User Guide
Quick-Muscle™ Skeletal - SeV Kit (Small)
Catalog Number: SM-SeV-S

Introduction
The Quick-Muscle™ Skeletal - SeV Kit 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 temperaturesensitive Sendai virus to produce a highly pure population of skeletal muscle cells without a genetic footprint.
Quick-Muscle™ Skeletal differentiated cell cultures display typical skeletal muscle morphology and markers, such as
myosin heavy chain (MHC).
Scale:The Quick-Muscle™ Skeletal - SeV Kit (Small) contains a set of reagents for use with a total of 4
wells of a 24-well plate.

Related Products:

Quick-Muscle™ Skeletal - Maintenance Medium, Catalog Number: SM-MM

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

VolumeStorage

Thaw

QMS-SeV110 μl-80°C

On iceMesendoderm RNA

4.5 μl-20°C or -80°C

On iceComponent P

50 μl-20°C or -80°C

Room temperature

Coating Material A

15.7 μl-20°C or -80°C

On ice or 4°C*IMPORTANT! This kit contains Sendai virus (SeV)
particles that are active at 33°C and become
inactive at 37°C. SeV is non-pathogenic in humans,and humans are not natural hosts of SeV; however,
Biosafety Level 2 (BSL-2) containment is requiredfor its use. Please use a biological safety cabinet,
laminar flow hood, and proper personal protective
equipment in order to prevent mucosal exposure.
More information on BSL-2 guidelines can be foundat www.cdc.gov/labs/BMBL.html.

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

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 25, 2023

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1Required Consumables
Item

VendorCatalog Number

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

Fisher Scientific07-200-740

Minimum Essential Medium (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

β-mercaptoethanol

ThermoFisher

21985023

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

Opti-MEM, reduced serum

ThermoFisher31985062

Lipofectamine MessengerMax

ThermoFisher

LMRNA015

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

ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

InvivoGen

ant-pr-1

Puromycin (10 mg/ml)

Source hPSC Culture Conditions
The Quick-Muscle™ Skeletal- SeV Kit (Small) gives the best differentiation results when source human pluripotent stem
cells (hPSCs) have been maintained in StemFit® Basic04, StemFit® AK02N, StemFlex™ Medium, or other similar culture
media which enable the maintenance of cultures 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 or well 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-C, NI511S).
● Differentiation should not be performed until the cells are at least 14 days post-thaw.
● We recommend preparing a minimum of 4.4 x 105 viable hPSC for use with this kit. This is usually obtained by using
1 well of a 6-well plate at 50-70% confluency.
● 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 0.5 and 2 µg/ml. 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 yourselected concentration so as to confirm that the puromycin is effective at killing the untransfected cells in your experiment.
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2Workflow
IMPORTANT! This workflow requires a humidified 33°C, 5% CO2 incubator. Before starting this protocol, please makesure the temperature is stable at 33°C.
Note: This protocol assumes that Day 0 is a Monday.

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

Preparation
10 mM β-mercaptoethanol
1. Mix 80 µl 55 mM β-mercaptoethanol with 360 µl PBS.
2. Filter, sterilize, and store at 4°C.
10 mM ROCK inhibitor Y27632 (iROCK)
3. Dissolve 10 mg ROCK inhibitor Y27632 in 3.12 ml DMSO.
4. Make aliquots of a convenient volume (e.g., 100 μl).
5. This solution, hereafter referred to as iROCK, can be stored at -20°C.
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.
Medium M
1.

Prepare Medium M using the reagents listed in the table below.
○ Warm all reagents at room temperature for 20-30 minutes.
○ Keep Medium M, and any subsequent media made with it, protected from light.
○ Store Medium M for up to 2 weeks at 4°C.
Reagents

VolumeMinimum Essential Medium (MEM) α, no nucleosides

13.5 ml

KnockOut Serum Replacement

750 μl100 mM Sodium Pyruvate

150 μlNon-essential amino acids (100x)

150 μlGlutamax (100x)

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

150 μl10 mM β-mercaptoethanol

150 μlelixirgensci.com

3Medium M(P)1.

Prepare Medium M(P) using the reagents listed in the table below.
○ Thaw Component P for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Warm Medium M at room temperature for 30 minutes.
○ Tap the Component P tube 3 times and then briefly spin it down before use.
○ Store Medium M(P) for up to 2 weeks at 4°C.
○ The rest of Medium M should be stored at 4°C for later use.
○ Leftover Component P can be discarded or saved at 4°C.
Reagents

VolumeMedium M

4.2 mlComponent P

2.1 μlDay -3< 1 hourNote: This protocol assumes that Day 0 is a Monday so Day -3 is Friday.
Plate Preparation1.

Prepare diluted Coating Material A by mixing together the following components in a 15 ml conical tube.
○ Thaw Coating Material A for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Keep Coating Material A on ice.
○ Make sure chilled PBS is used for this mixture.
Reagents

VolumeCoating Material A

6.0 µlChilled PBS

1.8 ml2. Add 400 µl diluted Coating Material A to each new well of 4 wells.
3. Incubate the plate at 4°C.
Note: For best results we recommend precoating the plate 1 day or up to a week before use and keeping at 4°C.
Alternatively plates can be precoated on Day 0 and placed at 37°C for at least 2 hours before use.

Day 04-6 hours

Note: This protocol assumes that Day 0 is a Monday.
Treatment
1.

Prepare Medium iM by mixing together the following components in a 15 ml conical tube.
○ Warm Medium M, iROCK, and Solution D1 at room temperature for at least 1 hour.
○ The rest of Medium M should be stored at 4°C for later use.
Reagents

VolumeMedium M

2.2 mliROCK

2.2 µlelixirgensci.com

42. Thaw QMS-SeV on ice.
3. Aspirate old medium from hPSC culture and add 2 ml PBS to each well being harvested.
4. Rock the plate 3 times, aspirate PBS from the culture, and add 300 μl of the cell dissociation reagent Solution D1 toeach well.
5. 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).

6. Carefully pipet out Solution D1 from the culture and add 1 ml Medium iM to the well.
○ Follow steps 7-8 one well at a time if multiple wells are used.
7. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
8. Using the same pipet tip, collect the cell suspension in a 1.5 ml tube.
Plating1.

Following this user guide, users will plate the hPSCs into 4 wells with 250 µl Medium iM per well. Please refer to thetable below for our plating recommendations for each of the 4 wells. If the volume of the cell suspension needed toget the desired cell density is greater than 250 µl, centrifuge the required volume of cell suspension at 200 x g for 4minutes, remove the supernatant, and resuspend the pellet into 250 µl Medium iM.
2. Aspirate diluted Coating Material A from only one coated well at a time and add 250 µl cell suspension to the well.
○ Most of the diluted Coating Material A should be aspirated but not completely to prevent the coated wellfrom drying before adding the cell suspension. The cell suspension should be added to the wellimmediately after diluted Coating Material A is removed.
3. Repeat Step 2 for the rest of the cell suspensions.
4. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute treated cells in thecultures.
IMPORTANT! Before adding QMS-SeV, ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV with a pipettor;
SeV is highly sensitive to physical stress.
5. Add the indicated volume of QMS-SeV into each well using a P200 pipettor.
○ By pushing the plunger, a small drop should be formed. As the drop is formed it can touch the medium
surface. Repeat this process by making several drops to touch, e.g., 12, 3, 6 and 9 o’clock of the medium
surface or until the tip becomes empty.
Cell density per well

QMS-SeV

0.8 x 105 cells

20 μl

1.0 x 105 cells

25 μl

1.2 x 105 cells

30 μl

1.4 x 105 cells

35 μl

6. Incubate the cultures at 33°C, 5% CO2 overnight.

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5Day 1~4 hours

IMPORTANT! The duration of incubation after the medium change is critical for the second treatment. If the confluency of
any of the cultures is 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 any of the cultures have less than 50% confluency, the cultures should beincubated up to 4 hours. If the cultures have greater than 70% confluency, the first treatment should be done within 1 hour
after the medium change, but the differentiation efficiency is expected to be low.
Medium Change
1. Warm Medium M at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well and add 250 μl Medium M to each well.
3. Incubate the cultures at 33°C, 5% CO2 for 1-4 hours (see the note above).
Mesendoderm RNA treatment
1.

45 minutes before the above incubation is completed, thaw Mesendoderm RNA on ice for 30 minutes and warm
Opti-MEM at room temperature for 20-30 minutes.
2. Prepare Mesendoderm Booster by the following steps:○ Tap the Mesendoderm RNA tube 3 times and then briefly spin it down before use○ Prepare a 15 ml tube and a 1.5 ml tube with 110 μl Opti-MEM each. Label the 15 ml tube “Mix 1” and the 1.5ml tube “Mix 2”.
○ Add 4.5 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 4.5 μl Mesendoderm RNA
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 Mesendoderm Booster. Leave Mesendoderm Booster at room temperature for 5 minutes
and no longer.
Mix 1 Reagents

VolumeMix 2 Reagents

VolumeOpti-MEM

110 µl

Opti-MEM

110 µlLMM

4.5 µlMesendoderm RNA

4.5 µl3. Add 1 ml Medium M to Mesendoderm Booster (for a final volume of 1.2 ml) 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 300 μl of Mesendoderm Booster mixture
to each well. Repeat until Mesendoderm Booster mixture has been added to all wells.
5. Incubate the culture plate at 33°C, 5% CO2 for 2.5 hours.
Medium Change
1. Warm Medium M at room temperature for 20-30 minutes.
2. Pipet out the medium from each well and add 500 μl Medium M.
3. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 2< 1 hourMedium Change and Drug Selection
1. Warm Medium M at room temperature for 20-30 minutes.
2. Transfer 2 ml Medium M into a tube and add puromycin to it at the predetermined optimal concentration (see earlier
section on “Drug Selection”).

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63. Pipet out most of the medium from each well and add 500 μl Medium M with puromycin.
4. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 3< 1 hourMedium Change and Temperature Shift
Note: This should be performed in the late afternoon.
1. Warm Medium M at room temperature for 20-30 minutes.
2. Pipet out most of the medium from each well and add 500 µl Medium M.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 4< 1 hour

Maintenance
1. Warm Medium M(P) at room temperature for 20-30 minutes.
2. Pipet out most of the medium from each well and add 1 ml Medium M(P).
3. Incubate the cultures at 37°C, 5% CO2 for 3 days.

Day 7
Assay or Continuous Maturation●●Elongated, spindle-shaped muscle cells can be observed on Day 4. For more mature skeletal muscle, werecommend culturing cells until Day 7. From Day 7, users may maintain differentiated skeletal muscle in themaintenance medium best suited for their needs, though we recommend Quick-Muscle™ Skeletal - MaintenanceMedium, Catalog Number: SM-MM.
Differentiation into skeletal muscle can be confirmed with an anti-MHC (myosin heavy chain) antibody.

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7

Appendix

Figure 1. Representative images of Quick-Muscle™ Skeletal - SeV Kit cell culture (plated with 1.2 x 105 cells) on days 1, 2, 4, and 7
post-differentiation (scale bars = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Muscle™ Skeletal - SeV cell culture (plated with 1.2 x 105 cells) shows typical skeletal
muscle morphology and expression of myosin heavy chain (MHC) on day 7. Nuclei were counterstained with Hoechst 33324. (scalebars = 200 μm)

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