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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.


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).
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Figure 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).

Mentions: In order to establish that GFP expression and CellMask™ Red staining accurately reflected the pharmacological response of NS-1 cells to NGF, we treated cells with varying doses of NGF and measured percent neurite response as a function of NGF concentration. Following NGF treatment, the cells were imaged by both direct methods described in this paper, and the dose-response curves were compared to the curve generated with the standard antibody labeling method (Fig. 3). The resulting curves produced comparable EC50 values of 3.3 ng/ml for GFP expressing cells, 2.1 ng/ml for anti βIII-tubulin labeled cells, and 1.4 ng/ml for HCMR stained cells. These results are in agreement with each other and data reported for antibody detection of NGF induced neurite outgrowth in NS-1 in the vendor technical bulletin (Cellomics, Pittsburg, PA). The data also showed that NS-1 cells are approximately 5-fold to 8-fold more sensitive to recombinant mouse 2.5S NGF compare to literature values reported for the parent PC12 cells [16].


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

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

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

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

Figure 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).
Mentions: In order to establish that GFP expression and CellMask™ Red staining accurately reflected the pharmacological response of NS-1 cells to NGF, we treated cells with varying doses of NGF and measured percent neurite response as a function of NGF concentration. Following NGF treatment, the cells were imaged by both direct methods described in this paper, and the dose-response curves were compared to the curve generated with the standard antibody labeling method (Fig. 3). The resulting curves produced comparable EC50 values of 3.3 ng/ml for GFP expressing cells, 2.1 ng/ml for anti βIII-tubulin labeled cells, and 1.4 ng/ml for HCMR stained cells. These results are in agreement with each other and data reported for antibody detection of NGF induced neurite outgrowth in NS-1 in the vendor technical bulletin (Cellomics, Pittsburg, PA). The data also showed that NS-1 cells are approximately 5-fold to 8-fold more sensitive to recombinant mouse 2.5S NGF compare to literature values reported for the parent PC12 cells [16].

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.