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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.


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.
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Figure 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.

Mentions: The assay identified several compounds with ≥ 150% response compared to 2 ng/ml NGF alone in two separate runs. Among these were four compounds which have previously been shown to promote neurite outgrowth, namely zaprinast [20], etoposide [21], corticosterone [22] and serotonin [23]. Of these, six compounds yielded a response greater than 200%, our threshold for the assay, compared to background outgrowth in the presence of 2 ng/ml NGF (Fig. 5). The activity values of these compounds in the two independent tests were very similar. Using a threshold of two-fold enhancement of total neurites as cut off, the actives rate in this assay is 0.5%. Compiled values of the average window (defined as the plate mean of NGF-stimulated control values divided by the plate mean of non-stimulated control values) and the average of Z primes from a representative plate set (n =14) were 4.8 and 0.57 respectively.


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

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

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

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

Figure 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.
Mentions: The assay identified several compounds with ≥ 150% response compared to 2 ng/ml NGF alone in two separate runs. Among these were four compounds which have previously been shown to promote neurite outgrowth, namely zaprinast [20], etoposide [21], corticosterone [22] and serotonin [23]. Of these, six compounds yielded a response greater than 200%, our threshold for the assay, compared to background outgrowth in the presence of 2 ng/ml NGF (Fig. 5). The activity values of these compounds in the two independent tests were very similar. Using a threshold of two-fold enhancement of total neurites as cut off, the actives rate in this assay is 0.5%. Compiled values of the average window (defined as the plate mean of NGF-stimulated control values divided by the plate mean of non-stimulated control values) and the average of Z primes from a representative plate set (n =14) were 4.8 and 0.57 respectively.

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.