A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.
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.
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.
Miho
M
Suzuki
Department of Regulatory Biology, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
Ichiro
I
Sakata
Department of Regulatory Biology, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
Takafumi
T
Sakai
Department of Science Education, Graduate School of Education, Saitama University, Saitama 338-8570, Japan.
Hiroaki
H
Tomioka
Department of Functional Materials and Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
Koichi
K
Nishigaki
Institut de Génétique et Développement de Rennes, UMR 6290, CNRS, Université Rennes 1, 35065 Rennes, France.
Marc
M
Tramier
Institut Jacques Monod, Complexes Macromoléculaires en Cellules Vivantes, UMR 6290, CNRS, Université Paris Diderot, 75013 Paris, France.
Maïté
M
Coppey-Moisan
D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000032:analysis* / Q000737:chemistry; D053453:Caspase 9 / Q000032:analysis* / Q000737:chemistry; D003513:Cycloheximide / Q000633:toxicity; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D005456:Fluorescent Dyes; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D053148:Caspase 3; D053453:Caspase 9
Apoptosis; FRET; Flow cytometry; Fluorescence lifetime; High-throughput assay