High Content Ratio Imaging for Apoptosis Detection Figure 7. Adaptability for assaying multiparameter kinetics. For assaying DEVDase activity as a function of time, HeLa SCAT3 cells were treated with a panel of 42 drugs (in duplicates) and imaged at the time points: 1 hr, 12 hr, 22 hr, 36 hr, 72 hr post drug treatment for both DEVDase activity and chromatin condensation. (A) Heat map of kinetics of FRET ratio loss at the indicated time points. Quantitation of the same was used to plot bar charts for (B) DEVDase activity (C) and chromatin condensation at the indicated time points, showing 2 response groups with 24 positive hits. Mevinolin and Indomethacin, representative drugs of the two response groups are shown (D, E) with upper panel showing CFP/YFP merge, FRET ratio loss and chromatin condensation images and lower panels showing dot plot representations of FRET ratio loss and chromatin condensation at the indicated time points. Details of well positions of drugs used are given in Supplementary Figure S4. Additional details of drug concentration, known mechanism of action have been given in Supplementary Table 2. doi:10.1371/journal.pone.0020114.g007 therapies were discovered based on their anti-proliferative activity or cytotoxicity as end point detection in cell-based assays, with no pre-existing knowledge of their targets of action. This kind of a unified approach in drug discovery fails in revealing potential anticancer agents with varied modes of action as a result of which, none of the currently used drugs selectively and directly target the molecular lesions responsible for malignant transformation. Most importantly, considering the enormous evidence linking cancer and apoptosis, it appears that screens of apoptosis rather than anti- proliferative activity are far more superior screens for anticancer drug development. Accordingly, the present study set forth to develop a rational strategy based on live cell apoptosis detection as a useful tool to obtain a better mechanistic understanding of newer anticancer agents. The major highlight of this study is that the conclusions drawn on the response of the population are actually derived by recording the response of each single cell. Therefore the results bear very low noise. Interestingly, soon after drug addition, some wells showed a high DEVDase read out due to the intrinsic fluorescence interference of the drug itself, which remained equally intense upto the assay end point. To the advantage of this methodology, such a display makes the technique suitable for early detection of any spurious signal, thereby preventing the possibility of wrongly interpreting an otherwise negative hit. Before designing the HTS methodology, we validated the functionality of the probes in the stably generated cell lines by live cell imaging based fluorescence microscopy, FRET acceptor bleaching by confocal microscopy, immunoblotting for FRET Figure 8. Adaptability for comparing differential drug sensitivity among cell lines. To check for adaptability of the method to compare for differences among cell lines, stably generated U251 SCAT3 and HeLa SCAT3 cells were treated with a panel of 37 drugs (in duplicates) and the DEVDase activity was imaged and analysed at 48 hrs post treatment. 29 hits were identified that showed significant variation from untreated controls. (A) U251 SCAT3 and (B) HeLa SCAT3 cells are shown. doi:10.1371/journal.pone.0020114.g008 PLoS ONE | www.plosone.org 13 May 2011 | Volume 6 | Issue 5 | e20114