Optimization of fluorescence assay of cellular manganese status for high throughput screening

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.