title: Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry.
abstract: Sensing systems based on Förster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein-protein interactions, changes in conformation, and Ca2+ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.
authors.affiliation: Department of Functional Materials and Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. miho@fms.saitama-u.ac.jp
authors.forename: Miho
authors.initials: M
authors.lastname: Suzuki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tanaka
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
authors.forename: Makiko
authors.initials: M
authors.lastname: Inoue
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sakai
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
mesh_terms: D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism*; D003513:Cycloheximide / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D053148:Caspase 3