Mixed Neurons - elixirgenscientific.comDecember 13, 2019-Page 3-Mixed Neurons IV. Pre-Protocol Preparation • Prepare a neural differentiation medium by mixing following reagents

-Page 1- Mixed NeuronsDecember 13, 2019

Mixed Neurons

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

VI. Appendix A: Literature Reference 7VII. Appendix B: Literature References 8

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

User Manual

Catalog Number: EXGS-QNMSVF-CW_ID_-1M

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


I. IntroductionElixirgen Scientific’s Mixed Neurons contains a mixed population of a variety of neurons that will express neuron markers characteristic of different subtypes including tubulin beta 3 class III (TUBB3, a pan-neuronal marker), choline acetyltransferase (ChAT, a cholinergic neuron marker), tyrosine hydroxylase (TH, a dopaminer-gic neuron marker), glutamic acid decarboxylase (GAD67, a GABAergic neuron marker), vesicular glutamate transporter 1 (vGLUT1, a glutamatergic neuron marker), and tryptophan hydroxylase 2 (TPH2, a serotonergic neuron marker).

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 sufficient reagents for plating and maintaining neurons recovered from 1 vials of Mixed Neuron for up to 7 days. From Day 7, Mixed Neruons are good for any experimental utilization or users may maintain differentiated neurons in the maintenance medium best suited for their needs, though we recommend Quick-Neuron™ Mixed Main-tenance Medium, available at https://elixirgenscientific.com/store/ (Catalog Number: EXGS-QNMM).

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

List of Components

Reagents 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 P 14 µl -20 °C

Component K 25 µl -20 °C

III. Additional Materials RequiredThe 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 polystyrene 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

• Prepare a neural differentiation medium by mixing following reagents after thawing Componen 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 solu-tion (note: the concentration of laminin varies among different lots and is specified on the vial or its certificate 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.

No. Reagent Volume1 DMEM/F12 12 ml2 Neurobasal 12 ml3 200 mM Glutamax (100x) 125 µl4 Penicillin-Streptomycin (10000 units/ml; 100x) 250 µl5 Component N 775 µl

NOTE: RETINOIC ACID SUPPLEMENTS• Retinoic acid (RA) is known to induce the differentiation of forebrain neurons (Reference 1).

• Our immunocytochemistry (ICC) and quantitative reverse transcription PCR (qRT-PCR) results demonstrat-ed that addition of RA had no significant influence on the population of mixed neurons (data not shown).

• This kit separately provides RA as Component K. Users may supplement Medium iN and Medium N with Component K at a concentration of 1 µl Component K per 1 ml medium.


V. Protocol

Day 0 - Thawing/PlatingNew Plate Preparation1. 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

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

Plating Assay

Medium iN(P) Medium N(P)

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 19 below.

Thawing/Plating1. Warm Medium N at room temperature for at least 1 hour before use.

2. Take 4 ml Medium N into a new 15 ml conical tube and add 4 μl 10 mM ROCK inhibitor Y27632 to it. Mix them well. The medium is referred to as Medium iN.

3. Thaw Component P on ice for 20-30 minutes. Add 2 μl Component P to Medium iN. Mix them well. The medium is referred to as Medium iN(P). Keep the rest of Component P at 4°C for its use on Day 1.

4. (If adding retinoic acid is desired) Thaw Component K on ice for 20-30 minutes. Add 4 μl Component K to Medium iN(P). Mix them well. The medium is referred to as Medium iN(KP). Keep the rest of Component K at 4°C for its use on Day 1.

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

6. 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).


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

8. Take 4.5 ml Medium N into a new 15 ml conical tube. Keep the rest of Medium N at 4°C for its later use.

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

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

11. Gently transfer all cell suspension using the same pipette tip to the conical tube prepared at step 7.

12. 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 in the conical tube well by pipetting 3 times.

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

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

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

16. Add 1 ml Medium iN(P) or Medium iN(KP) to the conical tube using a P1000 pipettor and pipet up and down 2-3 times.

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

18. 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 2. Recommended cell plating density

Culture well size Plating volume (ml) Live cell number

6-well Plate 2.0 5 x 10⁵

24-well plate 0.4 1 x 10⁵

All volumes and cell numbers are per well

19. 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(P) or Medium iN(KP) to the plating volume required for cultures according to Table 2.

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

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

Day 1 - Feeding1. Warm Medium N at room temperature for 20-30 minutes.

2. Take 12 ml Medium N into a new conical tube and add 6 μl Component P to it. Mix them well. The medium is referred to as Medium N(KP) and is stable for up to 2 weeks at 4°C.

3. (If adding retinoic acid is desired) Add 12 μl Component K to Medium N(KP). Mix them well. The medium is referred to as Medium N(KP) 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(P) or Medium N(KP) to it according to Table 3.


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™ Mixed Mainte-nance Medium, available at https://elixirgenscientific.com/store/ (Catalog Number: EXGS-QNMM).

• Differentiation into multiple types of neurons can be confirmed with anti-TUBB3 (tubulin beta 3 class III, a global marker for neurons), anti-ChAT (choline acetyltransferase, a cholinergic neuron marker), an-ti-TH (Tyrosine hydroxylase, a dopaminergic neuron marker), anti-GAD67 (glutamic acid decarboxylase, a GABAergic neuron marker), anti-vGLUT1 (vesicular glutamate transporter 1, a glutamatergic neuron marker), and anti-TPH2 (Tryptophan hydroxylase 2, a serotonergic neuron marker) antibodies. Typical immunostaining images of TUBB3-positive and vGLUT1-positive neurons on Day 7 following this protocol are shown in Appendix A.

Table 3. Recommended culture medium volume

Culture Size Volume (ml)

6-well Plate 4

24-well plate 0.8

All volumes are per well

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

Day 3-6 - Feeding1. Warm Medium N(P) or Medium N(KP)Medium N or Medium N(K) 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(P) or Medium N(KP)Medium N or Medium N(K) to reach 100%.

3. Incubate the cultures at 37°C, 5% CO₂ for 2-3 days.

4. Repeat steps 1-3 until Day 7.

Day 7 - Ready for Assay


VI. Appendix A: Reference Pictures and Figures

Day 1

4x

10x

Day 2 Day 3 Day 6

Figure 1. Representative images of cultures by Mixed Neurons on Days 1 through 6 after thawing.

DAPI TUBB3 vGLUT1 Merged

10x

40x

Figure 2. Immunostaining images of cultures by Mixed 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-vGLUT1 primary antibody (Abcam, Catalog Number: AB6427, 1:3000 dilution) in combination with a secondary antibody (Invitrogen, Catalog Number: A11034, Alexa 488 goat anti-rabbit, 1:500 dilution).


VII. Appendix B: Literature References

Cunningham TJ and Duester G (2015). Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat Rev Mol Cell Biol 16, 110–123.

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Mixed Neurons - elixirgenscientific.comDecember 13, 2019-Page 3-Mixed Neurons IV. Pre-Protocol Preparation • Prepare a neural differentiation medium by mixing following reagents