ArticleTheranostics2012
FLIM-FRET Imaging of Caspase-3 Activity in Live Cells Using Pair of Red Fluorescent Proteins.
Article in Theranostics, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Visualizing the Nucleome Using the CRISPR-Cas9 System: From in vitro to in vivo.Biochemistry. Biokhimiia · 2023Review
- Reconstruction of fluorophore absorption and fluorescence lifetime using early photon mesoscopic fluorescence molecular tomography: a phantom study.Journal of biomedical optics · 2022Article
- Application of Genetically Encoded Photoconvertible Protein SAASoti for the Study of Enzyme Activity in a Single Live Cell by Fluorescence Correlation Microscopy.Materials (Basel, Switzerland) · 2022Article
- Impact of Doxorubicin on Cell-Substrate Topology.International journal of molecular sciences · 2022Article
- Fluorescence Microscopy-An Outline of Hardware, Biological Handling, and Fluorophore Considerations.Cells · 2021Review
- Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids.Scientific reports · 2018Article
- Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.Journal of biophotonics · 2016Article
- Eliminating caspase-7 and cathepsin B cross-reactivity on fluorogenic caspase-3 substrates.Molecular bioSystems · 2016Article
- Article
- Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity.International journal of molecular sciences · 2015Article
- Estimating orientation factors in the FRET theory of fluorescent proteins: the TagRFP-KFP pair and beyond.Biophysical journal · 2015Article
- Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.Proteins · 2014Article
- Single cell optical imaging and spectroscopy.Chemical reviews · 2013Review
- Longitudinal bioluminescence imaging of the dynamics of Doxorubicin induced apoptosis.Theranostics · 2013Article
- Protease activity: meeting its theranostic potential.Theranostics · 2012Article
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.