dopaminergic neurons - elixirgen scientific, inc. · 2020-01-13 · dopaminergic neurons. they have...

-- Quick-NeuronTM Dopaminergic (Frozen) December 16, 2019

Dopaminergic Neurons

This protocol is intended for the recovery and the maintenance of dopaminergic neurons derived from hiPSCs

User Manual

Catalog Number: EXGS-QNDSVF-CW_ID_-1M

Elixirgen Scientific, LLC855 N. Wolfe St., Suite 619Baltimore, MD 21205Phone: (443) 869-5420Email: [email protected]

Table of Contents:I. Introduction 2II. Kit Contents 2III. Additional Materials Required 2IV. Pre-Protocol Preparation 3V. Protocol 4

VI. Appendix A: Reference Pictures and Figures 7

-- Quick-NeuronTM Dopaminergic (Frozen)December 16, 2019

I. IntroductionElixirgen Scientific’s Dopaminergic Neurons contains neurons that are expected to become tyrosine hydrox-ylase (TH) and dopamine (DA)-positive in 7 days after plating. These neurons are committed to becoming Dopaminergic neurons.They have been derived from human induced pluripotent stem cells (hiPSCs) and exhibit a typical neuronal morphology with outgrowing neurites when maintained as directed in this protocol. This kit contains suffi-cient reagents for plating and maintaining neurons recovered from 1 vial of Dopaminergic Neurons for up to 7 days. From Day 7, Dopaminergic Neurons are good for any experimental utilization or users may main-tain differentiated neurons in the maintenance medium best suited for their needs, though we recommend Quick-Neuron™ Dopaminergic Maintenance Medium, available at https://elixirgenscientific.com/store/ (Cata-log Number: EXGS-QNDM).

II. Kit ContentsUpon receipt of this kit, immediately store the vial of Frozen Cells in a liquid nitrogen tank and all other re-agents at their proper storage temperatures as described in the table below. All reagents are shipped via dry shipper.

III. Additional Materials Required

List of Components

Contents Amount Storage Conditions

Frozen Cells 1 vial (>1.0 million viable cells, 0.5 ml) Liquid nitrogen tank

Component N 840 µl -20 °C

Component D2 28 µl -20 °C





The following materials are needed but not supplied with this kit:

• 24-well tissue-culture-treated polystyrene plate, 35 mm culture dish or 6-well tissue-culture-treated poly-styrene plate

• DMEM/F12 (e.g., ThermoFisher, Catalog Number: 21331-020)

• Neurobasal (e.g., ThermoFisher, Catalog Number: 21103049)

• Glutamax (100x) (e.g., ThermoFisher, Catalog Number: 35050061)

• Penicillin-Streptomycin (e.g., ThermoFisher, Catalog Number: 15140-122)

• Poly-L-Ornithine (e.g., Sigma Aldrich, Catalog Number: P4957-50ML)

• Laminin Mouse Protein, Natural (e.g., ThermoFisher, Catalog Number: 23017015)

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

• ROCK inhibitor Y27632 (e.g., Selleckchem Catalog Number: s1049)

• Trypan blue solution, 0.4% (e.g., ThermoFisher, Catalog Number: 15250061)

• Dimethyl sulfoxide (DMSO; e.g., Sigma-Aldrich, Catalog Number: D8418)


IV. Pre-Protocol Preparation

No. Reagent Volume

1 DMEM/F12 12 ml

2 Neurobasal 12 ml

3 200 mM Glutamax (100x) 125 µl

4 Penicillin-Streptomycin (10000 units/ml; 100x) 250 µl

5 Component N 775 µl

• Prepare a neural differentiation medium by mixing following reagents after thawing Component N at 4°C overnight. The medium is called Medium N and stable for up to 2 weeks when stored at 4°C.

• Prepare a 10 mM ROCK inhibitor Y27632 stock solution in DMSO to prepare for Medium iN on Day 0. Preparation steps are as follows:

- Dissolve 10 mg ROCK inhibitor Y27632 in 3.1225 ml DMSO.

- Make aliquots of a convenient volume (e.g., 100 μl) and store at -20°C.

• Prepare 0.002% poly-L-ornithine solution in PBS for use on Day 0. This solution is referred to as Ornithine hereafter. Preparation steps are as follows:

- Take 300 μl 0.01% poly-L-ornithine solution and mix it with 1.2 ml PBS.

- Store the diluted solution at 4°C and use it within two weeks.

• Prepare 1 mg/ml laminin stock solution in PBS for use on Day 0. This solution is referred to as Laminin hereafter. Preparation steps are as follows:

- Thaw Laminin Mouse Protein, Natural at 4°C or on ice.

- Chill PBS at 4°C or on ice.

- Take cold Laminin Mouse Protein, Natural and mix it with chilled PBS to make 1 mg/ml stock solution (note: the concentration of laminin varies among different lots and is specified on the vial or its certif-icate of analysis. Calculate the volume needed to prepare 1 mg/ml stock solution accordingly).

- Make aliquots of a convenient volume (e.g., 15 μl) and store them at -20°C.

• We do not recommend additional freeze-thaw cycles of any reagents.

• Taking 4x and/or 10x images of cultures every day (or even after every medium change) is a good way to monitor your experiment.

• Customer service on the phone (+1-443-869-5420) and through email ([email protected]) is available to assist users in troubleshooting and interpretation of results.


Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8

Plating Assay

Medium iN Medium N(D2D3) Medium N(D4D6)Medium N(D4D5)

V. Protocol

Day 0 - Plating and Treatment

New Plate Preparation

1. Select a proper cell culture size suited for your experiment. Add Ornithine according to the volumes listed in Table 1 or scale the volume described in the protocol by the ratio between well sizes accordingly.

Table 1. Recommended volume of Ornithine and diluted Laminin for coating

Culture size Volume (ml)

6-well Plate 1.5

24-well Plate 0.3

All volumes are per well

2. Incubate the plate at 37°C, 5% CO₂ for at least 2 hours (or at 4°C overnight one day before).

3. Thaw Laminin on ice for 20-30 minutes. Do not thaw it at room temperature.

4. Take 1.5 ml ice-cold PBS into a tube and add 15 µl Laminin to it. Mix them well.

5. Aspirate the supernatant from each well and add 500 μl or 2 ml PBS to each of the 24 wells or 6 wells, respectively.

6. Repeat step 5.

7. Aspirate PBS from each well and add diluted Laminin to it according to the well size.

8. Incubate the plate at 37°C, 5% CO₂ for at least 2 hours.

9. When cells are ready for plating, repeat step 5 twice.

10. Follow step 18 below.

Thawing/Plating

1. Warm Medium N at room temperature for at least 1 hour before use.

2. Take 4.5 ml Medium N into a new 15 ml conical tube.

3. Take 1.5 ml Medium N into a new 1.5 ml tube and add 1.5 μl 10 mM ROCK inhibitor Y27632. Mix them well. The medium is referred to as Medium iN. Keep the rest of Medium N at 4°C for its later use.

4. Take out the vial of Frozen Cells from the liquid nitrogen storage tank.

5. Incubate the cryovial in a water bath set at 37°C (do not submerge the cap) until most of the content is thawed but a small ice crystal remains (approximately 2 minutes).

6. Wipe the vial with dry paper towel. Spray 70% ethanol to the vial and bring it inside a biosafety cabinet.

7. Take 0.5 ml Medium N from the tube at step 2 using a P1000 pipettor and add it into the cryovial drop-wise at 1 drop / 1-2 seconds.

8. Using the same pipette tip, gently pipet the cell suspension up and down once.

9. Gently transfer all cell suspension using the same pipette tip to the conical tube prepared at step 2.

10. Take 1 ml cell suspension from the conical tube using the same pipette tip, add it to the original cryovial and pipet up and down 2-3 times to transfer the whole content to the same conical tube. Mix the content


17. Take out the determined volume of the cell suspension from the previous step in a new tube and bring up the volume with Medium iN to the plating volume required for cultures according to Table 2.

18. Aspirate PBS from each coated well and add the cell suspension to it.

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

Day 1-2 - Feeding

1. Warm Medium N at room temperature for 20-30 minutes.

2. Thaw Component D2 on ice and Component D3 at room temperature for 20-30 minutes.

3. Take 6.25 ml Medium N into a new conical tube and add 12.5 μl Component D2 and 12.5 μl Component D3 to it. Mix them well. The medium is referred to as Medium N(D2D3) and is stable for up to 2 weeks at 4°C.

4. Pipet out the old medium from each well using a P1000 pipettor and add Medium N(D2D3) to it accord-ing to Table 3. Store the rest Medium N(D2D3) at 4°C for its use on Day 3.

Table 2. Recommended cell plating density

Culture size Plating volume (ml) Live cell number

6-well Plate 2.0 1 x 106

24-well Plate 0.4 2 x 105

All volumes and cell numbers are per well

in the conical tube well by pipetting 3 times.

11. Centrifuge the cell suspension at 200 xg for 4 minutes.

12. Aspirate most of the supernatant from the conical tube but leave a small volume of the supernatant (< 50 μl) to cover the pellet.

13. Simply tap the side of the conical tube several times to break the cell pellet.

14. Add 1 ml Medium iN to the conical tube using a P1000 pipettor and pipet it up and down 2-3 times.

15. Count cells and also determine the viability of cells by trypan blue staining.

16. Determine the volume of cell suspension needed to start a culture(s) according to the recommended cell plating density for different well sizes in Table 2.

Table 3. Recommended culture medium volume

Culture size Medium Volume (ml)

6-well Plate 4.0

24-well Plate 0.8

All volumes are per well

5. Incubate the cultures at 37°C, 5% CO2 for 2 days.

Day 3 - Feeding

1. Warm Medium N(D2D3) at room temperature for 20-30 minutes.

2. Pipet out the old medium from each well using a P1000 pipettor and add Medium N(D2D3) to it accord-ing to Table 2.

3. Incubate the cultures at 37°C, 5% CO2 overnight.


Day 4 - Feeding

1. Thaw Component D4 on ice and Component D5 at room temperature for 20-30 minutes.

2. Warm Medium N at room temperature for 20-30 minutes.

3. Take 2.2 ml Medium N into a tube and add 2.2 μl Component D4 and 4.4 μl Component D5 to it. Mix them well. This medium is referred to as Medium N(D4D5). Store the rest Component D4 at 4°C for its use on Day 4.

4. Pipet out the old medium from each well using a P1000 pipettor and add 50% Medium N(D4D5) to it according to Table 3.


Day 5-6 - Feeding

1. Thaw Component D6 at room temperature for 20-30 minutes.

2. Warm Medium N and Component D4 at room temperature for 20-30 minutes.

3. Take 6.25 ml Medium N into a tube and add 6.25 μl Component D4 and 6.25 μl Component D6 to it. Mix them well. This medium is referred to as Medium N(D4D6) and is stable for up to 2 weeks at 4°C.

4. Pipet out the old medium from each well using a P1000 pipettor and add 100% Medium N(D4D6) to it according to Table 3.

5. Incubate the cultures at 37°C, 5% CO2 for 2 days.

Day 7 - Feeding

1. Warm Medium N(D4D6) at room temperature for 20-30 minutes.

2. Pipet out 50% of the old medium from each well using a P1000 pipettor and add Medium N(D4D6) to reach 100%.


Day 8 - Ready for Assay

NOTE: OBSERVING NEURONS AND ASSAYING• Differentiated neurons can be observed on Day 1-2 under the microscope. For more mature neurons,

we recommend culturing cells until Day 7. From Day 7, users may maintain differentiated neurons in the maintenance medium best suited for their needs, though we recommend Quick-Neuron™ Dopaminergic Maintenance Medium, available at https://elixirgenscientific.com/store/ (Catalog Number: EXGS-QNDM).

• Dopaminergic neurons can be confirmed with anti-TUBB3 (tubulin beta 3 class III, a global marker for neurons), anti-TH (tyrosine hydroxylase, a dopaminergic neuron marker), and anti-DA (dopamine, a neurotransmitter of dopaminergic neuron) antibodies. Typical immunostaining images of TUBB3-positive and TH-positive neurons on Day 7 following this protocol are shown in Appendix A.


VI. Appendix A: Reference Pictures and Figures

Figure 1. Representative images of cultures by Dopaminergic Neurons on Days 1 through 6 after thawing (4x and 10x objective).

Day 1

4x

10x

Day 3 Day 4 Day 5 Day 6

Figure 2. Immunostaining images of cultures by Dopaminergic Neurons on Day 6 after thawing.

Staining conditions: Anti-β-III tubulin monoclonal antibody (R&D systems, Catalog Number: MAB1195, 1:10,000 dilution) in combination with a secondary antibody (Invitrogen, Catalog Number: A21424, Alexa 555 goat anti-mouse, 1:500 dilution). Anti-TH primary antibody (GeneTex, Catalog Number: GTX102424, 1:300 dilution) in combination with a secondary antibody (Invitrogen, Catalog Number: A11034, Alexa 488 goat anti-rabbit, 1:500 dilution).

DAPI TUBB3 TH Merged

10x

40x

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dopaminergic neurons - elixirgen scientific, inc. · 2020-01-13 · dopaminergic neurons. they have...

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