High Content Ratio Imaging for Apoptosis Detection Figure 9. Comparison of IETDase, LEVDase and DEVDase activity against MMP loss. Hela cells stably expressing SCAT3, SCAT8 and SCAT9, after 12 hrs treatment with a panel of drugs were imaged for IETDase, LEHDase and IETDase activity (A) Qualitative data was quantified and is represented as bar chart. SCAT3, SCAT8 and SCAT9 FRET ratio loss are represented side-by-side for comparison (B) Corresponding biparameteric dot plots showing comparison of IETDase, LEHDase and IETDase activity against MMP loss are shown. The drugs shown here are positive hits obtained from a larger panel of drugs used. doi:10.1371/journal.pone.0020114.g009 probe cleavage and corresponding caspase activation as well as by flow cytometry. We found the cleavage pattern of the transfected probe to be corresponding to that of the activation of the endogenous caspases, thereby showing their suitability for use in further experiments to assay caspase activity. In addition, since there was no significant variation in the percentage of apoptotic nuclei in the untransfected and transfected cell lines, the possibility of transfection-induced artifacts can be ruled out. In fact, we carried out mild selection and flow sorting, thereby being less invasive in enriching the transgene-containing cells, specifically to address this concern. With a simple methodology and using stably generated caspase sensor cell lines, we then developed a high content screening platform that could be adaptable for live cell multiparameter imaging, time kinetics study and cell line-to-cell line comparison of various apoptosis parameters such a mito- chondrial membrane potential (MMP) loss, chromatin condensa- tion, mitochondrial superoxide generation together with caspase activation. From our studies, we demonstrate the potential of this high content screening platform in giving integrated information of the varied effects of different drugs based on their mechanism of action. We propose that multiparameter death signaling signatures obtained with drugs of differing mechanisms of actions could be used for developing apoptosis centered algorithms for profiling large chemical libraries for unknown functions. PLoS ONE | www.plosone.org 14 May 2011 | Volume 6 | Issue 5 | e20114