A rapid, inexpensive high throughput screen method for neurite outgrowth.
A rapid, inexpensive high throughput screen method for neurite outgrowth.
复制标题
DOI：
10.2174/1875397301004010074
复制
发表时间：
2010-12-13
期刊：
影响因子：
--
通讯作者：
Ibeanu GC
中科院分区：
文献类型：
--
作者：
Yeyeodu ST;Witherspoon SM;Gilyazova N;Ibeanu GC
神经突生长测定是评估化学对神经元细胞的影响的最常见的表型筛选。目前的自动化测定涉及昂贵的设备、冗长的样品制备和处理、昂贵的试剂和缓慢的数据采集和分析速率。我们已经开发了一个高通量屏幕（HTS）神经突生长使用强大的神经元细胞模型耦合到快速和廉价的可视化方法，减少数据量和快速的数据分析。神经筛选1（NS-1）细胞，一个亚克隆的PC 12，具有快速生长和增强的敏感性，神经生长因子被用作模型神经元。该方法通过使用表达GFP的细胞或用HCS CellMask™ Red染色的天然细胞在多重30分钟固定和染色步骤中减少制备时间。与当前协议相比，2x2相机合并过程将图像数据文件和分析时间分别减少了75%和60%。此外，在蒙太奇生成期间消除自动对焦步骤减少了数据收集时间。通过NGF和SU 6656刺激和抑制神经突生长的药理学特征与利用微管蛋白的免疫荧光检测的当前标准方法相当。通过使用该方法测定的1，120个成员的Prestwick化合物文库的成员对NGF诱导的神经突生长的增强鉴定了六种分子，包括依托泊苷、醋酸异氟泼尼龙、醋酸氟氢可的松、硫代鸟苷、羟保泰松和赤霉酸，其使由2ng/ml NGF引发的神经突质量增加了一倍以上。这个简单的程序代表了一个重要的常规方法，在高通量筛选大型化学库使用的神经突生长表型作为衡量化学分子对神经元细胞的影响。
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.