the presently available techniques. On the other hand, population based studies inspite of giving robust results, bear noise as well as fail to give details of subcellular spatial and temporal apoptotic events as in single cell studies. Basic cancer research has given us remarkable insights into our understanding of the biology of cancer with the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype [20,21]. Cancer-generating mutations disrupt apoptosis, leading to tumor initiation, progression or metastasis. The mutations that suppress apoptosis also reduce the sensitivity to cancer treatment [22]. Considering the enormous evidence linking cancer and apoptosis, it appears that screens of apoptosis rather than the cell protein content status are far more superior for anticancer drug screening [23,24]. However, miniaturization of apoptosis detection screens for highthroughput assays remains a challenge. Caspase activation is an accepted read- out of apoptosis and proteinase activity and can be exploited for biomolecular screening [25]. Caspase activation can be detected by measurement of catalytic activity, immunoblotting, immuno- labeling using conformation-sensitive antibodies or affinity labeling followed by flow cytometry. Out of these, live cell real time detection of activity is possible only by a FRET based probe- cleavage study [26,27]. All the other methods either require cell- free protein extracts to be prepared or cell fixation done, thus being non-adaptable for a live cell high throughput (HTS) study. Current caspase activation HTS assays are based on fluorigenic substrates or FRET based probes, the former being an end-point detection. Also, the FRET based live cell HTS detection systems developed so far have failed to give quantitative information in a cell-to-cell specific, real-time manner, or are not adaptable for multiparameter analysis [28]. We have developed a novel highthroughput platform in cell lines of varying origins that could be adopted for real time, multiparameter apoptosis detection and high content drug screening. To enable simultaneous detection and analysis of multiple parameters using spectrally similar fluorophores, spatially separated segmentation masks were employed. By employing this approach, we have identified novel non-conventional apoptosis induction mechanisms of certain drugs. A high content screening platform to assay multiple death signals like caspase activation, MMP loss, chromatin condensation and mitochondrial superoxide generation simultaneously in live cells as is shown in our study helps to link the interrelationship of these critical events in the execution of cell death in HTS fashion. Through a time kinetics study we found two response groups of ‘sudden’ acting and ‘gradual’ acting drugs. This assay is useful for comparing caspase activation profiles among different cell lines as well as for that of initiator and effector caspase activity in the same cell line itself. Another important advantage of the platform described is long term fate analysis of non-responsive cells. Using this, we report a non-caspase 9 mediated pathway of death induction of 3 classes of compounds in the cell population that survived initial death induction by these same compounds. This is the first ever report of a highthroughput platform which records subcellular signals from individual cells so as to give a more accurate picture of the whole population and thus becomes useful in the real-time tracking of multiple apoptosis parameters in live cells, that can be adapted for direct screening of drugs for multiple cellular signaling. Materials and Methods Cell culture and drug addition All cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) containing 10% Fetal Bovine Serum High Content Ratio Imaging for Apoptosis Detection (FBS) and antibiotics in a humidified CO2 (5%) chamber at 37uC. For experiments, cells were seeded in 96 well plates at desired densities. After an overnight culture, medium was removed and 100 ml fresh phenol-red free DMEM containing 5% FBS with the compounds to be tested were added. Concentrated stocks of each drug was prepared in either DMSO or water according to solubility and stored at 280uC. The list of drugs used, known mechanism of action and the respective concentration have been given in supplemental information (Table S1). Expression vectors and generation of stable cell lines SCAT3 and SCAT9 vectors were kindly gifted by Dr. Masayuki Miura, University of Tokyo, Japan [30]. The former is linked with preferred caspase 3/7-specific DEVD recognition site and the latter with caspase 9-specific recognition site LEHD in between donor and acceptor. SCAT 8 was supplied by Dr. Markus Rehm, Royal College of Surgeons in Ireland, Ireland that is intercon- nected with short linker of preferred caspase-8/-10 recognition site IETD between donor and acceptor [16]. Cell transfection was performed using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions. Stably expressing clones were generated by selecting the cells in 800 mg/ml of G418 (Invitrogen, Carlsbad, CA) containing medium for 30–45 days. Following clonal propagation, the multiple clones with different levels of transgene expression were expanded and further sorted based on quantitative FRET, analysed by FACS Aria (Becton Dickinson) so as to obtain an enriched population of SCAT3, SCAT8 and SCAT9 expressing cells with optimum default FRET. Briefly, cells were excited with 407 nm laser and the FRET-high cells were identified from the scatter plot of ECFP emission collected using 450640 nm filter against EYFP emission collected using 530630 nm filter. FRET acceptor bleaching by Confocal laser scanning microscopy Cells seeded in chambered coverglass (Lab-TekTM, Nunc, Rochester, NY) were sequentially scanned using a laser scanning Leica TCS SP2 Confocal microscope (Leica Microsystems, Germany) for donor (ECFP) and acceptor (Venus) fluorescence signals under the 458 nm and 514 nm laser lines respectively to obtain pre-bleach donor intensity (Donorpre) and pre-bleach images. Following this, the acceptor was bleached off completely with high intensity 514 nm laser pulses after which, the donor intensity was obtained (Donorpost) and FRET efficiency was calculated using the formula: % FRET efficiency = (Donorpost2 Donorpre)/Donorpost. Pre-bleach and post-bleach donor/acceptor intensity images were used to correlate the results visually. Flourescence time lapse imaging Cells were grown on chambered cover glass (Lab-TekTM, Nunc, Rochester, NY), to which, the indicated drugs reconstituted in DMEM containing 5% FBS was added. For imaging, cells were stained as described and imaged in a live cell incubation chamber (Tokai Hit, Japan) that maintains optimum CO2, temperature and humidity. Imaging was carried out under a 206 Plan Apo 1 objective under an inverted fluorescence microscope (Nikon Eclipse, TE2000-E) equipped with a CARV II confocal imager (Becton Dickinson) either in confocal or non confocal mode and an automated excitation and emission filter wheel. The images were captured using EMCCD camera Andor iXON 885 using IPLab software (BD, USA) at regular intervals for the indicated time periods. To minimize photobleaching, the intensity of the light was reduced to 1% by intensity iris control. For ratio imaging PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20114