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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.


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.
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Figure 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.

Mentions: The cell fractions, which were relatively homogeneous after sorting, were tested in the neurite outgrowth assay. At this point the camera was able to autofocus with precision on the cells due to uniformity of the GFP fluorescence in the cell populations. The cell fractions were rapidly expanded and frozen for future use and we chose to continue our work with the cell population which showed moderate GFP fluorescence, because of slow growth characteristics observed with the high expressors. In cells with moderate expression, GFP fluorescence was clearly visible throughout the cytoplasm and neurite processes when viewed by fluorescence microscopy (Fig. 2A).


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

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

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

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

Figure 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.
Mentions: The cell fractions, which were relatively homogeneous after sorting, were tested in the neurite outgrowth assay. At this point the camera was able to autofocus with precision on the cells due to uniformity of the GFP fluorescence in the cell populations. The cell fractions were rapidly expanded and frozen for future use and we chose to continue our work with the cell population which showed moderate GFP fluorescence, because of slow growth characteristics observed with the high expressors. In cells with moderate expression, GFP fluorescence was clearly visible throughout the cytoplasm and neurite processes when viewed by fluorescence microscopy (Fig. 2A).

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.