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.

Keywords: NS-1; Nerve growth factor; PC12; SU6656.; high content screening; neurite outgrowth.

Fig. (1)

Fluorescence intensity profile of GFP expressing NS-1 cells. A flow cytometry histogram showing the profile of a mixed population of GFP expressing NS-1 cells (green shading) relative to the population of untransfected NS-1 cells (gray shading). In this example, 80% of the NS1-GFP population exhibited an intensity of at least 10 times that of the control population.

Fig. (2)

A comparison of fluorescent images generated by the three staining methods. GFP-NS-1cells (A), βIII-tubulin immunofluorescence (B) and HCS CellMask^™ Red (C) fluorescence images of cell bodies and neurites were acquired on the BD Pathway 855 Bioimager. Unaltered, exemplar TIFF files were retrieved with “ImageJ” software and the “Yellow Hot” look-up table was used to determine relative intensities; no other data manipulations were applied. A gray scale ramp is shown in panel C.

Fig. (3)

Comparison of NGF dose on neurite outgrowth response measured by three visualization methods. NS-1 cell neurites were visualized by transfection with GFP (GFP), staining with anti-βIII-tubulin followed by DyLight 488-conjugated secondary antibody (anti-βIII tubulin) or staining with CellMask Red (CellMask Red). Data were normalized using responses to 0ng/ml and 50ng/ml NGF as minimum and maximum total neurite length/cell, respectively. Calculated EC₅₀ values were 3.3, 2.1 and 1.4 ng/ml using GFP, anti-βIII-Tubulin and CellMask Red, respectively. Values plotted are means ± S.E.M. (n=4).

Fig. (4)

Inhibition of NGF-induced neurite outgrowth by Src-family kinase inhibitor SU6656. NS-1 cells were treated with varying doses of SU6656, and then NGF was added to a final concentration of 20ng/ml. Neurite outgrowth was analyzed at 48h by HCMR. The curve was fitted in GraphPad PRISM using a 4-parameter logistic model with recursive least squares weighting. Each point represents an average of three experiments in triplicate. The bars represent the standard deviation of the data from the means.

Fig. (5)

Active compounds exhibiting greater than 200% activity in two separate experiments. NS1 cells were treated with 2 ng/mL NGF in the presence of 1.5 µM compounds for 48 h and stained with a cocktail of Hoechst and HCS CellMask Red^™ for 30 min. Data was acquired on the BD Pathway855 imaging station and analyzed with BD Attovision^™ 1.6 software. The histograms represent the compound activity measured as total neurites per cell over background of 2 ng/mL NGF in two independent experiments; the error bars represent standard deviations.