serum Replacement (KSR)Gibco - Thermo FischerCatalog #10828028
Laminin (Lam)Sigma AldrichCatalog #L2020
L-ascorbic acid (AA)Sigma AldrichCatalog #A4403
LDN193189Cayman Chemical CompanyCatalog #Cay11802
Matrigel hESC-Qualified Matrix, LDEV-freeCorningCatalog #354277
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-freeCorningCatalog #356230
MEAA (MEM Non-Essential Amino Acids)Gibco - Thermo FischerCatalog #11140050
N2 supplementGibco - Thermo FischerCatalog #17502048
Neurobasal mediumGibco - Thermo FischerCatalog #21103049
Penicillin-StreptomycinGibco - Thermo FischerCatalog #15140122
Poly-L-Ornithine (PLO)Sigma AldrichCatalog #P3655
PurmorphamineCayman Chemical CompanyCatalog #Cay1000963410
HCl (Hydrochloric acid)Carl RothCatalog #9277
HSA (Human Serum Albumin)Sigma AldrichCatalog #A6784
SHH (recombinant human Sonic Hedgehog C24II N-Terminus)R&D SystemsCatalog #1845-SH
Synth-a-Freeze Cryopreservation MediumGibco - Thermo FischerCatalog #A1254201
TGFβ3 (recombinant human Transforming Growth Factor-beta 3)R&D SystemsCatalog #243-B3
Y-27632 dihydrochloride (Y)Cayman Chemical CompanyCatalog #Cay10005583
Supplement K (BrainXell)
1.1 Reagent preparation and storage
1.1.1 General instructions
- Warm reagents stored at-20 °C toRoom temperature before reconstitution.
- Reconstitute reagents under sterile conditions in a laminar flow hood following the instructions and dilution reagents below. Go to: Dilution of stock solutions.
- After reconstitution, aliquot stock solutions in sterile Safe-Lock tubes and store at-20 °C .
- Take note and control the expiration time of reagents after reconstitution.
Note: reagents in solution might have a shorter expiration time as lyophilized reagents.
- Once thawed, reconstituted reagents can be kept for up to 5 days at4 °C .
1.1.2 Protect from the light
- Db-cAMP
- L-ascorbic acid
- LDN193189
1.1.3 Minimize exposure to air
- L-ascorbic acid
1.1.4 Reconstitution of reagents
- BDNF: Reconstitute BDNF in 0.1% HSA/PBS to obtain a stock concentration of20 µg/mL .
- CHIR99021: Reconstitute CHIR99021 in DMSO to obtain a stock concentration of4 mM .
- DAPT: Reconstitute DAPT in DMSO to obtain a stock concentration of10 mM .
- Db-cAMP: Reconstitute db-cAMP in deionized sterile water to obtain a stock concentration of200 mM . Filter the stock solution with a 0.22 µm pore size hydrophilic PVDF membrane. Protect from the light.
- Fibronectin: Reconstitute fibronectin in deionized sterile water to obtain a stock concentration of1 mg/mL .
- FGF-8b: Reconstitute FGF-8b in 0.1% HSA/PBS to obtain a stock concentration of100 µg/mL .
- GDNF: Reconstitute GDNF in 0.1% HSA/PBS to obtain a stock concentration of20 µg/mL .
- Laminin: No reconstitution required. Aliquot LamininOn ice . Store aliquots at-20 °C .
- L-ascorbic acid: Reconstitute l-ascorbic acid in deionized sterile water to obtain a stock concentration of0.2 M . Minimize exposure to air. Protect from the light.
- LDN193189: Reconstitute LDN193189 in DMSO to obtain a stock concentration of500 µM . Protect from the light.
- Matrigel: No reconstitution required. Aliquot MatrigelOn ice . Store aliquots at-80 °C . Dilute Matrigel matrix with ice-cold DMEM/F-12 medium for coating plates.
- Poly-L-Ornithine: Reconstitute poly-l-ornithine in PBS to obtain a stock concentration of10 mg/mL . Filter the stock solution with a 0.22 µm pore size hydrophilic PVDF membrane.
- Purmorphamine: Reconstitute purmorphamine in DMSO to obtain a stock concentration of2 mM .
- SHH: Reconstitute SHH in 0.1% HSA/PBS to obtain a stock concentration of100 µg/mL .
- SB431542: Reconstitute SB431542 in DMSO to obtain a stock concentration of10 mM .
- TGFß3: Reconstitute TGFβ3 in 0.1% HSA/4 mM HCl/PBS to obtain a stock concentration of20 µg/mL .
- Y-27632: Reconstitute Y-27632 in DMSO to obtain a stock concentration of10 mM .
2 Equipment
- 37°C/CO2 incubator
- Cell counter
- Centrifuge for 15 mL conical tubes
- Laminar flow hood
- Light microscope
- Pipette boy
- Vacuum aspirator and tips
- Water Bath
3 Materials
1.5 mL Safe-Lock TubesEppendorfCatalog #5409331
2 mL Cryogenic vialsNalgeneCatalog #V5007 (Sigma Distributor)
6-well plategreiner bio-oneCatalog #657160
15 mL conical centrifuge tubegreiner bio-oneCatalog #188271
50 mL conical centrifuge tubegreiner bio-oneCatalog #227261
Cell Carrier Ultra 96 blackPerkin ElmerCatalog #6055308
P1000, P200, P20, P10, P2.5 pipettes and filter tipsEppendorf
Sterile 50, 25, 10, 5 mL serological pipettes greiner bio-oneCatalog #768180
Safety warnings
Fatal if swallowed. Suspected of causing cancer. Toxic by ingestion
- Mitomycin-C
May form combustible dust concentrations in air
- L-ascorbic acid
Toxic by inhalation and ingestion. Cause skin and eye irritation
- 2-Mercaptoethanol
- CHIR99021
- DMSO
- Y-27632
Before start
Schematic representation of the optimized derivation of mDA neurons from iPSCs. Created with BioRender.com
Differentiation of iPSCs into midbrain dopaminergic (mDA) neurons
Differentiation of iPSCs into midbrain dopaminergic (mDA) neurons
Note: This protocol is optimized for mDA neuron differentiation in 6-well plate format.
Medium change schema:
Day 0-20:4 mL per well
Day 21-65:3 mL per well
Day 0-15: daily media changes
Day 16-20: media changes every 2 days
Day 21-65: media changes every 2-3 days
Day -2: Coating of 6-well plates with Geltrex
Day -2: Coating of 6-well plates with Geltrex
1d
Thaw Geltrex Overnight On ice and dilute to 100x in ice cold DMEM/F-12.
12h
Coat 6-well culture plates with1 mL per well of Geltrex solutionOvernight at37 °C 5 % CO2 .
12h
Day -1: Single cell dissociation and seeding for differentiation
Day -1: Single cell dissociation and seeding for differentiation
1m
Discard Geltrex solution and add2 mL per well of E8 Flex medium supplemented with10 µM Y-27632 avoiding the coating to dry out.
Keep the plate at37 °C for00:05:00 before seeding cells.
5m
Warm PBS, Accutase and E8 Flex medium toRoom temperature .
15m
Add1 mL per well of Accutase to iPSCs in 6-well plate.
Dissociate cells with a P1000 pipette by pipetting the cell suspension up and down for 3-6 times.
Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.
Transfer the cell suspension to a 15 mL conical centrifuge tube. A total volume of12 mL hould be obtained from a full 6-well plate.
Centrifuge the cell suspension at300 x g, 23°C, 00:05:00 .
5m
Discard the supernatant carefully and resuspend the cell pellet in1 mL E8 Flex medium supplemented with10 µM Y-27632 by gently pipetting the cell suspension up and down for 3-6 times with a P1000 pipette to obtain a homogeneous cell suspension.
Adjust the volume with E8 Flex medium supplemented with10 µM Y-27632 to12 mL . Mix well.
Perform two separate live-cell counts using a hemocytometer or an automated cell counter.
Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting.
Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter.
Expected result
A cell viability of 95-99% should be obtained.
Note: Working with a different brand of Accutase might impact the cell viability when following the dissociation protocol described above. If cell viability is lower than expected, reduce the incubation time with Accutase to00:20:00 and the number of pipetting to dissociate the cell colonies to 3-4 times.
Seed 800,000 cells per well in a total volume of4 mL per well on 6-well plates coated with Geltrex.
Rock the plate back-forth and side-to-side for00:00:10 to achieve an even spread of cells in the plate well.
10s
Keep cells intactOvernight at37 °C 5 % CO2 .
12h
Day 0: Start differentiation
Day 0: Start differentiation
1h
Before starting:
a) Prepare enough amount of Knockout Serum Replacement (KSR) medium.
For 500 mL of KSR medium, add:
413.5 mL Knockout DMEM/F-12 medium
75 mL Knockout Serum Replacement
5 mL MEM Non-Essential Amino Acids
5 mL GlutaMAX
500 µL 2-mercaptoethanol
1 mL Penicillin-Streptomycin
Storage: KSR medium can be stored for 5 days at4 °C or for up to one month at-20 °C .
b) Reconstitute lyophilized reagents following the instructions and stock concentrations indicated in Materials (1.1.4 Reconstitution of reagents).
Use the following final concentrations:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF8
4 µM CHIR99021
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µM or10 nM DAPT
Storage: Once thawed, the stocks of small molecules and growth factors can be stored for up to 5 days at4 °C .
Notes: Small molecules and growth factors must be freshly added immediately before each medium change. It is strongly advised to avoid mixing different lots of reagents in the same differentiation.
Perform a quick microscope inspection to the cells to check confluency.
Critical step: Start the differentiation with a 100% confluent cell culture, meaning that the culture area of the plate should be completely covered with a cell monolayer. Not confluent cell cultures might affect differentiation efficiency. If cell lines did not achieve 100% confluency on day 0, the number of cells seeded per cm2 on day -1 should be adjusted accordingly.
Prepare differentiation medium:
Warm KSR medium at37 °C .
Add:
500 nM LDN193189
10 µM SB431542
Mix well.
Critical step: Do not add small molecules to cold medium to avoid inadequate dissolution.
15m
Perform full media change:
Discard old culture medium and add4 mL per well of differentiation medium very carefully avoiding touching the bottom of the well.
Critical step: To prevent cells from drying out during full media changes, change the medium of one 6-well plate at each time. Add differentiation medium very gently (dropwise) to avoid perturbation of the cell layer. Cell detachments might affect the differentiation efficiency. In case of cell detachment, a confluency above 95% is desired to continue the differentiation.
Media changes during differentiation
Media changes during differentiation
From day 1, change 75% of the differentiation medium daily until day 15, and then, every 2 days until day 20.
Note: to perform 75% medium change of a working volume of4 mL per well,, discard 3 mL of old medium and add4 mL per well of fresh prepared differentiation medium.
Day 1 and 2
Day 1 and 2
15m
Warm KSR medium at37 °C .
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Day 3 and 4
Day 3 and 4
15m
Warm KSR medium at37 °C .
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Day 5 and 6
Day 5 and 6
1h
Before starting:
Prepare enough amount of N2 medium.
For 500 mL of N2 medium, add:
479 mL Neurobasal medium
10 mL B27 supplement without vitamin A
5 mL N2 supplement
5 mL GlutaMAX
1 mL Penicillin-Streptomycin
Storage: N2 medium can be stored for 5 days at4 °C or for up to one month at-20 °C .
Warm KSR and N2 medium at37 °C .
Mix:
75 % KSR medium
25 % N2 medium
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
2 µM Purmorphamine
100 ng/mL FGF-8b
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
Day 7 and 8
Day 7 and 8
15m
Warm KSR and N2 medium at37 °C .
Mix:
50 % KSR medium
50 % N2 medium
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Day 9 and 10
Day 9 and 10
15m
Warm KSR and N2 medium at37 °C .
Mix:
25 % KSR medium
75 % N2 medium
Add:
500 nM LDN193189
10 µM SB431542
100 ng/mL SHH
4 µM CHIR99021
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Day 11 and 12
Day 11 and 12
1h
Before starting:
Prepare enough amount of NB/B27 medium.
For 500 mL NB/B27 medium, add:
484 mL Neurobasal medium
10 mL B27 supplement without vitamin A
5 mL GlutaMAX
1 mL Penicillin-Streptomycin
Storage: NB/B27 medium can be stored for 5 days at4 °C or for up to one month at-20 °C .
Warm NB/B27 medium at37 °C .
Add:
4 µM CHIR99021
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µM DAPT
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
Day 13 -15, 17 and 19
Day 13 -15, 17 and 19
15m
Warm NB/B27 medium at37 °C .
Add:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 µM DAPT
Mix well.
Perform medium change: 4 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Day 20: Replating of mDA neuron precursors at high cell density
Day 20: Replating of mDA neuron precursors at high cell density
2d
Note: At day 20 of differentiation, mDA neuron precursors can be replated as describe below or cryopreserved.
Before starting:
Coating of 6-well plates step 1
Coat 6-well culture plates with1 mL per well 0.1 mg/mL Poly-L-Ornithine (PLO) in PBS. Incubate platesOvernight at37 °C . Wash plates three times with PBS. Discard PBS and proceed to coating step 2.
Coating of 6-well plates step 2
Coat 6-well culture plates with1 mL per well 10 µg/mL Laminin plus 2 µg/mL Fibronectin, both diluted in PBS. Incubate platesOvernight at37 °C . Do not store coated plates. Proceed with preparation of plates for seeding cells.
Preparation of 6-well plates for seeding cells
Warm NB/B27 medium at37 °C .
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
10 µM Y-27632
Discard coating reagents and add2 mL per well of NB/B27 complete medium.
Keep the plate at37 °C for00:15:00 before seeding cells.
2d
Warm PBS, Accutase and NB/B27 medium toRoom temperature .
Discard old culture media and wash cells once with1 mL per well of PBS.
Discard PBS and add1 mL per well of Accutase.
Incubate cells at37 °C for00:15:00 .
15m
After incubation, block Accutase with1 mL per well NB/B27 medium supplemented with10 µM Y-27632.
Dissociate cells with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.
Perform a quick microscope inspection of the cells. A single cell suspension should be obtained.
Transfer the cell suspension to a conical tube. A total volume of12 mL should be obtained from a full 6-well plate.
Centrifuge the cell suspension at300 x g, 23°C, 00:05:00 .
5m
Discard the supernatant carefully and add1 mL NB/B27 complete medium. Resuspend the cell pellet very gently with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.
Complete the volume to12 mL with NB/B27 complete medium and mix well.
Perform two separate live-cell counts using a hemocytometer or an automated cell counter.
Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting.
Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter.
Expected result
A cell viability of 90-99% should be obtained. If lower, reduce the number of pipetting to dissociate the cell clumps to 3-4 times.
Seed desired density (300,000 to 500, 000 cells) on MatTek dishes or 6-well plate coated with PLO/Laminin.
Rock the plate back-forth and side-to-side for00:00:10 to achieve an even spread of cells in the plate well.
10s
Keep cells intact Overnight at37 °C 5 % CO2 .
12h
Day 23: Treatment with antimitotic inhibitor (Supplement K)
Day 23: Treatment with antimitotic inhibitor (Supplement K)
15m
Before starting:
Warm NB/B27 medium at37 °C .
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Prepare enough amount of NB/B27 complete medium supplemented with1 Parts per Million (PPM) Stock: 1000x Supplement K (BrainXell). Mix well.
15m
Discard old culture medium and add2 mL per well of NB/B27 complete medium supplemented with Supplement K.
Place cells back at37 °C 5 % CO2 .
Every 5 days: Change medium for terminal differentiation of mDA neurons
Every 5 days: Change medium for terminal differentiation of mDA neurons
15m
Warm NB/B27 medium at37 °C .
Add:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Mix well by 20x full inversions of the conical tube or flask.
Perform medium change: 2 mL per well .
Place cells back at37 °C 5 % CO2 .
15m
Every 10 days: Supplement differentiation medium with Laminin
Every 10 days: Supplement differentiation medium with Laminin
15m
Warm NB/B27 medium at37 °C .
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
Cool down NB/B27 complete medium toRoom temperature .
Add:
10 µg/mL Laminin
Mix well.
15m
Discard old culture medium. Perform medium change as following:
3 mL per well in 6-well plates
200 µL per well in 96-well plates
Place cells back at3 °C 5 % CO2 .
Cryopreservation of mDA neurons on day 20
Cryopreservation of mDA neurons on day 20
5m
Before starting:
Thaw Synth-a-Freeze cryopreservation medium from -20C.
Perform cells dissociation and previously describedgo to step #1.5 .
After determining the number of live cells, centrifuge the cell suspension at400 x g, 23°C, 00:05:00 .
5m
Resuspend the cell pellet very gently in Synth-a-Freeze medium to 3-5x10^6 cells/mL.
Distribute500 µL of the cell suspension in cryogenic vials.
Transfer cryogenic vials in a container at-80 °C Overnight .
Transfer cells to the vapor phase of a liquid nitrogen storage facility.
Thawing cryopreserved mDA neurons
Thawing cryopreserved mDA neurons
12h
One day before thawing:
Coat 6-well culture plates with Geltrex diluted in ice cold DMEM/F-12 mediumOvernight at37 °C .
12h
Day of thawing
Day of thawing
15m
Before starting
Prepare:
Wash medium
Warm NB/B27 medium at37 °C .
Add: 10 µM Y-27632.
Recovery medium
Warm NB/B27 medium at37 °C .
Make NB/B27 complete medium by adding:
20 ng/mL BDNF
0.2 mM Ascorbic acid
20 ng/mL GDNF
0.5 mM db-cAMP
1 ng/mL TGFβ3
10 nM DAPT
10 µM Y-27632
Cool down NB/B27 complete medium toRoom temperature .
6-well plates
Discard coating reagent and add2 mL per well of recovery medium.
Keep plates at37 °C for00:15:00 before seeding cells.
Conical centrifuge tubes
Label 15-mL conical tubes and fill with5 mL wash medium. Keep atRoom temperature .
Thaw cryopreserved mDA neurons by placing the cryogenic vial containing cells in a37 °C water bath for approximately00:01:00 or until no ice is visible but the liquid is still cold.
1m
Fill a 5-mL serological pipette with4 mL wash medium and collect the thawed cell suspension very carefully.
Transfer the cell suspension dropwise to the 15-mL conical tube containing5 mL of wash medium.
Centrifuge the cell suspension at400 x g, 23°C, 00:05:00 .
5m
Discard the supernatant carefully and resuspend the cell pellet with1 mL recovery medium by gently pipetting up and down 3-6 times to obtain a homogeneous cell suspension.
Seed cells in a total volume of2 mL per well on 6-well plates coated with Geltrex.
Treat cells with Supplement K following the same procedure described on day 23 of differentiationgo to step #5 .
Rock the plate back-forth and side-to-side for00:00:10 to achieve an even spread of cells in the plate well.
10s
Day 7 after thawing: Replate mDA neurons
Day 7 after thawing: Replate mDA neurons
1m
Replate mDA neurons 7 days after thawing on MatTek or 6-well plates at appropriate cell densities following the procedure described for replating mDA neurons on day 20 go to step #4.15 .
For long-term culture of mDA neurons, change differentiation medium every 5 days as described previouslygo to step #6.1 .