FRET-Based Measurement of Apoptotic Caspase Activities by High-Throughput Screening Flow Cytometry
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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 wordsApoptosis Cancer Caspases Flow cytometry Förster resonance energy transfer (FRET) High-throughput screening (HTS)
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.
- 9.Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A, Lohner K, Zweytick D (2011) In search of a novel target—phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim Biophys Acta 1808(11):2638–2645. doi: 10.1016/j.bbamem.2011.07.026 CrossRefPubMedCentralPubMedGoogle Scholar
- 18.Hellwig CT, Kohler BF, Lehtivarjo AK, Dussmann H, Courtney MJ, Prehn JH, Rehm M (2008) Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation. J Biol Chem 283(31):21676–21685. doi: 10.1074/jbc.M802889200 CrossRefPubMedGoogle Scholar
- 20.Delgado ME, Olsson M, Lincoln FA, Zhivotovsky B, Rehm M (2013) Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution. Biochim Biophys Acta 1833(10):2279–2292. doi: 10.1016/j.bbamcr.2013.05.025 CrossRefPubMedGoogle Scholar
- 26.Laussmann MA, Passante E, Hellwig CT, Tomiczek B, Flanagan L, Prehn JH, Huber HJ, Rehm M (2012) Proteasome inhibition can impair caspase-8 activation upon submaximal stimulation of apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) signaling. J Biol Chem 287(18):14402–14411. doi: 10.1074/jbc.M111.304378 CrossRefPubMedCentralPubMedGoogle Scholar
- 28.Webb JL (1963) Effect of more than one inhibitor. In: Enzymes and metabolic inhibitors, vol 1. Academic, New York, pp 66–79Google Scholar