A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements
| Autor: | Ichiro Sakata, Marc Tramier, Maïté Coppey-Moisan, Koichi Nishigaki, Miho Suzuki, Hiroaki Tomioka, Takafumi Sakai |
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| Rok vydání: | 2015 |
| Předmět: |
Proteases
Green Fluorescent Proteins Biophysics Apoptosis Biology 7. Clean energy Biochemistry Flow cytometry Green fluorescent protein 03 medical and health sciences 0302 clinical medicine Fluorescence Resonance Energy Transfer medicine Humans Cycloheximide Molecular Biology Fluorescent Dyes 030304 developmental biology 0303 health sciences medicine.diagnostic_test Caspase 3 Tumor Necrosis Factor-alpha Cell Biology Cell sorting Flow Cytometry Molecular biology Fluorescence Caspase 9 Förster resonance energy transfer 030220 oncology & carcinogenesis Cytometry HeLa Cells Plasmids |
| Zdroj: | Analytical Biochemistry. 491:10-17 |
| ISSN: | 0003-2697 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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