High Content Ratio Imaging for Apoptosis Detection Figure 5. High content multiparameter analysis: FRET ratio loss, TMRM intensity and chromatin condensation. OVCAR8 cells stably transfected with SCAT3 was treated with 42 drugs (in duplicates) for 20 hrs. Following drug treatment, cells were imaged for FRET ratio loss, TMRM loss and chromatin condensation under the BD pathway imager as described in Materials and Methods. Polygon band segmentation was applied and the 19 positive hits obtained displaying variable MMP loss, FRET ratio loss and chromatin condensation response patterns are shown. (A Images of FRET ratio loss, TMRM and chromatin condensation are shown, (B) with the corresponding scatter plot representation obtained through data export and analysis in BD FACSDiva software. doi:10.1371/journal.pone.0020114.g005 from one carcinoma type to another, stable generation of these sensor probes in a large panel of cells will enable for comprehensive cell-to-cell comparative study in a kinetic mode. Z factor, which defines the screenability of any particular highthrouput assay, was found to be above 0.7 in all our experiments and for the entire cell lines used, thus demonstrating the usability in large compound library screening. The development of novel molecularly targeted cancer therapeutics remains slow and expensive with many late-stage failures. This is because most of the frequently used anti-cancer Figure 6. High content multiparameter analysis: FRET ratio loss, TMRM intensity, chromatin condensation and MitoSOX production. For simultaneous imaging and correlation between mitochondrial superoxide (SOX) generation and MMP loss along with FRET ratio loss together with chromatin condensation in the same cells, OVCAR8 SCAT3 cells were stained in separate wells with mitochondrial SOX indicator dye MitoSOX Red and TMRM dye. 8 positive hits were selected on the basis of robust variations (A) Images of FRET ratio loss, mitochondrial superoxide and chromatin condensation from the same well and the (C) corresponding TMRM pattern from a different well (B) Biparametric dot plot showing comparison of MitoSOX and FRET ratio loss. Encircled area shows the response patterns with each drug. doi:10.1371/journal.pone.0020114.g006 PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e20114