High Throughput Ratio Imaging to Profile Caspase Activity: Potential Application in Multiparameter High Content Apoptosis Analysis and Drug Screening Jeena Joseph, Mahendra Seervi, Praveen K. Sobhan, Santhoshkumar Thankayyan Retnabai* Integrated Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram, Kerala, India Abstract Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based. Citation: Joseph J, Seervi M, Sobhan PK, Retnabai ST (2011) High Throughput Ratio Imaging to Profile Caspase Activity: Potential Application in Multiparameter High Content Apoptosis Analysis and Drug Screening. PLoS ONE 6(5): e20114. doi:10.1371/journal.pone.0020114 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received December 14, 2010; Accepted April 25, 2011; Published May 27, 2011 Copyright: 2011 Joseph et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Innovative Young Biotechnologist Award, Dept. of Biotechnology, Govt. of India to STR, CSIR SRF, Govt. of India to JJ, UGC SRF, Govt. of India to MS and ICMR SRF, and Govt. of India to PKS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: trsanthosh@rgcb.res.in Introduction Since the identification of the first caspase in the early 1990s and later when better biological systems for apoptosis study were described, the field of cell death research has taken a giant surge. Today, this has led to the identification of molecular mechanisms of newer forms of physiological cell death other than apoptosis like paraptosis, autophagy, necrosis and necroptosis that cross-regulate each other and have common molecular intermediates in deciding and executing the ultimate cell fate [1–6]. Mitochondrial outer membrane permeabilization (MOMP), intermembrane space (IMS) protein release, activation of pro-apoptotic Bcl-2 and caspase family proteins have been identified as key events in the initiation, progression and execution of apoptosis [7–12]. But, there is still much that we do not understand about how cell death signaling is fine-regulated particularly, how the signaling within a single cell is coordinated for its ultimate orchestration at the population level. For example, there is no clarity about why cell- to-cell variability in death execution exists and whether the hierarchical and spatiotemporal events are integrated at the cellular level or at the population level [13–15]. This is because the techniques of study used thus far have been limited to either single cell studies or population level studies only. Recent single cell studies using time-lapse confocal microscopy of green fluorescent protein (GFP) tagged apoptotic intermediates like cytochrome C and FRET probes for caspase activation have given valuable insights into the initiation and progression of the hierarchical events of apoptosis signaling within single cells [16,17], thereby throwing light into the subcellular changes and spatio-temporal relationship between two events. Further, single cell studies have contributed in understanding the ‘point-of-no- return’ or the nature of two different cells that respond differently [18,19]. Given the advantages, single cell studies are still not sufficient in giving an integrated picture of the subcellular events and the effect of these in a single cell on the population as a whole. This is because, in spite of the fact that single cell studies give real time low-noise information in single cells, concomitant analysis of the response of the whole population is practically impossible using PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20114