Isolation and Culture of Spinal Cord Motor Neurons

Alice Klausmeyer1, D. Stern1, S. Wiese1

1 Institute for Cell Morphology and Molecular Neurobiology, Group for Cell Biology, Ruhr‐University, Bochum
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.9
DOI:  10.1002/0471143030.cb0109s66
Online Posting Date:  March, 2015
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Isolated spinal motoneurons are a powerful tool for studying basic mechanisms of neurite growth and survival. Since motoneurons are a minor population of developing spinal cord cells, they need to be purified and enriched to separate them from non‐neuronal cells. Therefore, the particular feature of embryonic motoneurons to express the low affinity neurotrophin receptor p75NTR is used to separate the motoneurons from other contaminating cells. Two ways are described to isolate embryonic motoneurons: the basic protocol taking advantage of the ability of p75NTR to bind lectin, and an alternative method using an antibody against p75NTR for a panning procedure. These protocols comprise suggestions for the cultivation of the isolated motoneurons for experiments regarding neural outgrowth and survival as well as instruction for the preparation of proteins of the cells. © 2015 by John Wiley & Sons, Inc.

Keywords: spinal cord; motoneuron; axon; lectin; p75NTR

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Table of Contents

  • Introduction
  • Basic Protocol 1: Lectin‐Based Isolation and Culture of Mouse Embryonic Motoneurons
  • Alternate Protocol 1: Enrichment of Embryonic Motoneurons by a p75NTR Antibody Panning Step
  • Support Protocol 1: Single Embryo Spinal Motoneuron Isolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Lectin‐Based Isolation and Culture of Mouse Embryonic Motoneurons

  • 0.5 mg/ml Poly‐DL‐ornithine hydrobromide (store at −20°C)
  • 150 mM borate buffer, pH 8.35 (store at room temperature)
  • 100% ethanol
  • 10 mg/ml laminin (Sigma) stock solution in HBSS (store at −20°C)
  • Hanks’ balanced salt solution (HBSS; appendix 2A)
  • 10 mM Tris·Cl, pH 9.5 ( appendix 2A; store at room temperature)
  • 10 μg/ml lectin (Sigma L9640) in HBSS (store at 4°C)
  • E12.5 pregnant mouse
  • 1% (w/v) trypsin in HBSS (store at −20°C)
  • Soybean trypsin inhibitor (SBTI) solution (see recipe)
  • Depolarization solution (see recipe)
  • Motoneuron culture medium (see recipe)
  • Glass coverslips
  • 4‐well culture dish (Greiner)
  • 10‐cm plastic cell culture dish
  • Two pairs of forceps
  • 1.5‐ml Eppendorf reaction tube (microcentrifuge tube)
  • 15‐ml disposable tube (e.g., BD Falcon)
  • Light microscope, inverted
  • Cell culture incubator (37°C, 6% CO 2)
  • Additional reagents and equipment for determining cell number (unit 1.1)

Alternate Protocol 1: Enrichment of Embryonic Motoneurons by a p75NTR Antibody Panning Step

  Additional Materials (also see Basic Protocol 1)
  • Anti‐p75NTR (Biosensis, M‐009‐100)
  • 10 mM Tris·Cl, pH 9.5 ( appendix 2A)

Support Protocol 1: Single Embryo Spinal Motoneuron Isolation

  Additional Materials (also see protocol 1Basic Protocol)
  • 24‐well cell culture dish
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Literature Cited

Literature Cited
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