Abstract
We have developed a high-content screening (HCS) assay to find activators of Wnt/Frizzled (Wnt/Fzd), a pathway known to be important in bone formation. Utilizing primary human preosteoblasts as a model, activation of the Wnt/Fzd pathway was detected by monitoring the stabilization and translocation of the transcription factor β-catenin from cytoplasm to the nucleus. Endogenous β-catenin was detected in preosteoblasts by immunofluorescent staining, and subcellular localization was determined by HCS using the Cellomics (Pittsburgh, PA) ArrayScan® IV. Positive controls, including Wnt3A-conditioned medium and inhibitors of glycogen synthase kinase-3β, resulted in increased nuclear β-catenin. The assay had a Z'-factor of 0.6 and was conducive to automation for high-throughput screening/HCS. By combining standard immunofluorescence technology with automated fluorescence microscopy, we demonstrate the capability of screening cellsignaling pathways in primary human cells.
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