Skip to Main content Skip to Navigation
Journal articles

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

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, 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.
Document type :
Journal articles
Complete list of metadatas

Cited literature [48 references]  Display  Hide  Download

https://hal-univ-rennes1.archives-ouvertes.fr/hal-01196602
Contributor : Laurent Jonchère <>
Submitted on : Wednesday, December 2, 2015 - 2:41:32 PM
Last modification on : Saturday, April 11, 2020 - 2:01:45 AM
Long-term archiving on: : Thursday, March 3, 2016 - 1:10:33 PM

File

A high-throughput direct FRET-...
Files produced by the author(s)

Identifiers

Citation

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, Elsevier Masson, 2015, 491, pp.10-17. ⟨10.1016/j.ab.2015.08.022⟩. ⟨hal-01196602⟩

Share

Metrics

Record views

626

Files downloads

1112