A Rapid, Inexpensive High Throughput Screen Method for Neurite Outgrowth

Abstract

Neurite outgrowth assays are the most common phenotypic screen to assess chemical effects on neuronal cells. Current automated assays involve expensive equipment, lengthy sample preparation and handling, costly reagents and slow rates of data acquisition and analysis. We have developed a high throughput screen (HTS) for neurite outgrowth using a robust neuronal cell model coupled to fast and inexpensive visualization methods, reduced data volume and rapid data analysis. Neuroscreen-1 (NS-1) cell, a subclone of PC12, possessing rapid growth and enhanced sensitivity to NGF was used as a model neuron. This method reduces preparation time by using cells expressing GFP or native cells stained with HCS CellMask™ Red in a multiplexed 30 min fixation and staining step. A 2x2 camera binning process reduced both image data files and analysis times by 75% and 60% respectively, compared to current protocols. In addition, eliminating autofocus steps during montage generation reduced data collection time. Pharmacological profiles for stimulation and inhibition of neurite outgrowth by NGF and SU6656 were comparable to current standard method utilizing immunofluorescence detection of tubulin. Potentiation of NGF-induced neurite outgrowth by members of a 1,120-member Prestwick compound library as assayed using this method identified six molecules, including etoposide, isoflupredone acetate, fludrocortisone acetate, thioguanosine, oxyphenbutazone and gibberellic acid, that more than doubled the neurite mass primed by 2 ng/ml NGF. This simple procedure represents an important routine approach in high throughput screening of large chemical libraries using the neurite outgrowth phenotype as a measure of the effects of chemical molecules on neuronal cells.

A high-content morphological screen identifies novel microRNAs that regulate neuroblastoma cell differentiation

“…Several HCS approaches based on quantification of neurite outgrowth have been described [33, 42-44]. However, quantifications of neurite outgrowth in these approaches were either based on cell lines engineered to express fluorescent reporters or involve staining of fixed cells [33, 42-44]. These approaches are generally time-consuming.…”

Differential intensity-dependent effects of magnetic stimulation on the longest neurites and shorter dendrites in neuroscreen-1 cells

“…NS-1 cells (ThermoFisher Scientific, Waltham, MA), subclones of rat pheochromocytoma (PC12), are known for their ideal sensitivity to neuron growth factor (NGF) and arborization of both dendrites and axons (Yeyeodu et al, 2010) and were chosen to investigate the effects of MS on neuronal morphology. NS-1 cells were subcultured twice a week in a flask of complete medium mixture of RPMI 1640 with 10% horse serum, 5% fetal bovine serum (FBS), 2 mM glutamine, 100 μg/ml penicillin/streptomycin (Life technologies, Grand Island, NY) and 5 ng/ml neurotrophic growth factor (NGF; R&D Systems, Minneapolis, MN) at 37°C in a 5% CO 2 and humidified incubator.…”

“…After blocking in phosphate-buffered saline (PBS) supplemented with 2% Bovine Serum Albumin (BSA) and 0.1% Triton X-100, NS-1 cells were stained with HCS CellMask Red (0.5 μg/ml, Invitrogen, Eugene, OR) (Yeyeodu et al, 2010) followed by a DAPI-containing mounting medium. Images were captured with the QCapture Pro software and neurite lengths and number were measured using ImageJ.…”

Modeling genetic epilepsies in a dish

“…Measuring neurite outgrowth in culture is perhaps the most common phenotypic assay to study neuronal maturation in vitro. 174 In addition to neurite length, the complexity of neurite branching, number of extensions per cell, and number of neurites per cell can be measured by image segmentation. Sholl analysis 175 is often used and provides the number of dendrite intersections against the radial distance from the soma center.…”