User Guide
Quick-Neuron™ 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 viable long-term and aresuitable 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 total of 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

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

VolumeStorageThaw

QN-SeV-P (undiluted)*

100 μl-80°C

On iceComponent N

3 x 840 μl-20°C or -80°C

On ice or 4°C

Component G1

3 x 20 μl-20°C or -80°C

On ice or 4°C

Component G2

60 μl-20°C or -80°C

On ice or 4°C

Component P

50 μl-20°C or -80°C

Room Temperature*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 biologicalsafety cabinet, laminar flow hood, andproper personal protective equipment inorder to prevent mucosal exposure. Moreinformation on BSL-2 guidelines can befound at www.cdc.gov/labs/BMBL.html.

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 03, 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

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
StemFit AK02N, or
StemFlex Medium

Elixirgen Scientific
TaKaRaThermoFisher

ASB04-C,
AK02N, orA3349401

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

D2650

Puromycin (10 mg/ml)

InvivoGen

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

11890* PBS should be used at room temperature unless otherwise specified.
** This is only required if you intend to cryopreserve the cells after differentiation.

Source hPSC Culture Conditions
The Quick-Neuron™ Excitatory - SeV Kit (Large) 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 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 culture dishes or wells precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 mlchilled PBS per well or dish 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).

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●
●
●

Differentiation should not be performed until the cells are at least 14 days post-thaw.
We recommend preparing a minimum of 3.3 x 106 viable hPSC for use with this kit. This is usually obtained by using
3 wells 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.

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 (or Tuesday if cells will be cryopreserved instead of passaged on day
3).

* From Day 10, users may maintain differentiated neurons in the medium best suited for their needs, though we recommend
Quick-Neuron™ Excitatory- Maintenance Medium, Catalog Number: EX-MM.

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.
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 StemFit AK02N or StemFlex.

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.

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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. 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., 90 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 Component N tubes 3 times and then briefly spin all tubes 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 Component N can be discarded or saved at 4°C for up to two weeks.
Required Volume
Reagents

Passaging on Day 3

Cryopreserving on Day 3

DMEM/F1237.2 ml

18.6 ml

Neurobasal Medium

37.2 ml

18.6 mlGlutaMAX

390 μl195 μlPenicillin-Streptomycin (10000 units/ml; 100x)

780 μl390 μlComponent N

2.4 ml1.2 mlDay -3< 1 hourNote: This protocol assumes that Day 0 is a Monday so Day -3 is Friday.
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.
○ Make sure chilled PBS is used for this mixture.
Reagents

VolumeiMatrix-511 silk

44.6 µl

Chilled PBS

13.5 ml

2. Add 2 ml diluted iMatrix-511 silk to each new well of a 6-well plate.
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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 0

~4 hours

Note: This protocol assumes that Day 0 is a Monday (or Tuesday if cells will be cryopreserved instead of passaged on day3) and that user’s hPSCs have already been used with a small size kit (Catalog number: EX-SeV-S) so that users arefamiliar with the experimental process and have optimized conditions for their particular cells.
Treatment
IMPORTANT! Source hPSC wells should be no more than 50-70% confluent thus requiring a minimum of 2 wells to
begin 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.
Required medium volume based on # of wells of
a 6-well plate
Reagents

2 wells

3 wells

Medium N

9.4 ml

10 ml

iROCK

9.4 µl10 µl

3. Referring to the table below, prepare the required volume of Medium S with iROCK in a new 15 ml
conical tube. Mix well and allow to warm at room temperature for 20-30 minutes.
Required medium volume based on # of wells of
a 6-well plate
Reagents

2 wells

3 wells

Medium S

3.5 ml

5 ml

iROCK

3.5 µl5 µl

4. Aspirate old medium from hPSC culture and add 1.5 ml of Medium iS 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-P 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 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 3.
8. 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).

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59. Carefully pipet out Solution D1 from the culture and add 1 ml Medium iN to the well.
○ Follow Steps 9-11 one well at a time if multiple wells are used.
10. Disperse the medium over the bottom surface of the well by pipetting 8-15 times to detach cells.
11. Using the same pipet tip, collect the cell suspension in a 15 ml tube.
IMPORTANT! In this protocol, users will treat hPSCs with QN-SeV-P 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-P should be mixed with 580 μl of a dense cell suspension to increase the chance that
QN-SeV-P 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.
12. Count cells and determine viability.
13. Determine the volume of cell suspension needed for 6 wells and include 10% extra (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 conicaltube.
14. 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 the supernatant, and resuspend the pellet into
580 μl Medium iN.
IMPORTANT! Before adding QN-SeV-P ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV with apipettor; SeV is highly sensitive to physical stress.
15. Add all the contents of the QN-SeV-P tube to the hPSCs and mix them by tapping with finger 2-3 times. Cap thetube loosely to allow gas exchange.
16. 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 diluted iMatrix-511 silk from only one coated well at a time and add 1 ml of cell suspension to each well.
○ Most of the diluted iMatrix-511 silk should be aspirated but not completely to prevent the coated well fromdrying before adding the cell suspension. The cell suspension should be added to the well immediately
after the diluted iMatrix-511 silk 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.

Day 1~2 hours

Medium Change
1.

Prepare Medium N(G1) by mixing together the following components in a 15 ml conical tube.
○ Thaw Component G1 for 20-30 minutes at the temperature indicated in the “Contents” table on page 1.
○ Warm Medium N at room temperature for 20-30 minutes.

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○
○

Tap the Component G1 tube 3 times and then briefly spin it down before use.
Keep the rest of Medium N, Component G1, and Medium N(G1) at 4°C for subsequent use.
Reagents

VolumeMedium N

20 ml

Component G1

40 µl

2. Transfer 9.5 ml Medium N(G1) into a tube and add puromycin to it at the predetermined optimal concentration (seeearlier section on “Drug Selection”).
3. Pipet out most of the old medium from each well and add 1.5 ml Medium N(G1) with puromycin. Incubate thecultures at 33°C, 5% CO2 overnight.

Day 2<1 hour

Medium Change and Temperature Shift
Note: This should be performed in the late afternoon.
1. Warm Medium N(G1) at room temperature for 20-30 minutes.
2. Transfer 9.5 ml Medium N(G1) 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.
3. Pipet out most of the old medium from each well and add 1.5 ml N(G1) with puromycin.
4. Incubate the cultures at 37°C, 5% CO2 overnight.

Day 3

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. Cells
can be cryopreserved instead of passaging by skipping to Appendix B at this point. Alternatively, cells can be live-passaged
as per the instructions below and any surplus cells can be cryopreserved following the instructions beginning at step 3 inAppendix B.
1.
2.
3.
4.
5.

Vortex ornithine briefly and centrifuge it at a maximum speed for 1 minute.
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.

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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 iN(G2P)

500 µl200 µl

35 µl

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

Medium N(G2P)

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

41 ml

45 ml

34 ml

Component G2

41 µl

45 µl

34 µl

Component P

20.5 µl22.5 µl

17 µl

Medium Preparation
1.

While the plate is incubating, prepare Medium N(G2P) using the volumes indicated in Table B.
○ Thaw Components G2 and Component P for 20-30 minutes at the temperature indicated in the “Contents”
table on page 1.
○ 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.
○ Keep the rest of Medium N at 4°C for later use.
2. Prepare Medium iN(G2P)○ Warm iROCK at room temperature for 20-30 minutes.
○ The rest of Medium N(G2P) should be stored at 4°C for later use.
Required medium volume per plate
Reagents

6-well plate24-well plate

96-well plate

Medium N(G2P)

11.5 ml

13 ml

10 ml

iROCK

11.5 µl

13 µl

10 µl

3. 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.
4. Aspirate most, but not all of, the PBS and add Medium iN(G2P) in the volume specified in Table A.
5. 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. Steps 2-9 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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1.
2.
3.
4.
5.
6.
7.
8.

Make sure that Solution D1 is at room temperature for at least 1 hour before use.
Pipet out the old medium from one well and add 1 ml PBS to the well.
Pipet out the PBS from the 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.
Carefully pipet out Solution D1 from the well and add 750 μl Medium iN(G2P) along the wall of the well.
Disperse the medium quickly over the bottom surface of the well by pipetting 6-8 times to detach cells.
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. Repeat steps 2-9 for the rest of the wells.
11. Gently pipet the cell suspension up and down up to 5 times to break the cell aggregates. Excessive pipetting candamage the already-suspended neuronal cells.
12. Count cells and determine viability.
13. Prepare specified amounts of a 1 x 10⁶ live cells/ml cell suspension using Medium iN(G2P) based on the tablebelow.
○ If there are leftover cells, freeze the cells down by following instructions beginning at step 3 in Appendix B
after plating cell suspensions on the new plate. Keep the leftover cells on ice until freezing.
14. 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

Required volume of cell suspension (1 x 10⁶ viable cells/ml)● (Vol of cell suspension/well x # of wells) + 10% extra

3.3 ml2.64 ml

1.6 mlVolume 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

15. Incubate the culture plate at 37°C, 5% CO2 overnight.

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9Day 4<1 hours

Medium Change
1. Warm Medium N(G2P) at room temperature for 20-30 minutes.
2. Pipet out the old medium from each well and add* room temperature Medium N(G2P) according to the table below.
Recommended volume per well
Reagents
Medium N(G2P)

6-well plate24-well plate

96-well plate

3 ml

800 µl150 µl*(Optional) Slowly add PBS according to the volumes in the table above alongside the wall of each well to avoid
lifting attached cells. Gently pipet out PBS before adding Medium N(G2P).
3. Incubate the culture plate at 37°C, 5% CO2 for 3 days.

Day 7<1 hours

Medium Change
1. Warm Medium N(G2P) at room temperature for 20-30 minutes.
2. Pipet out half the original volume of the medium from each well and add Medium N(G2P) according to the following
table.
Recommended volume per well
Reagents
Medium N(G2P)

6-well plate24-well plate

96-well plate

1.5 ml400 µl75 µl

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

Day 10
Assay or Continuous Maturation●●

Differentiated neurons can be observed on Day 4. 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 theirneeds, 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 and anti-vGLUT1 antibodies.

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10Appendix A

Figure 1. Representative phase contrast images of Quick-Neuron™ Excitatory - SeV cell cultures on days 1,3,4,6 and 10
post-differentiation (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Excitatory - SeV Kit cell cultures shows typical neurite growth and expression of
the pan-neuronal marker TUBB3 and the glutamatergic neuron-specific marker vGLUT1 on day 10 post-differentiation (scale bar = 258μm). 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 33342.

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Appendix B
Freezing cells down on Day 3
Note: After thawing frozen cells, over 50% of the cells will be viable.
Required volume
1. Prepare Medium iN by mixing together the following components in a 15 ml conical tube.
○ Warm Medium N and iROCK for 20-30 minutes at room temperature.
○ Keep the rest of Medium N at 4°C for later use.
Reagents

VolumeMedium N

5 ml

iRock

5 µl

2. Harvest cells as per the instructions in steps 1-12 of “passaging cells” on day 3 using Medium iN instead ofiN(G2P).
3. Determine the volume of the cell suspension and number of cryovials needed to freeze 0.1 ~ 2 x 106 cells per
cryovial.
4. Centrifuge at 310 x g for 4 minutes.
5. While waiting for the centrifugation, label each cryovial. We recommend writing the name of the PSC line used, thetype of neurons, harvesting day and date, and the number of cells in the vial.
6. Aspirate the supernatant and resuspend the pellet with 0.5 ml / vial STEM-CELLBANKER.
7. Distribute 0.5 ml of the suspension to each cryovial.
8. 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.
9. Loosely close the lid of Mr. Frosty with cryovials, put it into a -80°C freezer and leave it overnight or up to a few days.
10. Transfer the cryovials into a liquid nitrogen storage tank.
11. 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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