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.

下面介绍了最初招标的技术目标，以及与神经嵴细胞迁移测定相关的简短讨论。 1) 开发 384 微孔格式的工作检测（第二阶段的目标是使该检测适应 1536 孔格式），然后将细胞在 384 孔培养皿中培养至汇合； 在测试化合物存在的情况下，通过使用荧光显微镜对细胞进行细胞核成像来量化细胞向中心区域的迁移。也将利用线粒体健康状况进行评估。荧光染料（MitoTrackerOrange 或相关染料）将在单独的通道中成像。 2) 利用标准阳性和阴性对照，表征检测的灵敏度、特异性、可变性、重现性、信号：背景、动态范围和准确性，Z'值>0.5。标准化合物抑制细胞迁移或线粒体功能的效果为 0.50 或更高。要考虑的标准化合物是用于细胞迁移的细胞松弛素 D 和用于线粒体健康的 FCCP。 3) 通过表征其检测已知影响途径/细胞表型的化合物的作用的能力来展示该测定的实用性，吞吐量至少为 10,000 个样本/天 目标是实现每天至少 10,000 个样本的数据采集吞吐量，我们计划利用 Vala 专有的 IC200 显微镜工作站 CyteSeer'i#¿细胞图像分析程序和方法将在拟议的资助期内进一步开发。 4) 制定一份 SOP，提交给 NCA T 进行评估 将为提交给 NCAT 的检测制定一份详细的 SOP。