An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.