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Automated Ratio Imaging Using Nuclear-Targeted FRET Probe-Expressing Cells for Apoptosis Detection

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Part of the Methods in Pharmacology and Toxicology book series (MIPT)

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.

Key words

Apoptosis detection Caspase activation Cytotoxicity assay Drug screening Fluorescence resonance energy transfer (FRET) High-throughput screening Ratio imaging 

Abbreviations

BFP

Blue fluorescent protein

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

DsRed

Discosoma sp. red fluorescent protein

ECFP

Enhanced cyan fluorescent protein

EMCCD

Electron multiplying charge coupled device

EYFP

Enhanced yellow fluorescent protein

FACS

Fluorescence-activated cell sorting

FBS

Fetal bovine serum

FITC

Fluorescein isothiocyanate

FRET

Fluorescence resonance energy transfer

FSC

Forward-scattered light

GFP

Green fluorescent protein

IMD

Intensity modulation display

NCCS

National Centre for Cell Science

NCI

National Cancer Institute

NLS

Nuclear localization signal

PFS

Perfect focus system

QE

Quantum efficiency

ROI

Region of interest

SCAT

Sensor for activated caspases based on FRET

Notes

Acknowledgements

This study was supported by research grants from Department of Biotechnology, Government of India. KAM was supported by research fellowship from ICMR (Indian Council of Medical Research, Govt. of India), DI and JJ were supported by research fellowship from CSIR (Council of Scientific and industrial Research, Govt. of India), and SNV was supported by research fellowship from UGC (University Grants Commission, Govt. of India). We thank Professor M. Radhakrishna Pillai, Director, RGCB, for his constant support and encouragement. We are grateful to all members of cancer research program laboratory and staff of the institutional FACS facility for helping throughout the work. We thank Prof. Jeremy M. Tavare and Dr. Gavin Welsh (University of Bristol, UK) for kindly sharing the plasmid, pcDNA3 ECFP-DEVD-EYFP; and Dr. Masayuki Miura (University of Tokyo, Japan) for the plasmid, pcDNA NLS-SCAT3.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Cancer Research Program-1Rajiv Gandhi Centre for Biotechnology (RGCB)ThiruvananthapuramIndia

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