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FRET-Based Measurement of Apoptotic Caspase Activities by High-Throughput Screening Flow Cytometry

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

Abstract

Unwanted and excessive apoptosis contributes to various degenerative diseases, and apoptosis-inducing drugs are a mainstay of anticancer treatment regimens. The fields of pharmacology and toxicology consequently have a long history of investigating apoptotic cell death in the context of drug safety and efficacy studies. Canonical apoptotic cell death is crucially dependent on type II cysteinyl aspartate-specific proteases (caspases), and their activation is therefore widely used as a marker for this cell death modality. Here we describe a flow cytometric method for noninvasive, highly sensitive and reproducible FRET-based measurements of caspase activation. Compared to other flow cytometric techniques for apoptosis detection, this approach requires only minimal sample handling steps and provides a highly cost efficient option for large scale drug interaction studies and screens of compound libraries.

Key words

Apoptosis Cancer Caspases Flow cytometry Förster resonance energy transfer (FRET) High-throughput screening (HTS) 

Notes

Acknowledgements

This work was supported by a postdoctoral fellowship grant from the Irish Research Council (GOIPD/2013/102) awarded to Christian Hellwig, an NBIP Career Enhancement and Mobility Fellowship cofunded by Marie Curie Actions (EU FP7), the Irish HEA PRTLI cycle 4 and the Italian National Research Council, awarded to Agnieszka Ludwig-Galezowska, and by funding from the European Union (Horizon 2020 Marie S. Curie ETN MEL-PLEX) awarded to Markus Rehm.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Centre for Systems Medicine, Department of Physiology and Medical PhysicsRoyal College of Surgeons in IrelandDublin 2Ireland

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