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Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells

Nature Protocols volume 3, pages 835–839 (2008)Cite this article

Abstract

This protocol describes detailed procedures for the fluorescent high-throughput screening of small molecules that induce neurogenesis in cultures of skeletal muscle cells. The detection of neurogenesis relies on a fluorescent dye, FM 1-43, which is used to study the neuronal property of depolarization-induced synaptic vesicle recycling. Thus, small molecules with neurogenesis-inducing activity in skeletal muscle cells can be rapidly identified by measuring the fluorescence intensity of the treated cells using a fluorescent microplate reader. This protocol uses murine myoblast C2C12 cells for screening, which are readily available and relatively easy to culture. Neurogenesis of PC12 cells induced by nerve growth factor is employed as a positive control for this screening. The screening time for this protocol is 8 d, which also includes the procedure to detect depolarization-induced synaptic vesicle recycling using FM 1-43.

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Figure 1: Structures of neurodazine, a compound identified using the fluorescent high-throughput screening method and the fluorescent dye FM 1-43.
Figure 2
Figure 3: Phase contrast (left) and fluorescence microscopic images (right) of cells after treatment with FM 1-43.
Figure 4: Quantitative analysis of fluorescence intensities of the neurogenesis-inducing compound-treated C2C12 cells and PC12 cells.

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Acknowledgements

This work was supported by a grant of the NRL program (MOST/KOSEF). G.-H.K. thanks the BK 21 program (KRF).

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  1. Department of Chemistry, Yonsei University, Seoul, 120-749, Korea

    Darren R Williams, Gun-Hee Kim, Myung-Ryul Lee & Injae Shin

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Correspondence to Injae Shin.

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Williams, D., Kim, GH., Lee, MR. et al. Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells. Nat Protoc 3, 835–839 (2008). https://doi.org/10.1038/nprot.2008.47

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