Protocol abstract
Nature Protocols 3, - 835 - 839 (2008)
Published online: 17 April 2008 | doi:10.1038/nprot.2008.47
Subject Categories: Cell and tissue culture | Neuroscience | Pharmacology and toxicology
Fluorescent high-throughput screening of chemical inducers of neuronal differentiation in skeletal muscle cells
Darren R Williams1, Gun-Hee Kim1, Myung-Ryul Lee1 & Injae Shin1
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.
- Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
Correspondence to: Injae Shin1 e-mail: injae@yonsei.ac.kr
nature-products
A-Z product listing
- 100
Penicillin/streptomycin(Invitrogen) - 8-Channel micropipette(Nichirto)
- 96-Well plate(Nunc)
- C2C12 Mus musculus (mouse) myoblasts(American Type Culture Collection (ATCC)
- Calcium chloride(Sigma-Aldrich)
- Centrifuge(Eppendorf)