An automated fluorescence based assay of neurite formation.

The assessment of neurite-promoting activity of growth factors and neurotrophins in vitro is usually done by the laborious process of counting neuronal processes manually under a standard microscope. This often requires fixation of the cells so that samples can be counted over a period of days when large numbers of cells or samples are assessed. This, therefore, limits the investigator to a single time point for that plate of cells. In an effort to provide an assay that could be used to screen samples quickly to grade overall neurotrophic activity, and to study time-dependent responses easily, an assay is described that quantitates neurite formation based on automated fluorescence detection. Dissociated embryonic mouse dorsal root ganglion neurons were placed in the upper chamber of 1 microm pore size Fluoroblok (Falcon) transwell chambers. The porous membrane of these transwells is optically opaque. NGF placed in the lower chamber of the transwell induced the production of neurites that preferentially passed from the neuronal cell body in the upper chamber through the pores into the lower NGF-containing chamber. The degree of neurite production was assessed both in live cells and in fixed cells by fluorescent labeling of the neurons and neuronal processes. Quantitation of neurite formation was done with a multiwell fluorescence plate reader and verified by confocal microscopy. This method allows the screening and quantitation of neurotrophic activity in both primary neurons and neuronal cell lines. In addition, this also provides experimental access to neuronal processes that are optically separated from the cell body. The latter aspect may also be of great use where fluorescence measurements within neurites and growth cones, such as for Ca2+ release studies, need to be isolated from contaminating fluorescence or synaptic influences of cell bodies.