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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.


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.
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Figure 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.

Mentions: In order to minimize the GFP signal gradient and ensure reproducibility of the data, we sorted the cell populations by FACS analysis and collected samples based on fluorescence intensity-dependent gating (Fig. 1). About 80% of the sorted cell population exhibited fluorescence intensity of at least 10-fold compared to untransfected cells.


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

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

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

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

Figure 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.
Mentions: In order to minimize the GFP signal gradient and ensure reproducibility of the data, we sorted the cell populations by FACS analysis and collected samples based on fluorescence intensity-dependent gating (Fig. 1). About 80% of the sorted cell population exhibited fluorescence intensity of at least 10-fold compared to untransfected cells.

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.