Krupa Ann , Mathew and Deepa, Indira and Jeena, Joseph and Prakash , Rajappan Pillai and Indu , Ramachandran and Shankara , Narayanan Varadarajan and Santhoshkumar, T R (2016) Automated Ratio Imaging Using Nuclear-Targeted FRET Probe-Expressing Cells for Apoptosis Detection. Apoptosis Methods in Toxicology. pp. 131-161. ISSN ISBN 978-1-4939-3588-8

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Abstract

In recent years, innovative bioassays have been designed to detect intracellular caspase activation as a reliable read-out of apoptotic activity of bioactive compounds. Most anticancer drugs target cells by triggering caspase dependent protein cleavage, culminating in apoptotic cell death. Therefore, detection of caspase activation has been recognized as one of the best approaches for detecting cancer cell death as compared to assaying the ill-defined general cytotoxic activity that often manifests with off-target side effects. Among the available methods of detection, those with cells stably expressing FRET-based fluorescent probes are more suitable for studying live cells, because they yield reliable readouts due to minimal invasiveness and easy automated quantitation possibilities. We have recently reported a successful FRET-based high-throughput assay protocol for detecting caspase activation in live cells using stable cells expressing intracellular FRET probes. An important advantage of this approach with respect to other apoptosis assays is its ability to monitor caspase activation, real-time, in live cells. However, proper automated identification of individual cells (viz., segmentation) requires nuclear staining and complex image processing. Here, we discuss an advanced tool for detecting intracellular caspase activation, which surpasses the above disadvantages. The tool described here works by first generating stable cancer cell lines expressing the FRET probe inside nucleus, thereby eliminating the need for extra staining and complex processing. Such nuclear targeting enables accurate automated segmentation and quantification of caspase activation in a multipoint/multidrug manner, using automated microscopy. We then describe the step-by-step protocol for detecting caspase activation in live cells using such a nuclear-targeted FRET-based tool. In addition, we propose the adaptability of this method for high-throughput screening platforms, so as to single out potential anticancer compounds to aid further lead optimization.

Item Type:	Article
Uncontrolled Keywords:	Apoptosis detection Caspase activation Cytotoxicity assay Drug screening Fluorescence resonance energy transfer (FRET) High-throughput screening Ratio imaging
Subjects:	Cancer Research
Depositing User:	Central Library RGCB
Date Deposited:	01 May 2019 06:57
Last Modified:	01 May 2019 06:57
URI:	http://rgcb.sciencecentral.in/id/eprint/739

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