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.
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