Journal
JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY
Volume 27, Issue 1, Pages 42-49Publisher
WILEY-BLACKWELL
DOI: 10.1002/jbt.21457
Keywords
Fura-2; Manganese; High Throughput Screening; Neurotoxicity; Striatal; Neurons
Categories
Funding
- Public Health Service award from the National Institute of General Medical Studies for the Vanderbilt Medical Scientist Training Program [T32GM07347]
- NIH [P30 ES000267, RO1 ES016931, R01 ES010563]
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [R01ES016931, P30ES000267, R01ES010563] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM007347] Funding Source: NIH RePORTER
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The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra- and interplate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport. (C) 2012 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:42-49, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21457
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