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Anal Biochem. 2015 Dec 15;491:10-7. doi: 10.1016/j.ab.2015.08.022. Epub 2015 Sep 1.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Author information

1
Department of Functional Materials and Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan. Electronic address: miho@fms.saitama-u.ac.jp.
2
Department of Regulatory Biology, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
3
Department of Science Education, Graduate School of Education, Saitama University, Saitama 338-8570, Japan.
4
Department of Functional Materials and Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
5
Institut de Génétique et Développement de Rennes, UMR 6290, CNRS, Université Rennes 1, 35065 Rennes, France.
6
Institut Jacques Monod, Complexes Macromoléculaires en Cellules Vivantes, UMR 6290, CNRS, Université Paris Diderot, 75013 Paris, France.

Abstract

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

KEYWORDS:

Apoptosis; FRET; Flow cytometry; Fluorescence lifetime; High-throughput assay

PMID:
26334608
DOI:
10.1016/j.ab.2015.08.022
[Indexed for MEDLINE]
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