High Content Ratio Imaging for Apoptosis Detection Figure 10. Drug resistant cell execute caspase 9 independent cell death. (i) HeLa SCAT9 cells were treated with a large panel of drugs and subject to revisit imaging of the surviving cells. Panels A to F show parental cells just prior to drug treatment. G to L represent cell response 24 hrs after the first round of treatment with the indicated drugs. Here, white and red solid arrows indicate representative live adherent (FRET loss negative) and dead rounded (high FRET loss positive) cells respectively. M to R show colonies out of cells that survived the first round of drug treatment. S to X and Y to AD show the response of the same area of the surviving population after 24 hrs and 48 hrs respectively upon second treatment of the escaped population with the same drugs. In panels S to X, white and yellow solid arrows indicate representative live adherent (FRET loss negative) and dead rounded (high FRET loss negative) cells respectively. Heat map shows the representative qualitative low to high FRET loss index (ii) Histogram showing percentage of HeLa SCAT9 cells with detached rounded morphology (dead cells) of parental population (light grey solid bars) and escaped population (dark grey solid bars) at 24 hrs post drug treatment with the 6 indicated drugs belonging to 3 classes of mechanism of action. doi:10.1371/journal.pone.0020114.g010 Upon comparing Dym and mitochondrial superoxide (SOX) generation, we found a high correlation between the two in all the drugs tested, except Gossypol, which induced considerably very high SOX production when compared to Dym. Gossypol, a polyphenolic aldehyde is known to induce Bax/Bak independent cytochrome c release by mediating a conformational change in Bcl2 [32]. In addition, Gossypol has been shown to be mediating free radical toxicity in isolated rat islet cells [33], oxygen radical formation in rat liver microsomes and human sperms [34] as well as ROS mediated growth inhibition in prostate cancer cells. Our multiparameter HTS results indicate that the growth inhibitory and toxic activities of Gossypol without a correspondingly massive MMP loss could be due to the massive superoxide generation and an alternate superoxide-mediated, MMP loss-independent apop- tosis signaling mechanism. Our observation of certain cells that maintain MMP loss without caspase activation or chromatin condensation at a late stage post-drug treatment suggest that the actual ‘point-of-no- return’ of apoptosis is a much more complex event that could probably be ultimately dependent on the mode of action of the death inducer. Also, through this method, compounds that activate caspases independent of the mitochondria-dependent pathway could be detected. So far, simultaneous detection of apoptosis and mitochondrial superoxide production in live cells worked best with flow cytometry and confocal microscopy [35], which suffer from the disadvantage of either being cumbersome or time-consuming for large scale compound screening. Here we report the first ever comparison of quantitative data on mitochondrial superoxide generation and caspase activity derived from live single cell imaging and thus project the possibility of this methodology in large scale compound screening and generation of multiparameter cell signaling information with a single experiment. In our multiparameter kinetics analysis, we got two ‘response groups’ of drugs, with one showing gradual activation and the other showing sudden activation. Also, we have compared the IETDase, LEVDase and DEVDase activity against MMP loss in HeLa cells, thereby showing the advantage of this screening platform in giving a comprehensive picture of initator and effector caspase activation in the same cell line. A favourable improvement of this could be the use of a three chromophore FRET based system using for e.g, CFP-YFP-mRFP for the simultaneous detection of multiple caspases or other enzymes within the same cell [36]. Another major advantage of this method is the possibility of following up of the respective wells for subsequent fate, whereby we have identified rare escape of cells from caspase activation with certain antitumor agents. In a unique experiment, we could also show emergence of drug- escaped colonies and their subsequent response to a second cycle of drugs. Employing such a technique, we show that cells that escaped after treatment with cell cycle inhibitors stay more resistant to subsequent re-treatment compared to HDAC inhibitors, translation inhibitors. Most of the stable cell lines described here were clonally expanded and their stable expression have been followed up for more than 200 passages without any significant loss of function and expression. Therefore, they can form a ready-to-use method for HTS-based drug screening applications. However, if this needs to be adopted for other cell lines, stably integrated colonies with optimum FRET efficiency need to be generated as described here. As an extension to our results, we also suggest a future possibility of stable generation of sensor probes in the entire NCI cell line panel with simultaneous multiparameter signaling analysis will help to define the pathway signatures of each drug and may augment selection of best hits from the early screening effort. This is also expected to augment the identification of unusual non classical apoptosis inducers from large libraries in less time. Supporting Information Movie S1 Visualization of FRET loss over time. MCF7 SCAT3 cells grown on chambered cover glass was treated with Camptothecin and imaged every 10 minutes after drug addition in controlled temperature and humidity conditions. Imaging was done using Nikon inverted fluorescent microscope under 206 1.0 objective and Andor 885 camera. The frame rate is 30 frames per second. (AVI) Movie S2 Simultaneous visualization of TMRM loss and caspase activation. MCF7 SCAT3 cells stained with MMP indicator dye TMRM was treated with Actinomycin D and imaged every 10 minutes after drug addition. Imaging conditions are same as mentioned in Supplementary Movie S1. The frame rate is 30 frames per second. (AVI) Figure S1 Visualization of FRET loss over time by widefield ratio imaging. Images of Camptothecin treated MCF7 SCAT3 cells at 1 hr, 10 hr and 18 hr after drug addition taken from live cell imaging. Imaging conditions are same as mentioned in Supplementary Movie S1. Arrows indicate some representative cells that show FRET loss at the indicated time points. (TIF) Figure S2 Simultaneous visualization of TMRM loss and caspase activation. Confocal images of Actinomycin D treated MCF7 SCAT3 cells counterstained with TMRM for visualizing MMP loss at 4 hr, 16 hr and 20 hr after drug addition. Imaging conditions are same as mentioned in Supplementary Movie S1. Arrows indicate some representative cells that show FRET loss at the indicated time points. (TIF) Figure S3 Validation of FRET sensor probe: Caspase inhibition reverses FRET loss pattern. Panels A–D, E–H PLoS ONE | www.plosone.org 16 May 2011 | Volume 6 | Issue 5 | e20114