FY-2013 Contract HHSN271201300010C, Topic 004. A High Throughput Assay for Neur

2013 财年合同 HHSN271201300010C，主题 004。神经元的高通量检测

• 批准号: 8756515
• 负责人: PATRICK M MCDONOUGH
• 金额: $ 14.98万
• 依托单位: VALA SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8756515
• 关键词: Affect; Automation; Biological Assay; Cell Nucleus; Cells; Contracts; Cytochalasin D; D Cells; Dyes; Evaluation; Fluorescence Microscopy; Fluorescent Dyes; Funding; Goals; Health; Image; Image Analysis; Methods; Microscopy; Migration Assay; Mitochondria; Neural Crest Cell; Pathway interactions; Phase; Phenotype; Reproducibility; Sampling; Sensitivity and Specificity; Signal Transduction; Testing; Work; cell motility; cellular imaging; data acquisition; high throughput screening; monolayer; programs

The technical objectives from the original solicitation are presented below, along with a brief discussion relevant to the Neural Crest Cell Migration assay. 1) Develop a working assay in a 384 microwell format (the Phase II objective will be to adapt the assay to the 1536 well format). Cells will be cultured to confluence in a 384 well dish; the cell monolayer will then be "scratched" to create a cell-free region within the center of each well. Migration of cells into the central region will be quantified in the presence of test compounds by imaging the cells for nuclei using fluorescence microscopy. Mitochondrial health will also be assessed utilizing a fluorescent dye (MitoTrackerOrange, or related dye) which will be imaged in a separate channel. 2) Characterize the sensitivity, specificity, variability, reproducibility, signal: background, dynamic range, and accuracy of the assay, utilizing standard positive and negative controls, Z' values >0.5. The goal is to achieve Z' values of 0.50 or greater for the effect of standard compounds to inhibit cell migration or mitochondrial function. Standard compounds to be considered are cytochalasin D for cell migration, and FCCP for mitochondrial health. 3) Demonstrate the utility of the assay by characterizing its ability to detect the effects of compounds known to affect the pathway/cellular phenotype, with a throughput of at least 10,000 samples/day with workstation automation. The goal is to achieve a throughput of at least 10,000 samples/day for data acquisition which we plan to achieve utilizing Vala's proprietary IC200 microscopy workstation, CyteSeer'i#¿ cell image analysis program, and methods to be further developed in the proposed funding period. 4) Develop an SOP to be submitted to NCA Ts for evaluation . A detailed SOP will be developed for the assay for submission to NCAT.

下面介绍了原始招标的技术对象，以及与神经Crest细胞迁移测定法相关的简短讨论。 1）以384 Microwell格式开发工作测定（II期目标将是将测定法调整为1536井格式）。细胞将在384井盘中培养为汇合；细胞单层将是 “刮擦”以在每个孔的中心内创建一个无单元的区域。在存在测试化合物的情况下，通过使用荧光显微镜对细胞进行成像，将在测试化合物的存在下进行量化。线粒体健康还将使用荧光染料（Mitotrackerorange或相关染料）进行评估，该染料将在单独的通道中成像。 2）表征灵敏度，特异性，可变性，可重复性，信号：测定的背景，动态范围和精度，利用标准正面和负面对照，z'值> 0.5。目的是实现标准化合物抑制细胞迁移或线粒体功能的Z值0.50或更高。要考虑的标准化合物是用于细胞迁移的细胞切拉斯蛋白D，线粒体健康的FCCP。 3）通过表征其检测已知会影响途径/细胞表型的化合物的作用的能力，证明了测定的实用性，并具有至少10,000个样品/天的吞吐量 使用工作站自动化。目的是实现至少10,000个样本/天的吞吐量，以获取数据，我们计划利用Vala专有的IC200显微镜工作站，Cyteseer'i＃�细胞图像分析程序以及在拟议的融资期间进一步开发的方法。 4）开发一个SOP，要提交给NCA TS进行评估。将开发详细的SOP，以评估提交给NCAT。