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Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos

Nature Protocols volume 5, pages 31–38 (2010)Cite this article

Abstract

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75NTR-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75NTR-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.

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Figure 1: Flow diagram of the procedure for isolation and culture of lumbar spinal motoneurons from mouse embryos described in this protocol.
Figure 2: Dissection of the lumbar spinal cord from an E13 mouse embryo.
Figure 3: Characteristics of the cultured motoneurons.
Figure 4: Immunocytochemistry of cultured motoneurons.

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Acknowledgements

This work was supported by grants from the ACHSE Foundation, the DFG, SFB 581 project B4 and the Herrmann and Lilly Schilling Stiftung and the National Health and Medical Research Council of Australia.

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Author notes

  1. Stefan Wiese and Thomas Herrmann: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Clinical Neurobiology, Wuerzburg, Germany

    Stefan Wiese, Thomas Herrmann, Carsten Drepper, Sibylle Jablonka, Natalia Funk & Michael Sendtner

  2. Group for Molecular Cell Biology, Ruhr-University, Bochum, Germany

    Stefan Wiese & Alice Klausmeyer

  3. Department of Human Physiology, Flinders University, Adelaide, Australia

    Mary-Louise Rogers & Robert Rush

Authors
  1. Stefan Wiese
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  2. Thomas Herrmann
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  4. Sibylle Jablonka
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  5. Natalia Funk
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  6. Alice Klausmeyer
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  8. Robert Rush
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  9. Michael Sendtner
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Contributions

S.W. initially developed the basic method based on techniques established by M.S. T.H., C.D., S.J. and N.F. established the current protocol, and M.-L.R. and R.R. developed and newly contributed tools (p75NTR-monoclonal antibodies) for this protocol. T.H., C.D. and M.S. prepared the manuscript.

Corresponding author

Correspondence to Michael Sendtner.

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Competing interests

The p75NTR antibodies that we used (MLR2) were originally produced in the laboratory of Robert Rush and Mary-Louise Rogers (coauthors of this paper). The purified antibodies of this hybridoma cultures have been made commercially available through Biosensis, and Robert Rush profited financially from their sale.

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Wiese, S., Herrmann, T., Drepper, C. et al. Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos. Nat Protoc 5, 31–38 (2010). https://doi.org/10.1038/nprot.2009.193

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