NeuroXVivo Rat Motor Neuron Culture Kit

Refer to the product datasheet for complete product details.

Reagents Provided

Reagents provided in the NeuroXVivo^™ Rat Motor Neuron Culture Kit (Catalog # CDK016):

• Recombinant BDNF (1000X)
• Recombinant CNTF (1000X)
• Recombinant GDNF (1000X)
• Recombinant Laminin α4 (1000X)
• Motor Neuron Culture Media Supplement (10X)
• Reconstitution Buffer 1

Other Supplies Required

Reagents

• E14-15 Timed Pregnant Rat
• Cultrex^® Mouse Laminin I (R&D Systems^®, Catalog # 3400-010-01)
• Cultrex^® Poly-D-Lysine (R&D Systems^®, Catalog # 3439-100-01)
• Phosphate Buffered Saline
• Papain Dissociation System
• DNase-I
• Leibovitz’s L-15 Medium, or equivalent
• Earle’s Balanced Salt Solution (EBSS)
• OptiPrep^™ Density Gradient Medium, or equivalent
• Neurobasal^® Medium, or equivalent
• Cytosine arabinoside
• Fetal Bovine Serum (FBS)

Equipment

• Sterile dissection tools
• 37 °C, 5% CO₂ humidified incubator
• Laminar flow cell culture hood
• Centrifuge
• Hemocytometer
• 37 °C water bath
• Inverted microscope
• Dissection microscope

Motor Neuron Culture Media Supplement (10X) - Warm 10 mL Motor Neuron Culture Media Supplement to 37 °C.

Recombinant BDNF (1000X) - Add 100 µL of Reconstitution Buffer 1 to the vial of Recombinant BDNF to produce Recombinant BDNF (1000X).

Recombinant CNTF (1000X) - Add 100 µL of Reconstitution Buffer 1 to the vial of Recombinant CNTF to produce Recombinant CNTF (1000X).

Recombinant GDNF (1000X) - Add 100 µL of Reconstitution Buffer 1 to the vial of Recombinant GDNF to produce Recombinant GDNF (1000X).

Recombinant Laminin α4 (1000X) - Add 100 µL of Reconstitution Buffer 1 to the vial of Recombinant Laminin α4 to produce Recombinant Laminin α4 (1000X).

Neurobasal Media - Warm Neurobasal media to 37 °C.

Complete Motor Neuron Culture Media - Add Motor Neuron Culture Media Supplement (10X) at a final concentration of 1X to the desired amount of Neurobasal Media. Add Recombinant BDNF (1000X), Recombinant CNTF (1000X), Recombinant GDNF (1000X), and Recombinant Laminin α4 (1000X) to a final concentration of 1X to the desired amount of Neurobasal Media plus Supplement. (e.g., for 25 mL of Complete Motor Neuron Culture Media, add 2.5 mL of Motor Neuron Culture Media Supplement (10X) and 25 µL each of Recombinant BDNF (1000X), Recombinant CNTF (1000X), Recombinant GDNF (1000X), and Recombinant Laminin α4 (1000X) to 22.4 mL of Neurobasal Media.)

Procedure Overview

Please refer to the product datasheet for complete protocol details.

Rat motor neurons can be cultured on Laminin I, Poly-D-Lysine-coated glass coverslips or tissue culture plates. Using the following protocol, the quantity of components in this kit is sufficient to culture one 24-well plate of neurons plated on Laminin I, Poly-D-Lysine-coated 12 mm glass coverslips.

Dissection

1. Isolate E14-15 rat embryos.
2. Decapitate rat embryos and discard head.
3. Place rat embryo bodies, dorsal side up, into a dissection dish containing ice cold PBS.
4. Expose spinal cord by removing skin and tissue.
5. Open DRGs from both sides of the spinal cord.
6. Remove spinal cord adjacent tissue to expose the dorsal root ganglia (DRGs).
7. Separate DRGs from both sides of the spinal cord.
8. Collect isolated spinal cords and transfer into a petri dish containing ice-cold L-15 Media.
9. Separate the dorsal and ventral halves of the spinal column.
10. Discard dorsal spinal cord tissue.
11. Cut ventral spinal cord tissue into small pieces.
12. Transfer dissected spinal cord tissue and L-15 Media into a conical tube.

Motor Neuron Culture Protocol
Day 1

1. Prepare glass coverslips or cell culture plates by coating with Poly-D-Lysine and Laminin-I.

Day 2

1. Isolate spinal cord tissue from E14-15 rat embryos following the dissection protocol outline.
2. Digest the spinal cord tissue with 20 U/mL Papain and 100 U/mL DNase 1 for 15 minutes.
3. Stop the tissue digestion by adding 3 mL of FBS.
4. Centrifuge at 200 x g for 3 minutes.
5. Decant supernatant.
6. Resuspend in 6 mL of L-15 Medium.
7. Titrate the tissue pieces with a fire-polished pasteur pipette until the solution is homogenous.
8. Divide the homogenized solution among 6 tubes containing 9% OptiPrep^® solution.
9. Centrifuge at 900 x g for 15 minutes. Centrifuge breaks should be off.
10. Transfer the top 2 mL of solution from each tube into one fresh 50 mL conical tube.
11. Add fresh L-15 Medium.
12. Centrifuge at 300 x g for 10 minutes.
13. Decant supernatant.
14. Resuspend the spinal motor neurons in 250-500 µL of Complete Motor Neuron Culture Medium.
15. Count the cells.
16. Prepare pre-coated coverslips by applying 80 µL of Complete Motor Neuron Culture Media to each coverslip.
17. Seed neurons onto pre-coated coverslips by adding 20 µL of your motor neuron cell suspension to each coverslip.
18. Incubate for at least 2 hours in a 37 °C, 5% CO₂ humidified incubator.
19. Add 900 µL of fresh pre-warmed Complete Motor Neuron Culture Medium to each well containing a coverslip.
20. Exchange media every 3-4 days.