A high-throughput direct FRET-based assay for analysing apoptotic proteases using flow cytometry and fluorescence-lifetime measurements

Miho Suzuki (1) , Ichiro Sakata (2) , Takafumi Sakai (2) , Hiroaki Tomioka (3) , Koichi Nishigaki (1) , Marc Tramier (4) , Maïté Coppey-Moisan (5)

1 Department of Functional Materials and Science
2 Department of Regulatory Biology
3 Department of Science Education
4 IGDR - Institut de Génétique et Développement de Rennes
5 IJM (UMR_7592) - Institut Jacques Monod

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, since apoptotic inducers are potent anti-cancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tuneable 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.

Mots clés: FRET; Fluorescence lifetime; Apoptosis; Flow cytometry; High-throughput assay

Domaines: Sciences du Vivant [q-bio]; Biologie cellulaire

Miho Suzuki, Ichiro Sakata, Takafumi Sakai, Hiroaki Tomioka, Koichi Nishigaki, et al.. A high-throughput direct FRET-based assay for analysing apoptotic proteases using flow cytometry and fluorescence-lifetime measurements. Analytical Biochemistry, 2015, 491, pp.10-17. ⟨10.1016/j.ab.2015.08.022⟩. ⟨hal-01196602⟩