Kit Summary

Reagents for the culturing and maturation of primary rat motor neurons in culture.

Key Benefits

  • Features an optimized serum-free media for rat motor neuron cultures
  • Utilizes R&D Systems® Recombinant BDNF, CNTF, GDNF, and Laminin α4
  • Detailed protocol is included
  • Ideal for both non-experienced and experienced neural cell culture researchers
 

 

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.

 

Kit Contents

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.

Limitations

  • 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.

 

Data Examples
Rat Motor Neurons Express Motor Neuron-specific Markers and Form an Extensive Network of Neuronal Processes.
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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 Induced with Glutamate and Potassium Chloride.
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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).

Preparation and Storage
  • Stability & Storage
    Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, primary rat motor neurons are maintained and matured ex vivo with the NeuroXVivo Rat Motor Neuron Culture Kit using the following procedure:

  • Dissect prenatal rat spinal cord tissue
  • Dissociate rat spinal cord tissue
  • Isolate rat motor neurons
  • Suspend single cells in Complete Motor Neuron Culture Media
  • Plate cells onto coated coverslips
  • Exchange media every 3-4 days
 

 

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

 

Reagent Preparation

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

Isolate E14-15 rat embryos.

Decapitate rat embryos and discard head.

Isolate E14-15 rat embryos

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.

Place rat embryo bodies, dorsal side up, into a dissection dish containing ice cold PBS

Remove spinal cord adjacent tissue to expose the dorsal root ganglia (DRGs).

Separate DRGs from both sides of the spinal cord.

Remove spinal cord adjacent tissue to expose the DRGs

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.

Collect isolated spinal cords and transfer into a petri dish containing ice-cold L-15 Media

Transfer dissected spinal cord tissue and L-15 Media into a conical tube.

Transfer dissected spinal cord tissue and L-15 Media into a conical tube
 

Motor Neuron Culture Protocol

Day 1

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

Prepare glass coverslips ofr cell culture plates

Day 2

Isolate spinal cord tissue from E14-15 rat embryos following the dissection protocol outline.

Isolate spinal cord tissue

Digest the spinal cord tissue with 20 U/mL Papain and 100 U/mL DNase 1 for 15 minutes.

Digest the spinal cord tissue

Stop the tissue digestion by adding 3 mL of FBS.

Centrifuge at 200 x g for 3 minutes.

Decant supernatant.

Resuspend in 6 mL of L-15 Medium.

Stop the tissue digestion by adding 3 mL FBS

Titruate the tissue pieces with a fire-polished pasteur pipette until the solution is homogenous.

Titruate the tissue pieces

Divide the homogenized solution among 6 tubes containing 9% OptiPrep® solution

Centrifuge at 900 x g for 15 minutes. Centrifuge breaks should be off.

Divide the homogenized solution among 6 tubes

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.

Decant supernatant.

Transfer the top 2 mL of solution from each tube into one fresh 50 mL conical tube

Resuspend the spinal motor neurons in 250-500 µL of Complete Motor Neuron Culture Medium.

Count the cells.

Resuspend the spinal motor neurons

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.

Prepare pre-coated coverslips

Add 900 µL of fresh pre-warmed Complete Motor Neuron Culture Medium to each well containing a coverslip.

Exchange media every 3-4 days.

Add 900 µL of fresh pre-warmed medium

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