Why use NeuroXVivo™ Rat Motor Neuron Culture Kit to Culture Primary Rat Motor Neurons?
Spinal motor neuron cultures are an indispensable model system for studying neuronal development, disease, and regeneration. They have been pivotal in investigating molecular and functional mechanisms underlying motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The survival and development of motor neurons, both in vivo and in vitro, requires specific combinations of growth factors, adhesion molecules, peptides, and vitamins. The NeuroXVivo™ Rat Motor Neuron Culture Kit provides optimized reagents and a validated protocol to successfully culture primary rat motor neurons in serum-free media. Specifically, this kit contains optimized concentrations of three growth factors, an adhesion substrate, and a neuronal media supplement that are required to support the robust growth and maintenance of rat primary motor neuron cultures.
The NeuroXVivo™ Rat Motor Neuron Culture Kit is a simple and reliable system for culturing primary rat motor neurons for both experienced and non-experienced users. This kit contains enough reagents to prepare 100 mL of Complete Motor Neuron Culture Medium, which is enough to culture one 24-well plate of rat motor neurons plated on Laminin I, Poly-D-Lysine-coated 12 mm glass coverslips.
This kit contains the following reagents to maintain mouse pluripotent stem cells in culture:
Recombinant BDNF (1000X)
Recombinant CNTF (1000X)
Recombinant GDNF (1000X)
Recombinant Laminin α4 (1000X)
Motor Neuron Culture Media Supplement (10X)
Reconstitution Buffer 1
Stability and Storage
Store unopened kit at ≤-20 °C. Do not use past the kit expiration date.
FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
This reagent should not be used beyond the expiration date indicated on the label.
Results may vary due to variations among cells derived from different donors.
Rat Motor Neurons Express Motor Neuron-specific Markers and Form an Extensive Network of Neuronal Processes Embryonic day 15 primary rat motor neurons were cultured for 14 days in vitro using this kit. Cultures were stained for the motor neuron-specific markers, Neurofilament 145kDa (red) and Homeobox 9 (green), using the Anti-MNX1/HLXB9 Antibody (Novus Biologicals®, Catalog # NBP2-24691). Neurons were counterstained with DAPI (blue).
Depolarization Mediated Intracellular Calcium Response in Cultured Rat Motor Neurons Embryonic day 15 primary rat motor neurons were cultured for 14 days in vitro using this kit. Representative traces of intracellular Ca2+ response (F340/F380) in Fura-2 loaded rat motor neurons in culture upon addition of 3 µM Glutamate (yellow), 50 mM Potassium Chloride (blue), or HBSS control (grey).
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.)
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.
Isolate E14-15 rat embryos.
Decapitate rat embryos and discard head.
Place rat embryo bodies, dorsal side up, into a dissection dish containing ice cold PBS.
Expose spinal cord by removing skin and tissue.
Open DRGs from both sides of the spinal cord.
Remove spinal cord adjacent tissue to expose the dorsal root ganglia (DRGs).
Separate DRGs from both sides of the spinal cord.
Collect isolated spinal cords and transfer into a petri dish containing ice-cold L-15 Media.
Separate the dorsal and ventral halves of the spinal column.
Discard dorsal spinal cord tissue.
Cut ventral spinal cord tissue into small pieces.
Transfer dissected spinal cord tissue and L-15 Media into a conical tube.
Motor Neuron Culture Protocol
Prepare glass coverslips or cell culture plates by coating with Poly-D-Lysine and Laminin-I.
Isolate spinal cord tissue from E14-15 rat embryos following the dissection protocol outline.
Digest the spinal cord tissue with 20 U/mL Papain and 100 U/mL DNase 1 for 15 minutes.
Stop the tissue digestion by adding 3 mL of FBS.
Centrifuge at 200 x g for 3 minutes.
Resuspend in 6 mL of L-15 Medium.
Titruate the tissue pieces with a fire-polished pasteur pipette until the solution is homogenous.
Divide the homogenized solution among 6 tubes containing 9% OptiPrep® solution
Centrifuge at 900 x g for 15 minutes. Centrifuge breaks should be off.
Transfer the top 2 mL of solution from each tube into one fresh 50 mL conical tube.
Add fresh L-15 Medium.
Centrifuge at 300 x g for 10 minutes.
Resuspend the spinal motor neurons in 250-500 µL of Complete Motor Neuron Culture Medium.
Count the cells.
Prepare pre-coated coverslips by applying 80 µL of Complete Motor Neuron Culture Media to each coverslip.
Seed neurons onto pre-coated coverslips by adding 20 µL of your motor neuron cell suspension to each coverslip.
Incubate for at least 2 hours in a 37 °C, 5% CO2 humidified incubator.
Add 900 µL of fresh pre-warmed Complete Motor Neuron Culture Medium to each well containing a coverslip.
Exchange media every 3-4 days.
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