User Guide

Quick-Neuron™ Cholinergic - mRNA Kit (Large)
Catalog Number: CH-mRNA-L

Introduction
The Quick-Neuron™ Cholinergic - mRNA Kit (Large) facilitates rapid and efficient differentiation of human iPS or ES cells
into a population of cholinergic neurons in just 7 days. Our proprietary transcription factor-based stem cell differentiation
method uses synthetic mRNAs to produce highly pure populations of neurons without a genetic footprint. Quick-Neuron™
Cholinergic differentiated cell cultures display typical neurite outgrowth and express a variety of neuronal markers, such as
the pan-neuronal marker tubulin beta 3 class III (TUBB3) and the cholinergic marker choline acetyl-transferase (ChAT).
When handled and maintained according to the instructions in this user guide, cholinergic neurons are viable long-term andare suitable for a variety of characterization and neurotoxicity assays.
Scale:The Quick-Neuron™ Cholinergic - mRNA Kit (Large) contains a set of reagents for use with a totalof 6 wells of a 6-well plate.

Related Products:

Quick-Neuron™ Cholinergic mRNA Kit (Small), Catalog Number: CH-mRNA-S
Quick-Neuron™ Cholinergic - Human iPSC-derived Neurons, Catalog Number: CH-mRNA-CW
Quick-Neuron™ Cholinergic - Maintenance Medium, Catalog Number: CH-MM

Kit Contents
Upon receipt, store the reagents at the temperatures indicated in the table below. All reagents are shipped on dry ice.
Contents

VolumeStorage

QN-mRNA-P

4 x 33 μl-80°C

Component N1

2 x 830 μl-20°C or -80°C

Component P

2 x 14 μl-20°C or -80°C

Component A

1 x 38 μl-20°C or -80°C

Conditions 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: April 18, 2023

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Required Consumables
Item

VendorCatalog Number

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 Scientific™ 96 Well Black/Clear Bottom Plate)Fisher Scientific12-566-70

Lipofectamine MessengerMAX

ThermoFisher

LMRNA001

Opti-MEM I Reduced Serum Medium

ThermoFisher

31985062

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

GlutaMAX

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

StemFit Basic04 Complete Type, or
StemFlex Medium

Elixirgen Scientific
ThermoFisher

ASB04-C, or
A3349401

iMatrix-511 silk

Elixirgen Scientific

NI511S

TrypLE Select Enzyme (1X)*

ThermoFisher

12563011

0.02% EDTA in DPBS*

Sigma-Aldrich

E8008-100ML

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

D8418

Puromycin (10 mg/ml)

InvivoGen

ant-pr-1(Optional) STEM-CELLBANKER***AMSBIO

11890* Can be substituted with our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.
** Can be substituted with our Neuron Coating Solutions (Coating Materials B and C), Catalog Number: NCS.
*** This is only required if you intend to cryopreserve the cells after differentiation.

Source hPSC Culture Conditions
The Quick-Neuron™ Cholinergic - mRNA Kit (Large) gives the best differentiation results when source human pluripotent
stem cells (hPSCs) have been maintained in StemFit® Basic04, StemFlex™ Medium, or other similar culture media whichenable the maintenance of cultures by single-cell passaging. This protocol also assumes that the source hPSCs arecultured in two to three 35-mm culture dishes or two to three wells of a 6-well plate. If iMatrix-511 silk is routinely used as a
coating substrate, prepare one culture dish or well precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS for
this kit.

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●
●
●

The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04-C, NI511S).
Differentiation should not be performed until the cells are at least 14 days post-thaw.
For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

Drug Selection
Users should perform a puromycin kill curve for their cells to determine the minimum concentration required to kill all
non-treated cells within ~60 hours. Based on Elixirgen Scientific’s internal tests, the appropriate concentration ranges
between 0.25 and 3 µg/ml. We achieved successful results using 1 μg/ml puromycin for 16 hours after the second QN
treatment, and for 40-50 hours after the fourth QN treatment. We recommend maintaining 2 wells of untransfected iPSC
(with standard StemFit conditions), alongside the transfected wells, until after puromycin selection is performed. Treat 1 of
those wells with puromycin at your selected concentration so as to confirm that the puromycin is effective at killing theuntransfected cells in your experiment.

Workflow*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: CH-MM.

Media Preparation
Important Note! For the best possible delivery of QN-mRNA-P into cells, we recommend Lipofectamine MessengerMax. If
users prefer another transfection reagent, please make sure that the reagent provides a transfection efficiency of ≥80%
prior to using this kit. QN-mRNA-P mixed with Lipofectamine MessengerMax must be immediately applied to cultures andcannot be stored.
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.
StemFit Basic04 Complete Type (Medium S)*
1. Thaw StemFit Basic04 Complete Type bottle overnight or multiple nights at 4°C.
2. Make aliquots of a convenient volume (e.g., 40 ml).
3. This solution, hereafter referred to as Medium S, can be stored at -80°C. Once thawed, Medium S should be stored
at 4°C for up to 2 weeks.
○ After thawing users may choose to add Penicillin-Streptomycin at a 1:200 dilution (e.g., 200 μl in 40 ml ofMedium S) before using Medium S.
*Medium S can be substituted with StemFlex.

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0.5X TrypLE Select with EDTA (Solution D1)*
1. Mix 1.5 ml TrypLE Select Enzyme (1X) with 1.5 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.
*Can be substituted with our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.

0.002% Poly-L-Ornithine solution (ornithine)*
1. Take 2 ml 0.01% Poly-L-Ornithine solution and mix it with 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.
*Ornithine can be substituted with Coating Material B from our Neuron Coating Solutions, Catalog Number: NCS.

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., 90 μl) and store at -20°C.
*Laminin can be substituted with Coating Material C from our Neuron Coating Solutions, Catalog Number: NCS.

Medium N1(AP)1.

Prepare Medium N1(AP) using the reagents listed in the table below.
○ Thaw Component N1 on ice for 20-30 minutes.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Store Medium N1(AP) for up to 2 weeks at 4°C. The leftover reagents can be discarded or saved for otheruses.
Medium N1(AP) Reagents

VolumeDMEM/F1217.3 ml

Neurobasal

17.3 mlGlutaMAX

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

360 μlComponent N1

1.10 ml

Component A

36.3 μl

Component P

18.2 μl

Day 0~8 hours

Note: This protocol assumes that Day 0 is a Monday and that user’s hPSCs have already been used with a small size kit(Catalog number: CH-mRNA-S) so that users are familiar with the experimental process and have optimized conditions
for their particular cells.
Plate Preparation1.

Prepare diluted iMatrix-511 silk by mixing together the following components in a 15 ml conical tube.
○ Keep iMatrix-511 silk on ice and make sure chilled PBS is used for this mixture.

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2.
3.
4.
5.

Diluted iMatrix-511 silk Reagents

VolumeiMatrix-511 silk

43.2 µl

Chilled PBS

13 ml

Add 2 ml diluted iMatrix-511 silk to each new well of a 6-well plate.
Incubate the plate at 37°C, 5% CO2 for 2 hours (or 4°C overnight one day before Day 0).
Aspirate the supernatant from each well and add 500 µl PBS.
Incubate the plate at 37°C, 5% CO2 until the hPSCs are ready for plating.

Plating
IMPORTANT! Source hPSC wells should be no more than 50-70% confluent thus requiring a minimum of 2 wells to
begin differentiation.
Determine the number of wells required to get 3.6 x 106 cells from the source hPSC 6-well plate.
NOTE: Cells will be plated in a new 6-well plate at 3 densities (0.5 x 106 cells, 0.55 x 106 cells, and 0.6 x 106 cells),
with 2 wells per density.
2. Prepare Medium iS by mixing together the following components in a 15 ml conical tube.
○ Warm Medium S, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium S should be stored at 4°C for later use.
1.

Medium iS Reagents

Required medium volume based on # of wells of a
6-well plate
2 wells

3 wells

Medium S

12.7 ml

15.5 ml

iROCK

12.7 µl15.5 µl

3. Aspirate old medium from hPSC culture and add 1.5 ml of Medium iS to each well.
4. Incubate the culture at 37°C, 5% CO2 for 1 hour before harvesting cells.
○ This is to decrease cell death on Day 1 and minimize the loss of cells.
5. Aspirate old medium from hPSC culture and add 2 ml PBS to each well being harvested.
6. Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
○ Keep the rest of Solution D1 at 4°C for use on Day 4.
7. Incubate the culture plate at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope,
continue to incubate at 37°C, 5% CO2 in 1-2 minute increments (see images below).

8. Carefully pipet out Solution D1 from the culture using a P1000 pipettor and add 1 ml Medium iS to the well.
○ Follow Steps 8-10 one well at a time if multiple wells are used.
9. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
10. Using the same pipet tip, collect the cell suspension in a 15 ml tube.
11. Count cells and determine viability.

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512. Take out the volume of the cell suspension needed for 2 wells of each cell density, according to the note in step 1,
and include an extra 10% as a buffer. Place each in a new tube labeled with the corresponding density.
13. Bring the volume of the cell suspension in each tube up to 2.2 ml with Medium iS.
○ If the volume in the tube exceeds 2.2 ml, centrifuge the required volume of cell suspension at 200 x g for 4
minutes, remove the supernatant, and resuspend the pellet in 2.2 ml Medium iS.
14. Aspirate PBS from each coated well and add 1 ml cell suspension to each well.
15. Leave the plate flat at room temperature for 10 minutes.
16. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 1~8 hours

IMPORTANT! Observe all wells under a microscope and confirm that all 6 wells show 50-70% confluency fortransfections with QN-mRNA-P. If there are any wells that do not fall within the range of confluence, do not use them.
First Treatment
1.

Thaw 1 vial of QN-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium S at room temperature for
20-30 minutes.
2. Prepare QN by the following steps:○ Prepare a 15 ml tube and a 1.5 ml tube with 825 μl Opti-MEM each. Label the 15 ml tube “Mix 1” and the 1.5
ml tube “Mix 2”.
○ Add 16.5 μl Lipofectamine MessengerMax (LMM) to the Mix 1 tube and mix by brief vortexing. Leave it atroom temperature for 10 minutes (Mix 1). Keep the rest of LMM at 4°C for later treatments.
○ IMPORTANT! Immediately before 10 minutes pass (i.e., around 8 minutes), add the entire contents of the
QN-mRNA-P vial to the other 1.5 ml tube with Opti-MEM (Mix 2). Mix by tapping 5 times. Do not vortex.
○ 10 minutes after mixing LMM with Opti-MEM, add Mix 2 into Mix 1, and pipet up and down 8-10 times. This
mixture is called QN. Leave QN at room temperature for 5 minutes and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

825 µl

Opti-MEM

825 µl

LMM

16.5 µl

QN-mRNA-P~33 µl3. Add 6.6 ml Medium S to QN and pipet up and down 2-3 times to mix.
4. Working with up to 2 wells at a time, aspirate the old medium out and add 1.25 ml of QN mixture to each well.
Repeat until QN mixture has been added to all wells.
5. Incubate the culture plate at 37°C, 5% CO2 for 2.5 hours.
Second Treatment
1. Pipet out the medium from each well using a P1000 pipettor and add 1 ml Medium S.
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QN-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section.
Medium Change and Drug Selection
1.

Transfer 10 ml Medium S into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
2. Pipet out the medium from each well and add 1.5 ml Medium S with puromycin.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

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6Day 2~8 hours

IMPORTANT! Observe the QN-treated cultures to make sure that they are reaching confluency (≥90%). If the culturesare <50% confluent and show signs of cell death (e.g., many floating cells), users should skip the third and fourth
treatments on Day 2 and proceed directly to the steps described in Day 3. The protocol will then be accelerated by oneday.
Third Treatment
1.

Thaw 1 vial of QN-mRNA-P on ice for 30 minutes and warm Opti-MEM and Medium S at room temperature for
20-30 minutes.
2. Repeat Steps 2-5 of the previous “First Treatment” section.
Fourth Treatment
1. Pipet out the medium from each well and add 1 ml Medium S.
2. Incubate the culture plate at 37°C, 5% CO2 for 2 hours.
○ Put 1 vial of QN-mRNA-P on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 2-5 of the previous “First Treatment” section.
Medium Change and Drug Selection
1.

Transfer 10 ml Medium S into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
○ If more than 90% of the cells show resistance to puromycin at the concentration used on Day 1, consider
increasing its concentration.
2. Pipet out the medium from each well and add 1.5 ml Medium S with puromycin.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 3< 1 hourMedium Change and Drug Selection
1. Warm Medium N1(AP) at room temperature for 20-30 minutes.
2. Transfer 10 ml Medium N1(AP) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
3. Pipet out the medium from each well and add 1.5 ml Medium N1(AP) with puromycin.
4. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 4

4-6 hours

New Plate Preparation
IMPORTANT! This kit can accommodate replating to all wells of either a 6-well, a 24-well, or a 96-well plate. Refer to thetables for the recommended volumes. Please note that the volumes are per well in Table A and per plate in Table B.
1. Vortex ornithine* briefly and centrifuge it at a maximum speed for 1 minute.
2. Add ornithine to each well of a new plate in the volume specified in Table A.
3. Incubate the plate at 37°C, 5% CO2 for at least 2 hours (or at 4°C overnight one day before plating).
4. Thaw laminin* and chill specified amounts of PBS on ice for 20-30 minutes.
5. Add laminin to chilled PBS in the volume specified in Table B. Mix well.
○ All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
6. Aspirate the supernatant from each well and add PBS in the volume specified in Table A.
7. Repeat Step 6.
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8. Aspirate PBS from each well and add diluted laminin according to Table A.
9. Incubate the plate at 37°C, 5% CO2 for at least 2 hours.
*Ornithine and laminin can be substituted with Coating Materials B and C, respectively, from our Neuron Coating Solutions, Catalog
Number: NCS.

Table A. Recommended volumes per well for different plate formats.
Recommended volume per well
Reagents

6-well plate24-well plate

96-well plate

Ornithine

1.5 ml300 µl

50 µl

PBS

2 ml

500 µl100 µlDiluted laminin

1.5 ml300 µl

50 µl

Medium N1(AP)

2.5 ml700 µl

85 µl

Table B. Recommended volumes per plate for different plate formats.
Recommended volume per plate
Reagents
Diluted laminin

6-well plate24-well plate

96-well plate

Laminin

100 µl

80 µl

53 µl

Chilled PBS

10 ml

8 ml

5.3 mlMedium Preparation
1. Warm Medium N1(AP) at room temperature for 20-30 minutes.
2. After the laminin incubation, aspirate most, but not all, of the supernatant from each well of the new plate and add
PBS in the volume specified in Table A above. Add the PBS dropwise to each well.
3. Aspirate most, but not all of the PBS and add Medium N1(AP) in the volume specified in Table A above.
4. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care.
1.
2.
3.
4.
5.
6.

Make sure that Solution D1 is at room temperature for at least 1 hour before use.
Working one well at a time, pipet out the old medium from each well and add 1 ml PBS to the well.
Working one well at a time, pipet out the PBS from each well and add 300 µl Solution D1.
Rock the plate 3 times to spread the Solution D1 evenly.
Incubate the cultures at 37°C, 5% CO2 for 3 minutes.
Working one well at a time, carefully pipet out Solution D1 from each well and add 750 μl Medium N1(AP).

IMPORTANT! Steps 7-9 below are critical. Perform these steps one well at a time. Refer to the images below tosuccessfully manage cell treatment and dissociation.

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7.

Working one well at a time, disperse the medium quickly over the bottom surface of the well by pipetting 6-8 times
to detach cells using a P1000 pipettor.
8. Observe cells and/or cell aggregates floating in the well under a microscope. It is normal that 10-20% of cells
remain attached to the well bottom after pipetting. These clusters of cells are not supposed to be lifted. Do not
attempt to detach all of the cells remaining on the well bottom.
9. Collect 750 μl cell suspension from each well and transfer to a tube.
10. Gently pipet the cell suspension up and down up to 5 times to break the cell aggregates using a P1000 pipettor.
Excessive pipetting can damage the already-suspended neuronal cells.
11. Count cells and determine viability.
12. Prepare specified amounts of a 1 x 10⁶ live cells/ml cell suspension using Medium N1(AP) based on the table below.
○ If there are leftover cells, freeze the cells down by following instructions in Appendix B after plating cellsuspensions on the new plate. Keep the leftover cells on ice until freezing.
13. Add cell suspension to the center of each well. Since each well already has Medium N1(AP), the total volume of themedium in each well is indicated in the table below.
Recommended Amounts
6-well plate24-well plate

96-well plate5 x 105 cells

1 x 105 cells

1.5 x 104 cells

Req vol of cell suspension (1 x 10⁶ viable cells/ml)● (Vol of cell suspension/well x # of wells) +10% buffer

3.3 ml2.64 ml

1.6 mlVolume of cell suspension/well

500 µl100 µl

15 µl

Total volume/well● Medium N1(AP) + cell suspension

3 ml

800 µl100 µlViable cells/well

14. Incubate the culture plate at 37°C, 5% CO2 for 3 days.

Day 7
Assay or Continuous Maturation●●

Differentiated neurons can be observed on Day 4. For more mature neurons, we recommend culturing cells until
Day 7. 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: CH-MM.
Differentiation into Cholinergic neurons after using Quick-Neuron™ Cholinergic - mRNA Kit can be confirmed withthe markers TUBB3 and ChAT.

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9Appendix

Figure 1. Representative phase contrast images of Quick-Neuron™ Cholinergic - mRNA cell cultures on days 1-7 post-differentiation(scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Cholinergic - mRNA cell culture shows typical neurite growth and expressionof the pan-neuronal marker TUBB3 and CHAT on day 7 post-differentiation (scale bars = 50 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-CHAT primary antibody (Millipore Sigma, Catalog Number: AMAB91130, 1:500 dilution) was used in
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.

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