510 www.sbsonline.org © 2007 Society for Biomolecular Sciences High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry–based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. (Journal of Biomolecular Screenin