User Guide

Quick-MuscleTM Skeletal - mRNA Kit (Small)
Catalog Number: SM-mRNA-S

Introduction
The Quick-Muscle™ Skeletal - mRNA Kit (Small) facilitates rapid and efficient differentiation of human iPS or ES cells
into skeletal muscle cells in just 7 days. Our proprietary transcription factor-based stem cell differentiation method uses
synthetic mRNAs to produce myosin heavy chain (MHC)-positive muscle cells without leaving a genetic footprint.
Scale: 						The Quick-Muscle™ Skeletal - mRNA Kit (Small) contains a set of reagents for use with atotal of 4 wells of a 24-well plate.

Related Products:			

Quick-Muscle™ Skeletal - SeV Kit (Small) , Catalog Number: SM-SeV
Quick-Muscle™ Skeletal - Maintenance Medium, Catalog Number: SM-MM

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

VolumeStorage

QM1-mRNA

9 μl (4.4 μg)-80°C

QM2-mRNA

9 μl (4.4 μg) x 3-80°C

Component P

14 μl-20°C or -80°C

Coating Material A

15.7 μl-20°C or -80°C

This kit contains iMatrix-511 silk (Nippi, Inc.).

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

Last revised: January 22, 2021

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1Required Consumables
ItemVendor

Catalog Number

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

Fisher Scientific07-200-740

Lipofectamine MessengerMAX

ThermoFisherLMRNA001

Opti-MEM I Reduced Serum Medium

ThermoFisher

31985062

Minimum Essential Media (MEM) α, no nucleosides

ThermoFisher

12561056

KnockOut Serum Replacement

ThermoFisher

10828010

Sodium Pyruvate (100 mM)

ThermoFisher

11360070

MEM Non-Essential Amino Acids Solution (100X)

ThermoFisher

11140050

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122β-mercaptoethanol (β-ME)

ThermoFisher

21985023

StemFit Basic04 Complete Type, or
StemFit Basic04, or
StemFit Basic02, or
StemFlex Medium

Elixirgen Scientific
Elixirgen Scientific
Elixirgen Scientific
ThermoFisher

ASB04-C, or
ASB04-F, or
ASB02, orA3349401

TrypLE Select Enzyme (1X)*

ThermoFisher

12563011

Sigma-Aldrich

E8008-100ML

Phosphate-buffered saline (without Ca Mg )ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D8418

0.02% EDTA in DPBS++++

* Can be substituted with our Cell Dissociation Reagent (Solution D1), Catalog Number: CDR.

Source hPSC Culture Conditions
The Quick-Muscle™ Skeletal - mRNA Kit (Small) gives best differentiation results when source hPSCs have been
maintained in StemFit® Basic04, StemFlex™ Medium, or other similar culture media which enable the maintenance ofcultures by single-cell passaging. This protocol also assumes that the source hPSCs are cultured in a 35-mm culture
dish or one well of a 6-well plate. If iMatrix-511 silk is routinely used as a coating substrate, prepare one culture dish orwell precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS for this kit.
• The protocols and reagents for StemFit® Basic04 and iMatrix-511 silk culture conditions are available at Elixirgen
Scientific (Catalog Numbers: ASB04, NI511S).
• Differentiation should not be performed until the cells are at least 14 days post-thaw.

WorkflowMedia
Preparation

Plate
Preparation
Plating

1st Treatment

2nd Treatment
3rd Treatment

4th treatment

Day -1

Day 0

Day 1

Day 2

Day 3

Medium iS

Medium M

Maintenance...
Medium M(P)Day 7...

Day 10SM-MM*

From Day 7, users may maintain differentiated cells in the maintenance medium best suited for their needs, though we
recommend Quick-Muscle™ Skeletal - Maintenance Medium, Catalog Number: SM-MM.

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Media Preparation
IMPORTANT! For the best possible delivery of QM1-mRNA and QM2-mRNA 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. QM1-mRNA or QM2-mRNA mixed with Lipofectamine MessengerMax must be
immediately applied to cultures and cannot be stored.
10 mM β-ME
1. Mix 80 µl 55 mM β-ME with 360 µl PBS.
2. Filter sterilize and store at 4°C.
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 is hereafter referred to as iROCK and 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 is hereafter referred to as Medium S and can be stored at -80°C.
*Medium S can be substituted with StemFit Basic02/04 (with bFGF added) or StemFlex.
0.5X TrypLE Select with EDTA (Solution D1)*
1. Mix 1 ml TrypLE Select Enzyme (1X) with 1 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.
Medium M
1. Prepare Medium M using the reagents listed in the table below.
• Thaw all reagents at room temperature for 20-30 minutes.
2. Store Medium M for up to 2 weeks at 4°C. The leftover reagents can be discarded or saved for other uses.
Medium M Reagents

VolumeMinimum Essential Media (MEM) α, no nucleosides

18 ml

KnockOut Serum Replacement

1 ml

Sodium Pyruvate (100 mM)

200 μlMEM Non-Essential Amino Acids Solution (100X)

200 μl200 mM Glutamax (100x)

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

200 μl10 mM β-ME

200 μlMedium M(P)1. Prepare Medium M(P) using the reagents listed in the table below.
• Take out all required reagents and incubate at room temperature for 20-30 minutes.
2. Store Medium M(P) for up to 2 weeks at 4°C.
Medium M(P) Reagents

VolumeMedium M

9 ml

Component P

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~3 hrDay 0
Plate Preparation
1. Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
• Thaw Coating Material A on ice for 20-30 minutes (or at 4°C overnight one day before Day 0).
• Make sure chilled PBS is used for this mixture.

2.
3.
4.
5.

Diluted Coating Material A Reagents

VolumeCoating Material A

11.9 µl

Chilled PBS

3.6 ml

Add 400 µl diluted Coating Material A to each new well of 8 wells.
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
1. 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.

2.
3.
4.
5.

Medium iS Reagents

VolumeMedium S

5.5 mliROCK5.5 µl

Tap the Solution D1 tube 5 times with a finger and centrifuge at maximum speed for 1 minute.
Aspirate old medium from hPSC culture and add 2 ml PBS.
Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1.
Incubate the plate at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope, incubate at37°C, 5% CO2 for up to 5 more minutes in 1-2 minute increments (see images below).
Before treatment

Almost ready

Ready to remove

Treated too long6. Carefully pipet out Solution D1 from the culture using a P1000 pipettor and add 1 ml Medium iS to the well.
7. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
8. Using the same pipette tip, collect the cell suspension in a 1.5 ml tube.
IMPORTANT! In this protocol, users will plate the hPSCs into 8 wells with 500 µl Medium iS per well. However, if users
know the best plating density already, prepare a cell suspension enough to plate 5 wells (4 wells plus 1 extra to accountfor the pipetting/plating error). We recommend aiming for 50-70% initial cell confluency on Day 1, with 20-50 cells percolony. Our data indicate that cell counts ranging from 1.0 - 2.4 x 105 viable cells per well are suitable. Cell count mayvary based on cell health and the method used for cell counting.
If the confluency on Day 1 is• above the target range, the differentiation efficiency will decrease.
• below the target range, more cell death will be observed.

Note continued on next page4elixirgensci.com

For first time users, we recommend plating the following numbers of cells into each of 8 wells: 1.0, 1.2, 1.4, 1.6, 1.8, 2.0,2.2 and 2.4 x 105 cells. On Day 1, 4 wells showing 50-70% confluency should be selected for transfection.
To those who know the cell number that shows 50-70% confluency on Day 1 after plating, the range of cell numbers canbe narrower and more specific. For example, if you already know plating 1.2 - 1.4 x 105 live cells will result in 50-70%
confluency, our recommendation for preparing 8 wells is as follows: 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 and 1.7 x 105 live
cells.
9. Take out the volume of the cell suspension needed for each cell density, according to the note above, and placeeach in a new tube labeled with the corresponding density.
10. Bring the volume of the cell suspension in each tube up to 500 µl with Medium iS.
• If the volume in the tube exceeds 500 µl, centrifuge the required volume of cell suspension at 200xg for
4 minutes, remove the supernatant, and resuspend the pellet in 500 μl Medium iS.
11. Aspirate PBS from each coated well and add 500 µl cell suspension to each well using a P1000 pipettor.
12. Place the plate in the incubator and rock it front to back and side to side for 15 seconds to make sure that the cells
are evenly distributed.
13. Incubate the cultures at 37°C, 5% CO2 overnight.

~5-8 hr

Day 1IMPORTANT! In this protocol, users will identify the best 4 wells with confluency of 50-70% after replacing Medium iSwith Medium M in the morning. After the medium change, cells will exhibit a more flattened shape, thereby increasing
the confluency. The duration of incubation after the medium change is critical for the first treatment. If users can select 4cultures having a confluency falling between 50-70%, the first treatment can be started anytime (even immediately after
medium change) up to 1 hour after the medium change. However, if all of the cultures have less than 50% confluency,
the cultures can be incubated up to 4 hours until they start to exhibit 50-70% confluency. If all of the cultures havegreater than 70% confluency, the first treatment should be done immediately after the medium change, although thedifferentiation efficiency is expected to be low.
First Treatment
1. Thaw 1 vial of QM1-mRNA on ice for 30 minutes and warm Opti-MEM and Medium M at room temperature for
20-30 minutes.
2. According to the note above, select 4 cultures with 50-70% confluency. Aspirate the old medium from each welland add 500 μl Medium M. Repeat this process for each of the selected 4 wells. Do not let cells dry out during themedium change.
3. Incubate the cultures at 37°C, 5% CO2 as needed.
4. Prepare QM1 by the following steps:• Prepare two 1.5 ml tubes with 220 μl Opti-MEM each.
• Add 9 μl LMM to one of the 1.5 ml tubes and mix by brief vortexing. Leave it at room temperature for 10minutes (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 9 μl QM1-mRNA to theother 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. Thismixture is called QM1. Leave QM1 at room temperature for 5 minutes and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

220 µl

Opti-MEM

220 µlLMM

9 µl

QM1-mRNA

9 µl

5. Add 104 μl QM1 to each culture by adding QM1 dropwise with one hand while gently shaking the plate with theother hand.
6. Rock the plate front to back and side to side for 15 seconds to make sure that QM1 is evenly distributed in thecultures.
7. Incubate the cultures at 37°C, 5% CO2 for 2.5-3 hours.
8. Pipet out the medium with QM1 from each well using a P1000 pipettor and add 500 μl Medium M.
9. Incubate the cultures at 37°C, 5% CO2 overnight.

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~8 hrDay 2
Second Treatment

1. Observe the QM1-treated cultures to ensure they are at 60-80% confluency before proceeding. If the cultures havea lower confluency than 60%, cell death is expected at some degree after the following treatments. If the cultures
have a greater confluency than 80%, then the differentiation efficiency is expected to be lower.
2. Thaw 1 vial of QM2-mRNA on ice for 30 minutes and warm Opti-MEM and Medium M at room temperature for 2030 minutes.
3. Pipet out the medium from each of the 4 wells using a P1000 pipettor and add 500 μl Medium M.
4. Incubate the cultures at 37°C, 5% CO2 for at least 10 minutes.
5. Prepare QM2 by the following steps:• Prepare two 1.5 ml tubes with 220 μl Opti-MEM each.
• Add 9 μl LMM to one of the 1.5 ml tubes and mix by brief vortexing. Leave it at room temperature for 10minutes (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 9 μl QM2-mRNA to theother 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 QM2. Leave QM2 at room temperature for 5 minutes and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

220 µl

Opti-MEM

220 µlLMM

9 µl

QM2-mRNA

9 µl

6. Add 104 μl QM2 to each culture by adding QM2 dropwise with one hand while gently shaking the plate with theother hand.
7. Rock the plate front to back and side to side for 15 seconds to make sure that QM2 is evenly distributed in thecultures.
8. Incubate the cultures at 37°C, 5% CO2 for 2.5-3 hours.
Third treatment
1. Pipet out the medium from each of 4 wells using a P1000 pipettor and add 500 μl Medium M to the wells.
2. Incubate the cultures at 37°C, 5% CO2 for 2 hours.
• Put 1 vial of QM2-mRNA on ice when the incubation above reaches 1.5 hours and leave it for 30 minutes.
3. Repeat Steps 5-8 of the “Second Treatment” section.
4. Pipet out the medium from each well using a P1000 pipettor and add 500 μl Medium M to it.
5. Incubate the cultures at 37°C, 5% CO2 overnight.

Day 3~3 hr

Fourth treatment
1. Observe the QM2-treated cultures to ensure they are reaching almost 100% confluency before proceeding. If thecultures have a significantly lower confluency, e.g., less than 50%, cell death has been induced. If the cultures havegrown over confluent, the differentiation efficiency is expected to be lower.
2. Thaw 1 vial of QM2-mRNA on ice for 30 minutes and warm Opti-MEM and Medium M at room temperature for
20-30 minutes.
3. Pipet out the medium from each of the 4 wells using a P1000 pipettor and add 500 μl Medium M.
4. Incubate the cultures at 37°C, 5% CO2 for at least 10 minutes.
5. Repeat Steps 5-8 of the “Second Treatment” section.
6. Warm Medium M(P) at room temperature for 20-30 minutes.
7. Pipet out the medium with QM2 from each well using a P1000 pipettor and add 500 μl Medium M(P) to it.
8. Incubate the cultures at 37°C, 5% CO2 overnight.

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Day 4<1 hrMaintenance1. Observe the cultures under a microscope and confirm the presence of elongated, spindle-shaped cells in the 		culture. A certain degree of cell death is expected.
2. Warm Medium M(P) at room temperature for 20-30 minutes.
3. Pipet out the old medium from each well using a P1000 pipettor and add 1 ml Medium M(P).
4. Incubate the cultures at 37°C, 5% CO2 for 3 days.

Day 7
Assay or Continuous Maturation
Elongated, spindle-shaped cells can be observed on Day 4. For more mature skeletal muscle, we recommend culturing
cells until Day 7. From Day 7, users may maintain differentiated cultures in the maintenance medium best suited fortheir needs, though we recommend Quick-Muscle™ Skeletal - Maintenance Medium, Catalog Number: SM-MM.
Differentiation into skeletal muscle after using the Quick-Muscle™ Skeletal - mRNA Kit can be confirmed with an 		anti-MHC (myosin heavy chain, a marker for skeletal muscle) antibody.

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Appendix

Figure 1. Representative images of Quick-Muscle™ Skeletal - mRNA Kit cell cultures on days 1-6 post-differentiation(scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Muscle™ Skeletal - mRNA cell cultures shows typical skeletal muscle
morphology and expression of myosin heavy chain (MHC) on day 6 post-differentiation. Staining conditions: Anti-MHC
primary antibody (R&D Systems, Catalog Number: MAB4470, 1:250 dilution) in combination with a secondary antibody
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen,Catalog Number:A11032 , 1:500 dilution). Nuclei were counterstained with Hoechst 33324.

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