Motor neuron protocol: Sep 2022
Download protocol here
| Catalog. No. | Product Name | Format Conc. | Stock Conc. | Storage on Arrival | Thawing | Storage Once Thawed |
| ax0078 | Human iPSC-Derived Motor Neuron Progenitors (Healthy) | ≥2 million cells/vial | N/A | Liquid Nitrogen | Follow protocol | N/A |
| ax0073 | Human iPSC-Derived Motor Neuron Progenitors (Healthy – sibling (C9ORF72 extension) | ≥2 million cells/vial | N/A | Liquid Nitrogen | Follow protocol | N/A |
| ax0074 | Human iPSC-Derived Motor Neuron Progenitors (ALS (C9ORF72 extension)) | ≥2 million cells/vial | N/A | Liquid Nitrogen | Follow protocol | N/A |
| ax0072 | Motor Neuron Maintenance Medium | 200 mL | 1x | -80ºC | Overnight at 4ºC | Once thawed, aliquot and store at 4ºC for up to 1 week or for long term store at -80ºC. |
| ax139888 (5 µg) | Recombinant Human Ciliary-Neurotrophic Factor (CNTF) | 5 μg Lyophilized Powder | N/A | -20ºC | N/A | Reconstituted protein should be used immediately or stored in working aliquots at -20°C |
| ax139800 (10 µg) | Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) |
10 μg Lyophilized Powder | N/A | -20ºC | N/A | Reconstituted protein should be used immediately or stored in working aliquots at -20°C |
| ax139855 (10 µg) | Recombinant Human Glial-Derived Neurotrophic Factor (GDNF) |
10 μg Lyophilized Powder | N/A | -20ºC | N/A | Reconstituted protein should be used immediately or stored in working aliquots at -20°C |
| ax0179 | Motor Neuron Maturation Accelerator supplement |
1 mL | 100x | Aliquot into required amounts and store at -80oC |
Thaw at RT | Aliquots are single useAdditional Reagents |
| Product Name | Supplier | Product Code |
| Retinoic acid | Sigma-Aldrich | R2625 |
| Y-27632 2HCl (ROCK inhibitor) | Focus Biomolecules | 10-2301 |
| Poly-D-Lysine | Sigma-Aldrich | P7405 |
| Compound E | Abcam | ab142164 |
| Vitronectin (VTN-N) | Thermofisher | A14700 |
Important! Axol Neural Cell Culture Media
DOES NOT contain antibiotics or antifungal agents. Axol does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted
This protocol and it associated products are for research use only. It is not for diagnostic
or therapeutic use and not to be administered to humans or animals.
Preparation of Media and Coating Reagents
Motor Neuron Maturation Accelerator Supplement (ax0179)
- Upon receipt, aliquot to your required volume and store Motor Neuron Maturation Accelerator
supplement at -80°C protected from light. For example: for 10 mL media changes, aliquot into 100
µL aliquots. - When ready to use, thaw an aliquot of Motor Neuron Maturation Accelerator supplement overnight
at 4°C in the dark. Do not refreeze after use
Motor Neuron Maintenance Medium (ax0072)
- Upon receipt, aliquot and store Motor Neuron Maintenance Medium at -80°C protected from light.
- When ready to use, thaw an aliquot of Motor Neuron Maintenance Medium overnight at 4°C in the dark.
- Motor Neuron Maintenance Medium requires supplementing with Compound E once thawed
- Prepare Compound E (ab142164) by creating a stock concentration of 1 mM in DMSO.
- Add Compound E (ab142164) to create a final concentration of 0.2 µM
| Product Name | Stock concentration |
| Compound E (ab142164) | 0.2 µM |
- Once made-up Motor Neuron Maintenance Medium plus Compound E can be frozen in 30ml
aliquots for future use.
Motor Neuron Maintenance Medium – For Plating
- Motor Neuron Maintenance Medium plus Compound E requires supplementing with retinoic
acid and Y-27632 2HCl (Rock inhibitor) before use. - Prepare the retinoic acid by creating a stock concentration of 1 mM in DMSO.
- Prepare the Y-27632 by creating a stock concentration of 10 mM in sterilewater
- On the day of use, prepare Motor Neuron Maintenance Medium – For Plating by adding
retinoic acid to create a final concentration of 0.1μM (N.B this concentration is different in
Complete Motor Neuron Maintenance media) and Y-27632 2HCl to a final concentration of
10μM.
Complete Motor Neuron Maintenance Medium
- Prepare Complete Motor Neuron Maintenance Medium plus compound E by adding all the
following factors on the day of use. (N.B. the concentration of retinoic acid is different from Motor
Neuron Maintenance Medium – For Plating).
| Growth Factor | Stock Concentration | Final Concentration | Volume for 50mls |
| Recombinant Human Ciliary-Derived Neurotrophic Factor (CNTF) (ax139888) |
5 µg/mL | 10 ng/mL | 100 µL |
| Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) (ax139800) |
10µg/mL | 5 ng/mL | 25 µL |
| Retinoic acid | 1 mM | 0.5 µM | 25 µL |
| Recombinant Human Brain-Derived Neurotrophic Factor (GDNF) (ax139855) |
10µg/mL | 10ng/ml | 50 µL |
| Motor neuron accelerator supplement | 100X | 1x | 500 µL |
Coating of culture ware
- To enable adherence of the cells to cultureware, we recommend the following methodology.
- Please note the coating concentration may need to be adjusted depending on the material type of
the flasks, plates or imaging slides being used.
Vitronectin + Poly-D-Lysine (PDL)
- Calculate the total surface area that requires coating.
- Prepare PDL by reconstituting 5mg of PDL in 10ml of sterile water
- Add 300 µL per cm² of 1x PDL solution (0.5 mg/mL) to the culture vessel. Incubate for 1 hour
at 37°C, remove the solution through aspiration, and thoroughly rinse the surface twice with
sterile water. Allow the culture vessel to fully dry out for at least 2 hours at 37°C, 5% CO2
before proceeding onto Vitronectin coating. - Calculate the total surface area that requires coating.
- Dilute the Vitronectin stock solution (50x) in D-PBS (1x) (without calcium or magnesium)
to make 1x working solution e.g., 10μL in 0.5mL. - Coat the surface of your culture vessel with 300μL per cm² of Vitronectin 1x working
solution. - Incubate overnight at 37°C, 5% CO2.
- Do not wash the culture vessel after coating with Vitronectin.
- Do not let the Vitronectin coating dry out before plating the cells.
Culturing Human iPSC-derived Motor Neuron Progenitors
Thawing and Plating
- Thaw an aliquot of Motor Neuron Maintenance Medium overnight at 4°C. On the day of
thawing the progenitors, prepare Motor Neuron Maintenance Medium – For Plating by adding
Y-27632 2HCl to a final concentration of 10μM and retinoic acid to a final concentration of
0.1μM. - Transfer the vial of cells from storage by transporting the vial buried in dry ice. Remove the
vial from dry ice and transfer it to a 37°C water bath. - Quickly thaw the vial of cells in a 37°C water bath. Do not completely submerge the vial (only
up to 2/3rd of the vial). Remove the vial before the last bit of ice has melted. - Do not shake the vial during thawing.
- Take the vial of cells to a biological safety cabinet, spraying the vial and working surface of
the cabinet thoroughly with 70% ethanol and wiping with a sterile paper towel before placing
the vial in the cabinet. - Using a P1000 pipette, gently addthe cell suspensiondropwise into a 15mL sterile conical tube
containing 8 mL Motor Neuron Maintenance Medium – For Plating. Gently rinse the cryovial
with 1 mL of fresh Motor Neuron Maintenance Medium – For Plating and transfer the medium
with remaining cells into the 15 mL sterile conical tube. - Centrifuge cells at 200 x g for 5 minutes at room temperature.
- Carefully aspirate and discard the supernatant using a 10 mL pipette.
- Using a P1000 pipette, gently resuspend the cell pellet in 1 mL of Motor Neuron
Maintenance Medium – For Plating until they are in a single cell suspension. - Perform a cell count to ensure optimal seeding density and dilute the cell suspension as
required for the chosen seeding density. - Recommended cell density is 130,000-180,000 cells/cm2.
- Note: For high density cultures you can plate cell densities up to 240000 cells/cm2
- Remove the coating solution and add an appropriate volume of medium and cells to the
culture vessel. Do not let the culture vessel dry out. - Incubate at 37°C, 5% CO2 for 24hrs, then replace the cell culture medium with Complete
Motor Neuron Maintenance Medium.
Maturation and Maintenance of Human iPSC-derived Motor Neurons
- To maintain a healthy culture, perform a full volume medium exchange every other day with
fresh pre-warmed Complete Motor Neuron Maintenance Medium (including all the factors in
the table above). - The motor neurons should be maintained in the above medium for a minimum of two weeks
to achieve strong expression of HB9, CHAT and ISL-1. - Repetitive firing of action potentials can be observed from week 2.
