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In vitro methods to prepare astrocyte and motoneuron cultures for the investigation of potential in vivo interactions

Nature Protocols volume 2, pages 1499–1507 (2007)Cite this article

Abstract

This protocol details methods to isolate and purify astrocytes and motoneurons (MNs) from the chick lumbar spinal cord. In addition, an approach to study the influences of astrocyte secreted factors on MNs is provided. Astrocytes are isolated between embryonic days 10 and 12 (E10–12), propagated in serum (2–3 h) and differentiated in chemically defined medium (3–4 h). When prepared according to this protocol, astrocyte cultures are more than 98% pure when assessed using the astrocyte-specific markers glial fibrillary acidic protein (GFAP) and S100β. MNs are isolated between E5.5 and 6.0 (3–4 h) using a procedure that takes selective advantage of the large size of these cells. These cultures can be maintained using individual trophic factors, target-derived factors or astrocyte-derived factors, the preparation of which is also described (5–6 h). All or part of these techniques can be used to investigate a variety of processes that occur during nervous system development and disease or after injury.

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Figure 1: Isolation of embryonic days 10–12 chick spinal cord.
Figure 2: Isolation of embryonic day 5.5 chick spinal cord.
Figure 3: Purification of chick astrocytes.
Figure 4: Purification of chick motoneurons (MNs).

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Acknowledgements

This work was supported by National Institutes of Health grant NS036081 and by funds from the WFUSM Brian White ALS Foundation (C.E.M.). The authors would like to thank David Prevette and William Safrit for their assistance with the generation of dissection images.

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  1. Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston Salem, 27157, North Carolina, USA

    Anna R Taylor, Mac B Robinson & Carol E Milligan

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Correspondence to Carol E Milligan.

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Taylor, A., Robinson, M. & Milligan, C. In vitro methods to prepare astrocyte and motoneuron cultures for the investigation of potential in vivo interactions. Nat Protoc 2, 1499–1507 (2007). https://doi.org/10.1038/nprot.2007.208

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