Abstract
High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some specifics vary according to the assay type, each will apply some degree of image processing and feature extraction followed by a data analysis pipeline to identify the perturbations (small molecules, etc.) of interest. We describe and discuss the advantages and limitations of high content assays and screens using the specific example of assaying mitochondrial dynamics in primary neurons. We provide a detailed description of our culturing methods, imaging and data analysis techniques and provide an open source, ready to use CellProfiler pipeline for high-throughput image segmentation and quantification tool for mitochondrial parameters.