User Guide

Quick-NeuronTM Excitatory - SeV Kit (Large)
Catalog Number: EX-SeV-L

Introduction
The Quick-Neuron™ Excitatory - SeV Kit (Large) facilitates rapid and efficient differentiation of human iPS or ES cells
into excitatory neurons in just 10 days. Our proprietary transcription factor-based stem cell differentiation method uses
Sendai virus to produce highly pure populations of neurons without a genetic footprint. Quick-Neuron™ Excitatory 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 glutamatergic neuron marker vesicular glutamate transporter 1(vGLUT1). When handled and maintained according to the instructions in this user guide, excitatory neurons are viablelong-term and are suitable for a variety of characterization and neurotoxicity assays.
Scale: 			
			

The Quick-Neuron™ Excitatory - SeV Kit (Large) contains a set of reagents for use with a totalof 6 wells of a 6-well plate.

Related Products:
						

Quick-Neuron™ Excitatory - SeV Kit (Small), Catalog Number: EX-SeV-S
Quick-Neuron™ Excitatory - Human iPSC-derived Neurons, Catalog Number: EX-SeV-CW
Quick-Neuron™ Excitatory - Maintenance Medium, Catalog Number: EX-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

QN-SeV (undiluted)*

110 μl-80°C

Component N

3x 840 μl-20°C or -80°C

Component G1

2x 10 μl

-20°C or -80°C

Component G2

4x 16 μl

-20°C or -80°C

Component P

2x 14 μl

-20°C or -80°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 isrequired for its use. Please use a biological safety cabinet,
laminar flow hood, and proper personal protective equipment inorder to prevent mucosal exposure. More information on BSL-2
guidelines can be found at www.cdc.gov/labs/BMBL.html.

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 9am-5pm EST).

Last revised: January 22, 2021

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1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

DMEM/F12

ThermoFisher

21331020

Neurobasal Medium

ThermoFisher

21103049

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

iMatrix-511 silk

Elixirgen Scientific

NI511S

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

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

STEM-CELLBANKERAMSBIO

11890

Source hPSC Culture Conditions
The Quick-Neuron™ Excitatory - SeV Kit (Large) gives the best differentiation results when source hPSCs have been
maintained in StemFit® Basic04, 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 2-3 35-mm
culture dishes or 2-3 wells of a 6-well plate. If iMatrix-511 silk is routinely used as a coating substrate, prepare 2-3
dishes or wells 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.
• For optimal differentiation, hPSC confluency should be around 50% to 70%. Do not use wells more than 90%
confluent.

WorkflowMedia
Preparation

Plate Preparation
Treatment
Medium Change
Plating

Day -1/0

Day 0

Day 1

Plate Preparation
Passaging Cells(Optional) Freeze cells
Day 2Medium N(G1)

Medium iN

33°C

Day 3

Day 4
Medium iN(G2P)

Maintenance

...

...

Day 10Medium N(G2P)

EX-MM*

37°C*From Day 10, users may maintain differentiated neurons in the maintenance medium best suited for their needs,
though we recommend Quick-Neuron™ Excitatory - Maintenance Medium, Catalog Number: EX-MM.
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Media 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 is hereafter referred to as iROCK and can be stored at -20°C.
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.
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. Store the 0.002% poly-L-ornithine solution (hereafter referred to as ornithine) 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., 90 μl) and store them at -20°C.
Medium N
1. Prepare Medium N using the reagents listed in the table below.
• Thaw Component N on ice for 20-30 minutes.
• Warm all other reagents at room temperature for 20-30 minutes.
2. Store Medium N for up to 2 weeks at 4°C. The leftover reagents can be discarded or saved for other uses.
Medium N Reagents

VolumeDMEM/F12

36 ml

Neurobasal Medium

36 ml

200 mM Glutamax (100x)

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

750 μlComponent N

2.33 ml

Medium N(P)
1. Prepare Medium N(P) by mixing together the following components in a 100 ml bottle.
• Warm Medium N at room temperature for 20-30 minutes.
• Thaw Component P at room temperature for 20-30 minutes.
2. Store Medium N(P) for up to 2 weeks at 4°C.
Medium N(P) Reagents

VolumeMedium N

53 ml

Component P

26.5 µl

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~5 hrDay 0
Plate Preparation
1. 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.

1.
2.
3.
4.

Diluted iMatrix-511 silk Reagents

VolumeiMatrix-511 silk

44.6 µl

Chilled PBS

13.5 ml

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

Treatment
IMPORTANT! We do not recommend using only 1 well of source hPSC as harvesting enough cells from 1 well likely
means that the well was over confluent and not ideal for differentiation.
1. Determine the number of wells required to get 3.3 x 106 cells from the source hPSC 6-well plate.
2. Prepare Medium iN by mixing together the following components in a 15 ml conical tube.
• Warm Medium N and iROCK at room temperature for at least 1 hour.
• The rest of Medium N should be stored at 4°C for later use.

Medium iN Reagents

Req medium vol based on# of wells of a 6-well plate
2 wells

3 wells

Medium N

9.4 ml10.5 ml

iROCK

9.4 µl10.5 µl

3. Referring to the table below, prepare the required volume of hPSC maintenance medium with iROCK in a new 15 ml
conical tube. Mix well and allow to warm at room temperature for 20-30 minutes.
Reagents for
hPSC treatment

Req medium vol based on# of wells of a 6-well plate
2 wells

3 wells

hPSC maintenance medium

3 ml

4.5 ml

iROCK

3 µl

4.5 µl4. Aspirate old medium from hPSC culture and add 1.5 ml of hPSC maintenance medium with iROCK to each well.
5. 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.
• During the incubation, start thawing QN-SeV on ice and Solution D1 at room temperature.
6. Aspirate old medium from hPSC culture and add 2 ml PBS to each well being harvested.
7. Rock the dish/plate 3 times, aspirate PBS from the culture, and add 300 μl Solution D1 to each well to begin celldissociation treatment.
• Keep the rest of Solution D1 at 4°C for its use on Day 3.
8. Incubate the culture at 37°C, 5% CO2 for 5 minutes. If all the cells are not rounded under a microscope, incubate at
37°C, 5% CO2 for up to 5 more minutes in 1-2 minute increments (see images on next page).

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Before treatment

Almost ready

Ready to remove

Treated too long

10. Carefully pipet out Solution D1 from the culture using a P1000 pipettor and add 1 ml Medium iN to each well.
• Follow Steps 10-12 one well at a time if multiple wells are used.
11. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
12. Using the same pipette tip, collect the cell suspension in a 15 ml conical tube.
IMPORTANT! In this protocol, users will treat hPSCs with QN-SeV in a tube and then plate the cells onto 6 wells with
1 ml Medium iN (0.5 x 106 cells) per well. However, we recommend preparing a suspension of 6.6 ml to avoid
insufficiency. First, QN-SeV should be mixed with 580 μl of a dense cell suspension to increase the chance that
QN-SeV finds its host cells. After 10 minutes incubation at 33°C, the total volume will be brought up to 6.6 ml with
Medium iN. Cell count may vary based on cell health, the method, and instrument used for cell counting.
13. Count cells to determine the volume of cell suspension needed for 6 wells and include a 10% buffer (a total of 3.3 x
106 cells to plate 0.5 x 106 cells in each of the 6 wells). Transfer the determined volume of the cell suspension into a
15 ml conical tube. Adjust the volume to 580 μl with Medium iN. If the volume of the cell suspension needed to get
3.3 x 106 cells exceeds 580 μl, centrifuge the required volume of cell suspension at 200xg for 4 minutes, remove thesupernatant, and resuspend the pellet into 580 μl Medium iN.
IMPORTANT! Before adding QN-SeV, ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV with apipettor; SeV is highly sensitive to physical stress.
14. Add 105 µl QN-SeV to the hPSCs and mix them by tapping with finger 2-3 times. Cap the tube loosely to allow gas
exchange.
15. Incubate the cell suspension at 33°C, 5% CO2 for 10 minutes with intermittent mixing, by finger tapping, every 2
minutes.
Plating
1. Bring up the volume of cell suspension to 6.6 ml with Medium iN and mix 2-3 times with serological pipet.
2. Aspirate PBS from only one coated well at a time and add 1 ml of cell suspension to each well. Most of the PBS
should be aspirated but not completely to prevent the coated wells from drying before adding the cell suspension.
Likewise, the cell suspension should be added to the well immediately after PBS is removed. Handle one well after
another.
3. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute treated cells in thecultures.
4. Incubate the cultures at 33°C, 5% CO2 overnight.

~1 hr

Day 1
Medium Change
1. Prepare Medium N(G1) by mixing together the following components in a 15 ml conical tube.
• Warm Medium N at room temperature for 20-30 minutes.
• Thaw Component G1 on ice for 20-30 minutes. Spin down before use.
• Keep the rest Medium N(G1) at 4°C for its use on Day 2.
Medium N(G1) Reagents

VolumeMedium N

10 ml

Component G1

20 µl

2. Pipet out most of the old medium from each well using a P1000 pipettor and add 1.5 ml Medium N(G1).
3. Incubate the cultures at 33°C, 5% CO2 overnight.
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<1 hrDay 2
Temperature Shift

1. In the afternoon, transfer the cultures to 37°C, 5% CO2 without changing medium and incubate them overnight.

4-6 hrDay 3
New Plate Preparation

IMPORTANT! Cells can be plated on 6-well, 24-well, or 96-well plates depending on the desired format. This kit canaccommodate replating to all wells of either a 6-well, a 24-well, or a 96-well plate. Refer to the tables at the bottom of
this page for the recommended volumes. Please note that the volumes are per well in Table A and per plate in Table B.
Surplus cells can be frozen following the instructions in the Appendix.
1.
2.
3.
4.
5.

Thaw ornithine at room temperature for 20-30 minutes.
Vortex ornithine briefly and centrifuge it at a maximum speed for a few seconds.
Add ornithine to each well of a new plate in the volume specified in Table A.
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 specified amounts of PBS on ice for 20-30 minutes. 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.
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 or until cells are ready for plating.
10. While the plate is incubating, prepare Medium iN(G2P) using the volumes indicated in Table B.
• Thaw/warm Medium N(P) and iROCK at room temperature for 20-30 minutes.
• Thaw Component G2 on ice for 20-30 minutes.
• Keep the rest of Medium N(P) and Component G2 at 4°C for later use.
11. After the laminin incubation, aspirate most, but not all of, the supernatant and add PBS in the volume specified inTable A. Add the PBS dropwise to each well.
12. Aspirate most, but not all of, the PBS and add Medium iN(G2P) in the volume specified in Table A.
13. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Table A. Recommended volumes per well for different plate formats.
Required 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 iN(G2P)

500 µl200 µl

35 µl

Table B. Recommended volumes per plate for different plate formats.

Diluted laminin

Medium iN(G2P)

6

Required volume per plate

Reagents

6-well plate24-well plate

96-well plate

Laminin

100 µl

80 µl

53 µl

Chilled PBS

10 ml

8 ml

5.3 mlMedium N(P)15.2 ml

13 ml

12 ml

Component G2

15.2 µl

13 µl

12 µl

iROCK

15.2 µl

13 µl

12 µl

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Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care.
1. Warm Solution D1 at room temperature for at least 1 hour before use.
2. Working one well at a time, pipet out the old medium from each well using a P1000 pipettor and add 1 ml PBS andgently rock the plate.
3. Working one well at a time, pipet out the PBS from each well using a P1000 pipettor and add 300 µl Solution D1.
4. Rock the plate 3 times to spread the Solution D1 evenly.
5. Incubate the cultures at 37°C, 5% CO2 for 3 minutes.
6. Working one well at a time, gently pipet out Solution D1 from each well using a P200 pipettor and add 750 µl 		Medium iN(G2P) to each well along the wall of the well.
IMPORTANT! Steps 7-9 are critical. Perform these steps one well at a time. Refer to the images below to successfully
manage cell treatment and dissociation.
Before Solution D1 treatment

During Solution D1 treatment

After dissociation

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. The clusters of cells are not supposed to be lifted. Do not attempt
to detach all of the cells remaining on the well bottom.
9. Gently pipet the cell suspension up and down in the well up to 5 times to break the cell aggregates using a P1000
pipettor. Excessive pipetting can damage the already-suspended neuronal cells.
10. Collect all of the cell suspension from each well in a tube using the same P1000 pipette tip.
11. Count cells and determine viability.
12. Prepare 1 x 10⁶ viable cells/ml cell suspension using Medium iN(G2P) based on the table below.
• If there are leftover cells, freeze the cells down by following instructions in the Appendix after plating cellsuspensions to 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 iN(G2P), the total volume of
the medium 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 mlVol of cell suspension/well

500 µl100 µl

15 µl

Total volume/well• Medium iN(G2P) + cell suspension

1 ml

300 µl

50 µl

Viable cells/well

14. Incubate the cultures at 37°C, 5% CO2 for 1 hour.
15. Observe each well under the microscope to make sure that the cells are attached to the well.
16. Incubate the cultures at 37°C, 5% CO2 overnight.

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7Days 4-9

~1 hr

Maintenance
1. Prepare Medium N(G2P) using the volumes indicated in the table below.
• Warm Medium N(P) at room temperature for 20-30 minutes.
• Thaw Component G2 on ice for 20-30 minutes.
Required volume for each format

Reagents

6-well plate24-well plate

96-well plate

Medium N(P)26.4 ml

42 ml

32 ml

Component G2

26.4 µl

42 µl

32 µl

2. Pipet out the old medium from each well and add Medium N(G2P) according to the table below.
Reagents
Medium N(G2P)

Required volume per well
6-well plate24-well plate

96-well plate

2 ml

800 µl150 µl3. Incubate the cultures at 37°C, 5% CO2 for 2-3 days.
4. Repeat Steps 1-3 until Day 10.

Day 10
Assay or Continuous Maturation
Differentiated neurons can be observed on Day 5. For more mature neurons, we recommend culturing cells until Day
10. From Day 10, users may maintain differentiated neurons in the maintenance medium best suited for their needs,
though we recommend Quick-Neuron™ Excitatory - Maintenance Medium, Catalog Number: EX-MM. Differentiation into
excitatory neurons after using the Quick-Neuron™ Excitatory - SeV Kit can be confirmed with anti-TUBB3 (tubulin beta
3 class III, a global marker for neurons) and anti-vGLUT1 (vesicular glutamate transporter 1, a glutamatergic neuron
marker) antibodies.

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Appendix

Figure 1. Representative images of Quick-Neuron™ Excitatory - SeV Kit cell cultures on days 1-10 post-differentiation(scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Excitatory - SeV Kit cell cultures shows typical neurite growthand expression of the pan-neuronal marker TUBB3 and the glutamatergic neuron-specific marker vGLUT1 on day 10
post-differentiation (scale bar = 100 μm). Staining conditions: Anti-β-III tubulin monoclonal antibody (R&D Systems,
Catalog Number: MAB1195, 1:250 dilution) in combination with a secondary antibody (Invitrogen,Catalog Number:
A32723, Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, AlexaFluor Plus 488, 1:500 dilution).
Anti-VGLUT1 Rabbit Polyclonal primary antibody (Synaptic Systems, Catalog Number: 135 303, 1:100 dilution) in combination with a secondary antibody (Invitrogen, Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33324.
Freezing cells down on Day 3
Note. After thawing frozen cells, approximately 50% of viable cells will be recovered.
1. Determine the volume of the cell suspension and number of cryovials needed to freeze 0.1 ~ 2 x 106 cells per 		cryovial.
2. Centrifuge at 200 xg for 4 min.
3. While waiting for the centrifugation, label each cryovial. We recommend writing the name of the iPSC line used, thetype of neurons, harvesting day and date, and the number of cells in the vial.
4. Aspirate the supernatant and resuspend the pellet with 500 µl/vial STEM-CELLBANKER..
5. Distribute 500 µl of the suspension to each cryovial.
6. Make sure that the caps are closed tightly and transfer the cryovials into a Mr. Frosty Freezing Container. Make surethat Mr. Frosty contains 250 ml isopropanol.
7. Loosely close the lid of Mr. Frosty with cryovials, put it into a -80°C freezer and leave it overnight or a few days.
8. Transfer the cryovials into a liquid nitrogen storage tank.
9. Follow the thawing process in the user guide of Quick-Neuron™ Excitatory - Human iPSC-derived Neurons, 		Catalog Number: EX-SeV-CW.
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