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.