User Guide
Quick-Glia™ Astrocyte - SeV Kit (Large)
Catalog Number: AS-SeV-L

Introduction
The Quick-Glia™ Astrocyte - SeV Kit (Large) facilitates rapid and efficient differentiation of human iPS or ES cells into
astrocyte cells in just 28 days. Our proprietary transcription factor-based stem cell differentiation method uses the
Sendai virus to produce astrocytes without a genetic footprint. Quick-Glia™ Astrocyte differentiated cell cultures
display typical astrocyte morphology and markers such as S100 Calcium Binding Protein β (S100β), Chondroitin Sulfate
Proteoglycan 8 (CD44), Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1), and mature astrocyte marker Glial
Fibrillary Acidic Protein (GFAP). When handled and maintained according to the instructions in this user guide,
astrocytes are viable long-term and are suitable for a variety of characterization and assays.
Scale:The Quick-Glia™ Astrocyte - SeV Kit (Large) contains a set of reagents for use with a total of 6
wells of a 6-well plate.

Related Products:

Quick-Glia™ Astrocyte - SeV Kit (Small), Catalog Number: AS-SeV-S
Quick-Glia™ Astrocyte - Human iPSC-derived Astrocytes, Catalog Number: AS-SeV-CW

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

VolumeStorageThaw

QGA-SeV (undiluted)*

100 μl-80°C

On iceComponent N1

2 x 830 μl-20°C or -80°C

On ice or 4°C

Component GA1

65 μl-20°C or -80°C

On ice or 4°C

Component GA2

65 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 is required for
its use. Please use a biological safetycabinet, laminar flow hood, and proper
personal protective equipment in order toprevent
mucosalexposure.
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: September 19, 2023

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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

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

iMatrix-511 silk

Elixirgen Scientific

NI511S

ScienCell Astrocyte Medium Kit:
• Basal Medium• Astrocyte Growth Supplement
• FBS
• P/S

ScienCell ResearchLaboratories

1801

ThermoFisher

12563011

Sigma-Aldrich

E8008-100ML

Phosphate-buffered saline (without Ca Mg )*ThermoFisher

20012050

ROCK inhibitor Y27632

Selleckchem

S1049

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

Geltrex hESC-Qualified, Ready-To-Use,
Reduced Growth Factor Basement Membrane Matrix

ThermoFisher

A1569601AMSBIO

11890

TrypLE Select Enzyme (1X)
0.02% EDTA in DPBS++

(Optional) STEM-CELLBANKER**++* 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-Glia™ Astrocyte - 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 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 culture dishes or wells precoated with 0.25 μg/cm2 iMatrix-511 silk diluted in 2 ml chilled PBS per well ordish 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 2 x 106 viable hPSC for use with this kit. This is usually obtained by using
2 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.

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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 28, users may maintain differentiated cells in the maintenance medium best suited for their needs. We recommend
continuing with the ScienCell Medium without FBS for an additional 2 weeks for more mature astrocytes.

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, hereafter referred to as iROCK, 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.
Medium N1
1.

Prepare Medium N1 using the reagents listed in the table below.
○ Thaw Component N1 for 20-30 minutes at the temperatures indicated in the “Contents” table on page 1.
○ Warm all other reagents at room temperature for 20-30 minutes.
○ Tap each Component tube 3 times and then briefly spin them down before use.
○ Keep Medium N1, and any subsequent media made with it, protected from light.
○ Store Medium N1 for up to 2 weeks at 4°C.
○ Leftover reagents can be discarded or saved at 4°C for up to 2 weeks.
Reagents

VolumeDMEM/F12

21.5 ml

Neurobasal

21.5 mlGlutaMAX

225 μlPenicillin-Streptomycin (10000 units/ml; 100x)450 μlComponent N1

1.35 mlelixirgensci.com

3Day -3~4 hours

Note: 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 new 6-well plate.
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.
Treatment
1. Determine the number of wells required to get 2.0 x 106 cells from the source hPSC 6-well plate.
2. Prepare Medium iN1 by mixing together the following components in a 15 ml conical tube.
○ Warm Medium N1, iROCK, and Solution D1 at room temperature for at least 1 hour protected from light.
○ The rest of Medium N1 should be stored at 4°C for later use.
Required medium volume based on # of wells of
a 6-well plate
Reagents

1 well2 wells

Medium N1

8.3 ml9.4 mliROCK

8.3 µl

9.4 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.
Required volume per # of wells of a 6-well plate
Reagents

1 well2 wells

hPSC maintenance medium

1.5 ml3.0 mliROCK

1.5 µl

3.0 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.

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4○

During the incubation, start thawing QGA-SeV (undiluted) on ice and warming 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 toeach well.
○ Keep the rest of Solution D1 at 4°C for use on Day 4.
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).

9. Carefully pipet out Solution D1 from the culture and add 1 ml Medium iN1 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 1.5 ml tube.
IMPORTANT! In this protocol, users will treat hPSCs with QGA-SeV (undiluted) in a tube and then plate the cells onto 6
wells with 1 ml Medium iN1 (3.0 x 105 cells) per well. However, we recommend preparing a suspension of 6.6 ml to avoid
insufficiency. First, QGA-SeV (undiluted) should be mixed with 340 μl of a dense cell suspension to increase the chancethat QGA-SeV (undiluted) finds its host cells. After 10 minutes incubation at 33°C, the total volume will be brought up to 6.6
ml with Medium iN1. Cell count may vary based on cell health, the method, and instrument used for cell counting.
12. Count cells and determine viability.
13. Take out the volume of the cell suspension needed for 6 wells and include 10% extra (a total of 1.98 x 106 cells toplate 0.3 x 106 cells in each of the 6 wells). Transfer the determined volume of the cell suspension into a 15 ml
conical tube.
14. Bring the volume of the cell suspension up to 340 μl with Medium iN1.
○ If the volume of the cell suspension needed to get 1.98 x 106 cells exceeds 340 μl, centrifuge the required
volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet into
340 μl Medium iN1.
IMPORTANT! Before adding QGA-SeV (undiluted), ensure that it is fully thawed. Do not centrifuge, vortex, or mix SeV witha pipettor; SeV is highly sensitive to physical stress.
15. Add all the contents of the QGA-SeV tube to the hPSCs and mix them by tapping with a 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 iN1 and mix 2-3 times with a serological pipet.
2. Aspirate PBS from only one coated well at a time and add 1 ml 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.
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4. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 1< 1 hourMedium Change
1.

Prepare Medium A1 by mixing together the following components in a 50 ml conical tube.
○ Warm Medium N1 at room temperature for 20-30 minutes.
○ Thaw 4 vials of Component GA1 on ice for 20 minutes.
○ Thaw 4 vials of Component GA2, protected from light, at room temperature for 20 minutes.
○ The rest of Medium A1 should be stored at 4°C for its use on Day 2.
Reagents

VolumeMedium N1

30 ml

Component GA1

60 µl

Component GA2

60 µl

2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 1.5 ml
Medium A1.
3. Incubate the culture plate at 33°C, 5% CO2 overnight.

Day 2< 1 hourMedium Change and Temperature Shift
Note: This should be performed in the late afternoon.
1. Warm Medium A1 at room temperature for 20-30 minutes.
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 1.5 ml
Medium A1.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

Day 3< 1 hour

IMPORTANT! Following this protocol, users will encounter cell death in the infected cultures. It will be most noticeable the
day after making the temperature shift. When monitoring the health of infected cultures, please refer to the images in the
appendix showing the recovery trajectory of a typical hPSC culture subjected to QGA-SeV-mediated differentiation. It is
optional, but recommended, to include the PBS wash if cell death/floating cells are observed.
Maintenance
1. Warm Medium A1 at room temperature for 20-30 minutes.
2. Pipet out the medium from each well, leaving a small volume behind to avoid the cells drying out, and add 1.5 ml
Medium A1.
*(Optional) Slowly add 1.5 ml PBS alongside the wall of each well to avoid lifting attached cells. Gently pipet out
PBS before adding Medium A1.
3. Incubate the culture plate at 37°C, 5% CO2 overnight.

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6Day 4< 2 hours

Medium Change
1.

Prepare ScienceCell Medium with 2% FBS by mixing together the following components in a 50 ml conical tube.
○ Warm Basal Medium, Astrocyte Growth Supplement (AGS), and Pen/Strep (P/S) from the ScienCell kit atroom temperature for 1 hour away from light.
○ Thaw FBS from the ScienCell kit at room temperature for 1 hour.
○ Aliquot and store unused AGS and FBS at -20°C and the Basal Medium and P/S at 4°C.
Reagents

VolumeBasal Medium

80 ml

AGS

830 µlP/S

830 µl

FBS

1.67 ml

2. Pipet out the old medium from each well , leaving a small volume behind to avoid the cells drying out.
3. Add 2.5 ml of ScienCell Medium with 2% FBS to each well, slowly along the wall of the well.
4. Incubate the cultures at 37°C, 5% CO2 for 3 days.

Days 7 -16< 1 hour

Maintenance
1.
2.
3.
4.
5.

Warm ScienCell Medium with 2% FBS at room temperature for 30 minutes.
Pipet out 1.25 ml of the old medium from each well.
Add 1.25 ml of ScienCell Medium with 2% FBS to each well, slowly along the wall of the well.
Incubate the cultures at 37°C, 5% CO2.
Repeat steps 1-4 every 2-3 days.

Day 17~ 2 hours

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 plate in Table A and per well in Table B.
Surplus cells can be frozen following the instructions in Appendix B.
1. Aliquot the volume of Geltrex specified in Table A to a prechilled 15 ml conical tube and keep on ice.
2. Add Geltrex to wells according to Table B.
3. Incubate the plate at 37°C, 5% CO2 for at least 1 hour or until cells are ready for plating. Alternatively, coating can be
performed by incubating the plate at 4°C overnight.
4. Warm ScienCell Medium (without FBS) at room temperature for 30-40 minutes.
5. After the Geltrex incubation, aspirate most, but not all of, the supernatant and add ScienCell Medium (without FBS)
in the volume specified in Table B.
6. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.

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

6-well plate24-well plate

96-well plate

Geltrex

10 ml

8 ml

5.3 mlScienCell Medium (without FBS)

16.5 ml

20 ml

12 ml

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

6-well plate24-well plate

96-well plate

Geltrex

1.5 ml300 µl

50 µl

ScienCell Medium (without FBS)

1 ml

400 µl

34 µl

Passaging Cells
IMPORTANT! For the following steps, gently pipet and add solutions. Differentiating cells are delicate and should be
handled with great care. Steps 2-10 below are critical. Perform these steps one well at a time. Refer to the images below
to successfully manage cell treatment and dissociation.

1.
2.
3.
4.
5.
6.

Warm Solution D1 at room temperature for at least 1 hour before use.
Pipet out the old medium from one well and add 1 ml PBS and gently rock the plate.
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 5 - 10 minutes.
Gently pipet out Solution D1 from the well and add 1 ml ScienCell Medium (without FBS) to the well along the wallof the well.
7. 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. Do not attempt to detach all of the cells remaining on the wellbottom.
9. Collect 1 ml cell suspension from the 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 0.5 x 106 viable cells/ml cell suspension using ScienCell Medium (without FBS) based on the table below.
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a. If there are leftover cells, freeze the cells down by following instructions (beginning at step 2) in Appendix B
after plating cell suspensions to 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 ScienCell Medium (without FBS), thetotal volume of the medium in each well is indicated in the table below.
Note: The cell plating density recommended should result in confluent cultures in about two weeks. If users desire
confluence in a shorter or longer period they should adjust the plating density accordingly. In addition, the density
recommended may not be optimal for all hPSC cell lines as growth rates can vary depending on the hPSC cell line.

Recommended amounts
6-well plate24-well plate

96-well plate

2.5 x 105 cells

5 x 104 cells

8 x 103 cells

Required volume of cell suspension (0.5 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

16 µl

Total volume/well○ ScienCell Medium (without FBS) + cell suspension

1.5 ml500 µl

50 µl

Viable cells/well

15. Move the plate in 5 cycles of quick back-and-forth and side-to-side motions to evenly distribute treated cells in thecultures.
16. Allow cells to attach by incubating the plate at room temperature for 10 minutes.
17. Incubate the cultures at 37°C, 5% CO2 overnight.

Day 18< 1 hour

Maintenance
1.
2.
3.
4.

Warm ScienCell Medium (without FBS) at room temperature for 30-40 minutes.
Pipet out the old medium from each well, leaving a small volume behind to avoid the cells drying out.
Add ScienCell Medium (without FBS) to each well according to the table below.
Incubate the cultures at 37°C, 5% CO2 for 2-3 days.
Required volume per well
Reagents
ScienCell Medium (without FBS)

Days 20-27

6-well plate24-well plate

96-well plate

2.5 ml

1 ml

0.25 ml< 1 hour

Maintenance
1.
2.
3.
4.
5.

Warm ScienCell Medium (without FBS) at room temperature for 30-40 minutes.
Pipet out half of the volume of old medium from each well (see Table above).
Add an equal volume of fresh ScienCell Medium (without FBS) to each well.
Incubate the cultures at 37°C, 5% CO2.
Repeat steps 2-4 every 2-3 days, making more ScienCell Medium as needed following the instructions above.

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9Day 28
Assay or Continuous Maturation
●
●

●
●

CD44, S100β, GFAP, and ALDH1L1-positive cells can be detected on Day 28.
For more mature astrocytes with increased expression of GFAP and ALDH1L1, we recommend culturing cells until
Day 42. When cells approach confluence they can be passaged following the instructions on Day 17, though
maintaining them at higher confluency results in higher GFAP expression.
From Day 28, users may maintain differentiated astrocytes in the maintenance medium best suited for their needs,
though we recommend ScienCell Astrocyte Medium without FBS.
Cells can be frozen following the instructions in Appendix B.

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10Appendix A

Figure 1. Representative images of Quick-Glia™ Astrocyte - SeV Kit (Small) cell cultures on days 1, 4, 7, 14, 21, 28, 35 and42 post-differentiation (scale bar = 100 μm). User’s cultures may display a slightly lower level of confluency on eachday due to minor differences between small and large Quick-Glia™ Astrocyte - SeV Kit formats.

Figure 2. (A) Gene expression profiles of iPSCs and Quick-Glia™ - Astrocyte SeV Culture on day 28 were comparedwith the profile of human primary astrocytes and the results are shown as scatter plots. The horizontal axis indicates theexpression levels of genes in human primary astrocytes purchased from ScienCell (Catalog Number: 1800-5), whereas
the vertical axis indicates the expression levels of genes in iPSCs (left) and in Quick-Glia™ - Astrocyte SeV Culture on
day 28 (right). The levels of gene expression are shown based on transcripts per million (TPM) in the log10 scale. Blue andgreen dots represent upregulated and downregulated genes (FDR<0.05), respectively, relative to their levels in human primary astrocytes. (B) Similarities of gene expression profiles of human iPSCs and Quick-Glia™ - Astrocyte SeV
Culture on days 14, 28 and 42 to the profile of human primary astrocytes are shown as a bar chart. The vertical axis
indicates Pearson correlation (r) based on median-subtracted logTPM.

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Figure 3. Real-time quantitative PCR analysis of expression levels of astrocyte-associated genes CD44, GFAP, S100β
and ALDH1L1 were examined. Graphs show comparison of gene expression in Quick-Glia™ - Astrocyte SeV Culture
on day 28 (A) and day 42 (B) with gene expression in human brain total RNA (TaKaRa, Catalog Number: 636530). The
relative gene expression is normalized to phosphoglycerate kinase 1 (PGK1), and then calculated as a fold induction
relative to undifferentiated hPSCs as a control. Error bars show standard deviation. Primers used are listed in Table 1.
Table 1. List of PCR primers used in Figure 3
Gene

Forward primer

Reverse Primer

Primer Concentration

CD44

CTGCCGCTTTGCAGGTGTA

CATTGTGGGCAAGGTGCTATT

250 nM

GFAP

ATCGAGAAGGTTCGCTTCCTG

TGTTGGCGGTGAGTTGATCG

250 nM

S100β

GGCTGGTCTCAAACTTCCTG

TCCACAACCTCCTGCTCTTT

250 nM

ALDH1L1TCACAGAAGTCTAACCTGCC

AGTGACGGGTGATAGATGAT

250 nM

PGK1

GTATGCTGAGGCTGTCACTCG

CCTTCCAGGAGCTCCAAACTGG

250 nMelixirgensci.com

12Appendix B
Freezing Cells Down
Note: After thawing frozen cells, approximately 80% of cells will be viable.
1.
2.
3.
4.

Follow steps 1-12 in the “Passaging cells” section of Day 17.
Determine the volume of the cell suspension and number of cryovials needed to freeze 1-4 x 106 cells per cryovial.
Centrifuge at 200 x g for 4 minutes.
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.
5. Aspirate the supernatant and resuspend the pellet with 0.5 ml/vial STEM-CELLBANKER.
6. Distribute 0.5 ml of the suspension to each cryovial.
7. 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.
8. 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.
9. Transfer the cryovials into a liquid nitrogen storage tank.
10. Follow the thawing instructions in the user guide for Quick-Glia™ Astrocyte - Human iPSC-derived Astrocytes,
Catalog Number: AS-SeV-CW.

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