User Guide
Quick-Neuron™ Motor - Human iPSC-derived Cells
Catalog Numbers:

MT-SeV-CW50065, MT-SeV-CW10149, MT-SeV-CW20300, MT-SeV-CW50023,
MT-SeV-CW70067, MT-SeV-CW50025, MT-SeV-CW50113, MT-SeV-CW50114,
MT-SeV-CW50115, MT-SeV-CW50137, MT-SeV-CW50147, MT-SeV-CW60130,
MT-SeV-CW60231, MT-SeV-CW60236, MT-SeV-CW20026, MT-SeV-CW20090, or
MT-SeV-CW10130

Introduction
Elixirgen Scientific’s proprietary transcription factor-based technology allows rapid and reproducible differentiation ofhuman iPSCs into neurons without sacrificing the purity of the cells. Quick-Neuron™ Motor differentiated cell cultures
display typical neurite outgrowth and express a variety of neuronal markers, such as the pan-neuronal marker tubulin beta 3class III (TUBB3), the cholinergic marker choline acetyl-transferase (ChAT), and the homeobox transcription factor HB9
expressed in motor neurons. When handled and maintained according to the instructions in this user guide, motor neurons
are viable long-term and are suitable for a variety of characterization and neurotoxicity assays.

Scale:

Quick- Neuron™ Motor - Human iPSC-derived Cells are available in two sizes: (Small) 1 million
viable cryopreserved cells and (Large) 5 x 1 million viable cryopreserved cells. The instructions
outlined in this user guide are for seeding 1 million viable cells at approximately 5 x 104 cells/cm2
into 2 wells of a 6-well plate (5 x 105 cells/well), 10 wells of a 24-well plate (1 x 105 cells/well), or 62
wells of a 96-well plate (1.6 x 104 cells/well).

Related Products:

Quick-Neuron™ Motor - SeV Kit, Catalog Number: MT-SeV
Quick-Neuron™ Motor - Maintenance Medium, Catalog Number: MT-MM

Contents
Upon receipt, immediately store the items at the indicated temperatures. Be especially careful to keep the frozen cells on
dry ice until placing them in liquid nitrogen and avoid any temperature fluctuation and slight thawing.
Contents

Amount (Small Size)

Amount (Large Size)

StorageThaw

Cryopreserved cells>1 million viable cells,(1 vial, 500 μl)

5 x >1 million viable cells,(5 vials, 5 x 500 μl)

Liquid nitrogen

37°C

Component N1

2 x 830 μl

7 x 830 μl-20°C or -80°C

On ice or 4°C

Component A

80 μl

2 x 80 μl-20°C or -80°C

Room temperature

Component P

50 μl

2 x 50 μl-20°C or -80°C

Room temperature

Component K

20 μl

20 μl

-20°C or -80°C

Room temperature

Condition of Use
This product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).
Last revised: August 23, 2023

elixirgensci.com

1Required Consumables
Item

VendorCatalog Number(Optional) 6-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-80(Optional) 24-well tissue-culture-treated polystyrene plate(e.g., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-740(Optional) 96-well tissue-culture-treated polystyrene plate(e.g., Thermo ScientificTM 96 Well Black/Clear Bottom Plate)Fisher Scientific12-566-70

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

0.01% Poly-L-Ornithine

Sigma-Aldrich

P4957-50ML

Extracellular Matrix such as
Laminin Mouse Protein, Natural, or
Geltrex Basement Membrane Matrix

ThermoFisher

23017015 orA15696-01

Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650*PBS should be used at room temperature unless otherwise specified.

Workflow
Note: This protocol assumes that Day 0 is a Tuesday.

*From Day 7, users may maintain differentiated neurons in the medium best suited for their needs, though we recommend
Quick-Neuron™ Cholinergic - Maintenance Medium, Catalog Number: MT-MM.

Experiment Planning
Define the cell culture plate or dish format in advance and calculate the number of wells to be used for each format inadvance. For example, you may use only a certain number of wells of a 96-well plate. The following section describes
culture condition volumes per well as user needs may vary. When a 96-well plate is used, we recommend filling the edge
wells of the plate with an aqueous medium instead of cells and culture medium. This will maintain humidity on the entire
plate. If performing an image-based analysis with a 96-well plate, we have found plating approximately 1-1.5 x 104 cells/wellelixirgensci.com

2

to yield the best results. Please refer to the table below for plate formats and corresponding surface area of each well used
for calculating reagents in the following sections.
Plate format
Approximate cell growthsurface area per well

6-well plate24-well plate

96-well plate

9.5 cm2

1.9 cm2

0.32 cm2

Preparation
10 mM ROCK inhibitor Y27632 (iROCK)
1. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as iROCK, can be stored at -20°C.
0.002% Poly-L-Ornithine solution (ornithine)
1. Take 700 μl 0.01% Poly-L-Ornithine solution and mix it with 2.8 ml PBS.
2. The 0.002% Poly-L-Ornithine solution, hereafter referred to as ornithine, can be stored at 4°C for up to 2 weeks.
1 mg/ml laminin stock solution (laminin)
1. Thaw Laminin Mouse Protein, Natural and chill PBS at 4°C or on ice.
2. Mix the Laminin Mouse Protein, Natural and PBS to make the 1 mg/ml stock solution, hereafter referred to aslaminin.
○ Laminin concentration varies by lot, so use the number specified on the vial or CoA when making yourcalculations.
3. Make aliquots of a convenient volume (e.g., 35 μl) and store at -20°C.
Medium N1
1.

Prepare Medium N1 using the reagents listed in the table below. Use the appropriate volume for the plate format
you plan to use.
○ Thaw Component N1 for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Tap Component N tube(s) 3 times and then briefly spin down before use.
○ Keep Medium N1, and any subsequent media made with it, protected from light.
○ Store Medium N1 for up to 2 weeks at 4°C.
○ Leftover Component N1 can be discarded or saved at 4°C for up to two weeks.
Required volume for each format
6-well plate24-well plate or
96-well plate

DMEM/F12

9.6 ml16.8 ml

Neurobasal Medium

9.6 ml16.8 mlGlutaMAX

100 μl

175 μlPenicillin-Streptomycin (10000 units/ml; 100x)

200 μl350 μlComponent N1

600 μl1.05 ml

Reagents

elixirgensci.com

3Medium N1(AP)1.

Prepare Medium N1(AP) sufficient for the number of wells plated using the volumes indicated in the table below for
calculating plus an additional 1.1 ml for resuspension (volume in table x number of wells + 1.1 ). For example, 2wells of a 6-well plate needs 15.1 ml Medium N1 (7 x 2 + 1.1), while 10 wells of a 24-well plate needs 26.1 ml
Medium N1 (2.5 x 10 + 1.1).
○ Thaw Component A and Component P for 20-30 minutes at the temperature indicated in the “Contents”
table on page 1.
○ Warm Medium N1 at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin all tubes down before use.
○ Component A is added 1:1000 and Component P 1:2000.
○ This will produce enough Medium N1(AP) for 1 week of culture.
○ Store leftover Medium N1 and Medium N1(AP) at 4°C.
Required volume per well
Reagents

6-well plate24-well plate

96-well plate

Medium N1

7 ml

2.5 ml450 µlComponent A

7 µl

2.5 µl0.45 µl

Component P

3.5 µl

1.3 µl0.23 µl

Diluted Component K
1.

Prepare Dilute Component K for use in Medium N1(AKP) as follows:○ Thaw Component K for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Tap the tube of Component K 3 times and then briefly spin it down before use.
○ Prepare a diluted working solution of Component K by mixing together the following components.
○ Store for up to 2 weeks at 4°C.
Reagents

VolumeDMSO

154 μlComponent K

12.5 μlDay 05-6 hours

Plate Preparation
IMPORTANT! Cells can be plated in 6-well, 24-well, or 96-well plates depending on the desired format. Refer to the table forthe recommended volumes per well.
1.
2.
3.
4.

Vortex ornithine briefly and centrifuge it at a maximum speed for 1 minute.
Add ornithine to each well of a new plate in the volume specified in the table.
Incubate the plate at 37°C, 5% CO2 for at least 2 hours (or at 4°C overnight one day before plating).
Thaw laminin and chill PBS on ice for 20-30 minutes in the volumes calculated in the table. Add 1/100 volume oflaminin to chilled PBS. Mix well.
○ All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
5. Aspirate the supernatant from each well and add PBS in the volume specified in the table.
6. Repeat Step 5.
7. Aspirate PBS from each well and add diluted laminin in the volume specified in the table.
8. Incubate the plate at 37°C, 5% CO2 for at least 2 hours.

elixirgensci.com

4

9. While the plate is incubating, prepare Medium iN1(AP) using the volume of Medium N1(AP) indicated in the tablecalculated for the number of wells in use plus 1.1 ml for resuspension (volume in table x number of wells + 1.1 ). For
example, 2 wells of a 6-well plate needs 4.5 ml Medium N (1.7 x 2 + 1.1), while 10 wells of a 24-well plate needs 5.6
ml Medium N (0.45 x 10 + 1.1) for this step.
○ Thaw/warm Medium N1(AP), and iROCK at room temperature for 20-30 minutes.
○ Add 1/1000 of iRock to Medium N1(AP).
○ Keep the rest of Medium N1(AP) at 4°C for later use.
10. After the laminin incubation, aspirate most, but not all, of the supernatant and add PBS in the volume specified in
the table. Add the PBS dropwise to each well.
11. Pipet out most of the PBS, but not all, from each well.
12. Add PBS dropwise in the volume specified in the table.
13. Repeat step 11.
14. Add Medium iN1(AP) to each well in the volume specified in the table.
15. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Recommended volume per well
Reagents

Corresponding
steps

6-well plate24-well plate

96-well plate

Ornithine

1,2

1.5 ml300 µl

50 µl

PBS for laminin dilution

4

1.65 ml

330 µl55 µl

5, 6, 10, 12

2 ml

500 µl100 µlDiluted laminin

7

1.5 ml300 µl

50 µl

Medium N1(AP) for Medium iN1(AP)9

1.7 ml450 µl

80 µl

Medium iN1(AP)

14

1 ml

300 µl

35 µl

PBS

Thawing Cells
1. Warm Medium N1 to room temperature for 20-30 minutes.
2. Take out the vial of frozen cells from the liquid nitrogen storage tank.
3. Incubate the cryovial in a water bath set at 37°C (do not submerge the cap) until the most of the content is thawed
but a small ice crystal remains (~2 minutes).
4. Wipe the vial with a dry paper towel. Spray 70% ethanol to the vial and bring it inside a biosafety cabinet.
5. Transfer 4.5 ml room temperature Medium N1 to a new 15 ml conical tube.
6. Set a P1000 pipette to 1 ml but take approximately 500 μl Medium N1 from the 15 ml conical and add it to the
cryo-vial dropwise at 1 drop per 1-2 seconds.
○ IMPORTANT! Use the same pipette tip for Steps 6-10.
7. Gently pipet the cell suspension up and down once.
8. Gently transfer all of the cell suspension to the 15 ml conical tube prepared in Step 5.
9. Take 1 ml of the cell suspension from the conical tube and add it to the original cryovial and pipet up and down 2-3
times and then transfer the whole contents back to the same 15 ml conical tube.
10. Mix the contents in the conical tube by gently pipetting cell suspension up and down 3 times.
11. Centrifuge the cell suspension at 200 x g for 4 minutes.
12. Use an aspirator to remove most of the supernatant from the conical tube, leaving a small volume of thesupernatant (<50 μl) to cover the pellet.
13. Tap the side of the conical tube up to 10 times to break up the cell pellet.
14. Add 1 ml room temperature Medium iN1(AP) to the conical tube and pipet up and down no more than 2-3 times.
Plating Cells
1.

Count the cells to determine the volume of cell suspension needed for the chosen number of wells and include 10%
extra for cell number and volume volume (e.g., for a 24-well plate scenario, a total of 1.1 x 106 cells to plate 1 x 105

elixirgensci.com

5

cells in each of the 10 wells). If the volume of the cell suspension needs to be adjusted, centrifuge the required
volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet with Medium
iN1(AP) to reach the multiplied volume of cell suspension with the number of wells.
2. Add cell suspension to the center of each well. Since each well already has Medium iN1(AP), the total volume of themedium in each well is indicated in the table below.
3. Incubate at 37°C, 5% CO2 overnight.
Recommended amounts
6-well plate24-well plate

96-well plate5 x 105 cells

1 x 105 cells

1.6 x 104 cells

Required total volume of cell suspension/well● (Volume of cell suspension/well) + 10% extra

550 µl110 µl38.5 µl

Volume of cell suspension distributed/well

500 µl100 µl

35 µl

Total volume/well● Medium iN1(AP) + cell suspension

1.5 ml400 µl

70 µl

Viable cells/well

Day 1< 2 hourMedium Change
1.

Prepare Medium N1(AKP) sufficient for the number of wells plated using the volumes indicated in the table below
for calculating.
○ Warm Medium N1(AP) and Diluted Component K at room temperature for 20-30 minutes away from light.
○ This will produce enough Medium N1(AKP) for 1 week of culture. Store leftover Medium N1(AP) andMedium N1(AKP) at 4°C for additional medium changes throughout the week.
Required volume per well
Reagents

6-well plate24-well plate

96-well plate

Medium N1(AP)

4.4 ml

1.8 ml330 µlDiluted Component K

4.4 µl

1.8 µl0.33 µl

2. Pipet out the old medium from each well and add PBS to each well along its wall according to the table below.
○ IMPORTANT! To avoid lifting cells, leave a small volume of the old medium (i.e., just enough to cover thesurface of the well). PBS should be applied along the wall of the well very slowly.
Required volume per well
Reagents
PBS

6-well plate24-well plate

96-well plate

2 ml

500 µl100 µl3. Pipet out the PBS from each well and add Medium N1(AKP) to each well along its wall according to the following
table.
Required volume per well
Reagents
Medium N1(AKP)

elixirgensci.com

6-well plate24-well plate

96-well plate

2 ml

800 µl150 µl6

4. Incubate the culture at 37°C, 5% CO2 for 2 days.

Day 3+< 1 hourMedium Change
1. Warm Medium N1(AKP) at room temperature for 20-30 minutes.
2. Pipet out half of the old medium from each well and add Medium N1(AKP) according to the table below.
Required volume per well
Reagents
Medium N1(AKP)

6-well plate24-well plate

96-well plate

1 ml

400 µl75 µl

3. Incubate the plate at 37°C, 5% CO2 for 2-3 days.
4. Repeat Steps 1-3 until Day 7 or until ready to assay.

Day 7+< 1 hour

Assay or Continuous Maturation●●

Differentiated motor neurons will be ready for assays on day 7. From day 7 users may maintain differentiatedneurons in the medium best suited for their needs, though we recommend Quick-Neuron™ Motor MaintenanceMedium, Catalog Number: MT-MM.
Differentiation into motor neurons can be confirmed with the markers TUBB3, ChAT, and HB9.

elixirgensci.com

7

Appendix

Figure 1. Representative images of Quick-NeuronTM Motor - Human iPSC-derived Neurons on days 1 and day 7 post-thaw (scale bar =100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Motor - mRNA cell culture shows typical neurite growth and expression of the
pan-neuronal marker TUBB3 as well as HB9 on day 7 post-thaw (scale bars = 100 μm). Staining conditions: Anti- ß-III tubulin monoclonal
antibody (Cell Signaling Technology, Catalog Number: 5568, 1:250 dilution) was used in combination with a secondary antibody(ThermoFisher, Catalog Number: A32731, Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488,
1:500 dilution). Anti-HB9 primary antibody (Developmental Studies Hybridoma Bank, Catalog Number: 81.5C10, 1:50 dilution) was usedin combination with a secondary antibody (ThermoFisher, Catalog Number: PIA32742 Goat anti-Mouse IgG (H+L) Highly
Cross-Adsorbed Secondary Antibody, AlexaFluor Plus 594 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

elixirgensci.com

8