sensor probes having ECFP and Venus tags as the donor and acceptor fluorophores. A panel of cell lines with the transfected probes was stably generated as described in Materials and Methods (Table S2), which were then validated for the presence of the transfected gene product using different methods. Employing fluorescence time lapse imaging, FRET probe cleavage, which corresponded to an increase in cyan coloured (CFP channel) over green colored (YFP channel) cells was taken as the read out for caspase activation (Figure S1, Movie S1) with untreated cells not showing any such change. Once caspase activation was thus qualitatively confirmed by fluorescence imaging (Figure 1 C–F), quantitation of the same in single cells was performed by confocal microscope-aided FRET acceptor bleaching method as described in Materials and Methods. From this, in MCF7 SCAT3, MCF7 SCAT8 and MCF7 SCAT9 cells, untreated population gave FRET efficiencies of 23.2862.08%, 28.3766.94% and 22.1062.78% respectively, whereas the population treated for 12 hrs with Staurosporine (STA) gave reduced FRET efficiencies of 5.3260.69%, 6.764.04% and 8.2160.40% in the respective cell variants. Caspase inhibitors for the three caspases reversed the FRET loss information as seen by the reduction in FRET loss positive cells (Figure 1 G) (Supplementary Fig. S3). In order to confirm whether SCAT probe cleavage was comparable to the corresponding endogenous caspase activation pattern, we probed samples of HeLa cells treated with Apicidin, Anisomycin and Cisplatin for caspase full length protein cleavage as well as SCAT probe cleavage. The results displayed similar activation profiles between the two, thereby confirming the utility of the probes as a direct read-out for the endogenous caspase activation; SCAT3 for DEVDase, SCAT8 for IETDase and SCAT9 for LEHDase activity (Figure 1 J). Further, using flow cytometer-based FRET detection, we confirmed the results obtained through western blotting (Figure 1 K). Also, a kinetics analysis of the percentage of cells showing FRET loss upon treatment with few drugs (data of Cisplatin shown) for SCAT3/8/9 probe cleavage was done by flow cytometry, which showed a gradational activation pattern over a time period of 0 to 24 hrs studied, with a surge of activation after 8 hrs post treatment (Figure 1 L,M). This shows that the panel of cell lines developed could also be used to study kinetics of percentage of cells with active caspases over a period of time. Also, in order to address the issue of transfectioninduced artifacts in the apoptotic response, we looked for the percentage of apoptotic nuclei in untransfected and transfected cells (Figure 1N). It is noteworthy that cell reponse does not significantly vary in both the cases. All these methods thus confirmed the functionality of the probes in the stable cell lines generated both qualitatively as well as quantitatively. The HCS platform is adaptable for high content apoptosis analysis Once validation of the engineered cells were complete, a platform for high content imaging and apoptosis analysis using these cell lines was designed (Figure 2). A representation of the output data is also shown (Figure 3). First, in OVCAR8 SCAT3 cells we used a random panel of drugs (in duplicates) from that given in Supplementary information to check for the adaptability of the method for high content screening. For this, cells were imaged at 0, 6, 18, 36 and 48 hrs post drug treatment. Data was analysed using polygon segmentation, where, FRET signal was measured in the whole cell. By the segmentation applied, neighboring cells could be clearly demarcated and apoptotic bodies as well as spread out cells could be picked up together for analysis. The cells imaged with 363 montage acquisition as well as single montage image with respective segmentation masks are High Content Ratio Imaging for Apoptosis Detection represented in Figure 4A. In general for most comparative data generation, cells were imaged with 363 montage to cover large population of cells from each well. In the pattern of drug activity obtained, some drugs were found to be active very early after drug treatment, while others displayed a slow, gradational activation (Figure 4B). Slight caspase activation could be picked up even as early as 6 hrs post treatment, which could be neatly followed up upto the 48 hrs time point analysed. Thus, it was confirmed that this could be adapted as a sensitive and reliable assay methodology for high content caspase activation profiling of live cells. The HCS platform is adaptable for multiparameter apoptosis analysis Since a high content screening platform to assay multiple apoptosis parameters like caspase activation, MMP loss and chromatin condensation simultaneously in live cells has not been reported yet, we wanted to check for the adaptability of this platform for such a multiparameter apoptosis analysis. Initially spinning Disk confocal imaging was carried out to check the suitability for multiparamater imaging of MMP loss together with ratio analysis for caspase activation status in live cells. MCF7 SCAT 3 cells treated with Actinomycin D after staining with MMP indicator TMRM were imaged using CARV confocal imager by time lapse as described. The representative images from multiple time points and video of merged channels are shown in Figure S2, Movie S2. As seen from the figure and movie, temporal information of loss of TMRM fluorescence and FRET can be easily monitored visually. For high throughput analysis, OVCAR8 cells stably transfected with SCAT3 was treated with 42 drugs (in duplicates) for 20 hrs. Following drug treatment, cells were imaged for the above-mentioned parameters under the BD pathway imager as described in Materials and Methods. We obtained, among these, 19 positive hits displaying variable MMP loss, FRET ratio loss and chromatin condensation response patterns depend- ing on the mechanism of action of the drug (Figure 5). From the results, even at 20 hrs post treatment, the drugs Cyclophospha- mide, Mitoxantrone, Monastrol, Apigenin, 17 DMAG, Apoptosis Activator I and Indomethacin displayed only a loss in mitochon- drial membrane potential (MMP loss) that was not accompanied by a correspondingly elevated caspase activation or chromatin condensation. DNA damaging agents Cisplatin, Actinomycin D, and Camptothecin, HDAC inhibitor Trichostatin A, kinase inhibitors 5,6-dichloro 1,b-D ribofuranosyl benzimidazole and Staurosporine and Lactacystine were found to induce FRET ratio loss without a correspondingly massive MMP loss. This points towards a Dym- independent DEVDase activity possible with drugs that induce damage of DNA or associated machinery. On the other hand, FRET loss, MMP loss and chromatin condensa- tion were all found to be induced by translation inhibitors Cycloheximide, Anisomycin as well as Novobiocin and Gossypol at 20 hrs post treatment. Furthermore, starvation (0.5% serum in culture medium HBSS) condition for 20 hrs induced chromatin condensation and slight reduction in MMP, without significant caspase activation. Further, in order to look at the correlation between mitochon- drial superoxide (SOX) generation and MMP loss along with FRET loss and chromatin condensation in the same cells, we stained separate wells with mitochondrial SOX indicator dye MitoSOX Red and TMRM dye. Eight positive hits were selected on the basis of robust variations (Figure 6). We found a high correlation between MitoSOX gain and TMRM loss in all the drugs tested, except for Gossypol, which induced considerably very high SOX production when compared to the MMP loss. Our PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20114