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A rapid, inexpensive high throughput screen method for neurite outgrowth.

Yeyeodu ST, Witherspoon SM, Gilyazova N, Ibeanu GC - Curr Chem Genomics (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC. 27707, USA.

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.

No MeSH data available.


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.
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Figure 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.

Mentions: Because the HCMR dye method presented a much faster alternative to GFP expression for enabling neurite outgrowth assays and is potentially applicable to other neuronal cells, we used the HCMR assay for the remainder of our studies. To further validate HCMR as a viable method for accurate assessment of neurite outgrowth, we tested whether the method could accurately model the dose-response profile of SU6656, a Src-family kinase inhibitor [17] for the inhibition of neurite outgrowth [18, 19]. As shown in the sigmoidal curve (Fig. 4), SU6656 caused a dose-dependent inhibition of neurite outgrowth induced by 20 ng/mL NGF. This concentration of NGF, which correlates to the EC90 of NGF for neurite outgrowth in NS-1 cells, was determined from a dose response assay with escalating concentrations of NGF. The calculated SU6656 IC50 of 64 nM is well within the range of SU6656 IC50s for various Src-kinase family members.


A rapid, inexpensive high throughput screen method for neurite outgrowth.

Yeyeodu ST, Witherspoon SM, Gilyazova N, Ibeanu GC - Curr Chem Genomics (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040990&req=5

Figure 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.
Mentions: Because the HCMR dye method presented a much faster alternative to GFP expression for enabling neurite outgrowth assays and is potentially applicable to other neuronal cells, we used the HCMR assay for the remainder of our studies. To further validate HCMR as a viable method for accurate assessment of neurite outgrowth, we tested whether the method could accurately model the dose-response profile of SU6656, a Src-family kinase inhibitor [17] for the inhibition of neurite outgrowth [18, 19]. As shown in the sigmoidal curve (Fig. 4), SU6656 caused a dose-dependent inhibition of neurite outgrowth induced by 20 ng/mL NGF. This concentration of NGF, which correlates to the EC90 of NGF for neurite outgrowth in NS-1 cells, was determined from a dose response assay with escalating concentrations of NGF. The calculated SU6656 IC50 of 64 nM is well within the range of SU6656 IC50s for various Src-kinase family members.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC. 27707, USA.

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.

No MeSH data available.