User Guide
Quick-Neuron™ GABAergic- Human iPSC-derived Neurons
Catalog Numbers:

GA-mRNA-CW50065, GA-mRNA-CW10149, GA-mRNA-CW20300, GA-mRNA-CW50023,
GA-mRNA-CW70067, GA-mRNA-CW50025, GA-mRNA-CW50113, GA-mRNA-CW50114,
GA-mRNA-CW50115, GA-mRNA-CW50137, GA-mRNA-CW50147, GA-mRNA-CW60130,
GA-mRNA-CW60231, GA-mRNA-CW60236, GA-mRNA-CW20026, GA-mRNA-CW20090, orGA-mRNA-CW10130

Introduction
Elixirgen Scientific’s proprietary transcription factor-based stem cell differentiation method uses synthetic mRNAs toproduce highly pure populations of neurons without a genetic footprint. Our Quick-Neuron™ GABAergic - Human
iPSC-derived Neurons display typical neurite outgrowth and express a variety of neuronal markers, such as thepan-neuronal marker tubulin beta 3 class III (TUBB3) and the GABAergic marker glutamic acid decarboxylase 1 (GAD1).
When handled and maintained according to the instructions in this user guide, GABAergic neurons are viable long-term andare suitable for a variety of characterization and neurotoxicity assays.
Scale:

Quick-Neuron™ GABAergic - Human iPSC-derived Neurons 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 105cells/well), or 62 wells of a 96-well plate (1.6 x 104 cells/well).

Related Products:

Quick-Neuron™ GABAergic - mRNA Kit, Catalog Number: GA-mRNA
Quick-Neuron™ GABAergic - Maintenance Medium, Catalog Number: GA-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 N

2 x 840 μl

8 x 840 μl-20°C or -80°C

On ice or 4°C

Component G120 μl

4 x 20 μl-20°C or -80°C

On ice or 4°C

Component G260 μl

3 x 60 μl-20°C or -80°C

On ice or 4°C

Component P50 μl

4 x 50 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 contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: September 22, 2023elixirgensci.com

1Required Consumables
ItemVendor

Catalog 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., Corning Costar Flat Bottom Cell Culture Plates)

Fisher Scientific12-566-70

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

0.01% Poly-L-Ornithine

Sigma-Aldrich

P4957-50ML

Extracellular Matrix such asLaminin 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 maintenance medium best suited for their needs, though we
recommend Quick-Neuron™ GABAergic - Maintenance Medium, Catalog Number: GA-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/wellto 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 growth surface area per well

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6-well plate24-well plate

96-well plate9.5 cm2

1.9 cm2

0.32 cm22

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 N
1.

Prepare Medium N using the reagents listed in the table below.
○ Thaw Component N 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 the Component N tubes 3 times and then briefly spin them down before use.
○ Keep Medium N, and any subsequent media made with it, protected from light.
○ Store Medium N for up to 2 weeks at 4°C.
○ Leftover reagents can be discarded or saved at 4°C for up to two weeks.
Reagents

VolumeDMEM/F12

18.6 ml

Neurobasal

18.6 ml

200 mM Glutamax (100x)

195 μlPenicillin-Streptomycin (10000 units/ml; 100x)390 μl

Component N

1.2 mlDay 0

5-6 hours

Plate Preparation
IMPORTANT! Cells can be plated in 6-well, 24-well, and 96-well plates depending on the desired format. Refer to the table
at the bottom of this page for the 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 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.

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○5.
6.
7.
8.
9.

All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
Aspirate the supernatant from each well and add PBS in the volume specified in the table.
Repeat Step 5.
Aspirate PBS from each well and add diluted laminin in the volume specified in the table.
Incubate the plate at 37°C, 5% CO2 for at least 2 hours.
While the plate is incubating, Prepare Medium iN(G1P) using the reagents listed in the table below.
○ Thaw Components G1 and P for 20-30 minutes at the temperatures indicated in the “Contents” table onpage 1.
○ Thaw iROCK at room temperature for 20-30 minutes.
○ Warm Medium N at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin all tubes down before use.
○ Store Medium N(G1P) for up to 2 weeks at 4°C.
○ Leftover reagents can be stored at 4°C for up to two weeks.
VolumeReagents
6-well plate24-well plate

96-well plate

Medium N

4.5 ml

5.6 ml6 ml

Component G1

9 μl

11.2 μl

12 μl

Component P

9 μl

5.6 μl

6 μl

iROCK

4.5 μl5.6 μl

6 μl

10. After the laminin incubation, aspirate most, but not all, of the supernatant and add PBS in the volume specified inthe table. Add the PBS dropwise to each well.
11. Pipet out most of the PBS, but not all, from each well.
12. Repeat Steps 10-11.
13. Add Medium iN(G1P) to each well in the volume specified in the table.
14. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Recommended volumes per well for different plate formats.
Recommended volume per well
Reagents

Correspondingsteps

6-well plate24-well plate

96-well plate

Ornithine

1, 21.5 ml

300 µl

50 µl

PBS for laminin dilution

41.65 ml

330 µl55 µl

5, 6, 10, 112 ml

500 µl100 µlDiluted laminin

71.5 ml

300 µl

50 µl

Medium iN(G1P)

131 ml300 µl

35 µl

PBS

Thawing Cells
1. Warm Medium N at 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 most of the content is thawed but asmall ice crystal remains (~2 min).
4. Wipe the vial with a dry paper towel. Spray the vial with 70% ethanol and place it inside a biosafety cabinet.
5. Transfer 4.5 ml room temperature Medium N to a new 15 ml conical tube.
6. Set a P1000 pipette to 1 ml but take approximately 500 μl Medium N from the 15 ml conical and add it to the cryovial
dropwise at 1 drop per 1-2 seconds.
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○ 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 in the 15 ml conical tube 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 iN(G1P) 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 (e.g., for a 24-well plate scenario, a total of 1.1 x 106 cells to plate 1 x 105 cells ineach of the 10 wells). If the volume of the cell suspension needs to be adjusted, centrifuge the required volume ofcell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet with Medium iN(G1P) toreach 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 iN(G1P), the total volume of
the medium 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 iN(G1P) + cell suspension

1.5 ml400 µl

70 µl

Viable cells/well< 2 hour

Day 1
Medium Change
1.

Prepare Medium N(G2P) using the reagents listed in the table below.
○ Warm/Thaw Component G2 and P for 20-30 minutes at the temperatures indicated in the “Contents” tableon page 1.
○ Warm Medium N for 20-30 minutes at room temperature.
○ Tap each Component tube 3 times and then briefly spin all tubes down before use.
○ Store Medium N(G2P) for up to 2 weeks at 4°C.
VolumeReagents
6-well plate24-well plate

96-well plate

Medium N

11 ml

22 ml

24.6 ml

Component G2

11 μl

22 μl

24.6 μl

Component P

11 μl

22 μl

24.6 μl

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52. Pipet out the old medium from each well and add Medium N(G2P) to each well along its wall according to the tablebelow.
○ 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

6-well plate24-well plate

96-well plate

2 ml

800 µl150 µlMedium N(G2P)

2. Incubate the culture at 37°C, 5% CO2 for 2 days.
< 1 hourDay 3Medium Change

1. Warm Medium N(G2P) at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well and add Medium N(G2P) to each well along its wall according to the tablebelow.
○ 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

6-well plate24-well plate

96-well plate

2 ml

800 µl150 µl

Medium N(G2P)

3. Incubate at 37°C, 5% CO2 for 3 days.
< 1 hourDay 6
Medium Change

4. Warm Medium N(G2P) at room temperature for 20-30 minutes.
5. Pipet out half the old medium from each well and add Medium N(G2P) to each well along its wall according to thetable below.
Required volume per wellReagents
Medium N(G2P)

6-well plate24-well plate

96-well plate

1 ml

400 µl75 µl

6. Incubate at 37°C, 5% CO2.

Day 7

< 1 hour

Assay or Continuous Maturation●●Differentiated neurons can be observed on Day 1-2. For more mature neurons, we recommend culturing cells until
Day 7. From Day 7, users may maintain differentiated neurons in the maintenance medium best suited for theirneeds, though we recommend Quick-Neuron™ GABAergic - Maintenance Medium, Catalog Number: GA-MM.
Differentiation into GABAergic neurons can be confirmed with the markers TUBB3, PVALB, and GAD1.

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Appendix

Figure 1. Representative phase contrast images of Quick-Neuron™ GABAergic - Human iPSC-derived Neurons cell cultures on days 1,2, 5 and 7 post-thaw (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ GABAergic - mRNA Kit cell cultures shows typical neurite growth andexpression of the pan-neuronal marker TUBB3 and the GABAergic neuron-specific marker PVALB on day 10 post-differentiation (scalebar = 100 μm). Staining conditions: Anti-β-III tubulin monoclonal antibody (R&D Systems, Catalog Number: MAB1195, 1:250 dilution) incombination with a secondary antibody (Invitrogen,Catalog Number: A32723 Goat anti-Mouse IgG (H+L) Highly Cross-AdsorbedSecondary Antibody, AlexaFluor Plus 488, 1:500 dilution). Anti-PVALB primary antibody (Novus Biologicals, Catalog Number:NB120-11427 , 1:1000 dilution) in combination with a secondary antibody (Invitrogen, Catalog Number: A11037 Goat anti-Rabbit IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

Figure 3. Real-time quantitative PCR analysis of expression levels of GABAergic neuron-associated genes PVALB and GAD1 wereexamined for Quick-Neuron™ - GABAergic - mRNA Culture on day 10. The relative gene expression is normalized to phosphoglycerate

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7kinase 1 (PGK1), and then calculated as a fold induction relative to undifferentiated hPSCs as a control. Error bars show standard
deviation. Primers used are listed in Table 1.
Table 1. List of PCR primers used in Figure 3
Gene

Forward Primer

Reverse Primer

Primer Concentration

PVALB

TCGACCACAAAAAGTTCTTCTTTAGGATGAATCCCAGCTC

250 nMGAD1GTCGAGGACTCTGGACAGTAGGAAGCAGATCTCTAGCAAA

250 nM

PGK1

GTATGCTGAGGCTGTCACTCGCCTTCCAGGAGCTCCAAACTGG

250 nMelixirgensci.com

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