- 标题
- 摘要
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- 实验方案
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Green Fluorescent Protein-Based Glucose Indicators Report Glucose Dynamics in Living Cells
摘要： Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as "Green Glifons", to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50 μM, 600 μM and 4,000 μM), producing a ~7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 β cells. We propose that these indicators will facilitate and contribute to in vivo and multi-color imaging of energy metabolism.
关键词： biosensors,artificial sweeteners,dual-color imaging,C. elegans,live cell imaging,glucose,blood glucose level,fluorescent protein
更新于2025-11-21 11:24:58
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Measuring the interaction of transcription factor Nrf2 with its negative regulator Keap1 in single live cells by an improved FRET/FLIM analysis
摘要： Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. Detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualise and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured Instrument Response Function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
关键词： FRET,live cell imaging,fluorescence lifetime,FLIM,sfGFP,protein-protein interaction,global binning,Keap1,Instrument Response Function,Nrf2
更新于2025-11-21 11:08:12
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Luminescent rhenium(I) carbonyl complex with redox noninnocent ONS donor azo-phenol ligand: Synthesis, X-ray structure, photophysical properties and live cell imaging
摘要： Herein, we have synthesized a new fluorescent rhenium(I) carbonyl complex 1, bearing {Re(CO)3}+ core with ONS donor thioether containing azo-phenol redox noninnocent ligand. The distorted octahedral geometry of the complex is confirmed by single crystal X-ray diffraction method. Cyclic voltammogram in acetonitrile exhibits irreversible oxidation peak (Epa = 1.36 V) along with quasi-reversible reduction peak at E1/2 = -0.92 V (ΔE = 210 mV). The complex exhibits low energy emission band at 525 nm with high emission quantum yield (Φ = 0.115). Cytotoxicity of the complex is studied by MTT method with human breast cancer cell lines (MCF-7) and IC50 value is found to be 23.6 μM. In presence of the complex (10 μM) a bright green fluorescence image of MCF-7 cell lines is observed under fluorescence microscope.
关键词： Electrochemistry,ONS donor ligand,MTT assay,Rhenium(I) carbonyl complex,Live cell imaging
更新于2025-11-19 16:46:39
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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time
摘要： T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or in?ammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several ef?cient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.
关键词： Advanced microscopy,Cell tracking,Live cell imaging,T-cell migration
更新于2025-09-23 15:23:52
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[Methods in Molecular Biology] Autophagy Volume 1880 (Methods and Protocols) || Correlative Light and Electron Microscopy of Autophagosomes
摘要： Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.
关键词： Autophagosome,Phagophore,Electron tomography,Live-cell imaging,Serial sectioning
更新于2025-09-23 15:23:52
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A colorimetric and fluorometric oligothiophene-indenedione-based sensor for rapid and highly sensitive detection of cyanide in real samples and bioimaging in living cells
摘要： A new simple oligothiophene-indenedione-based sensor 3TI has been synthesized for the highly reactive and selective detection of cyanide anion (CN?) in 70% aqueous media. The sensor 3TI displays distinct colorimetric and fluorometric detection of CN? due to the addition of CN? to the electron-deficient indenedione-vinyl group of 3TI, which hampers intramolecular charge transfer (ICT) efficiencies. The recognition mechanism of 3TI for CN? was confirmed by the optical measurements, 1H NMR titration, FTIR spectra, HRMS analysis, and TD-DFT calculations. The sensor 3TI for CN? shows the outstanding advantages of high fluorescence brightness, fast response time (30 s), low detection limit (31.3 nM), minimal pH dependence in the physiologically relevant range, and excellent selectivity in presence of other competitive anions. Encouraged by these desirable sensing properties, the 3TI has been successfully used for determination of CN? in real water and food samples, silica-based sensing kits and fluorescence imaging in living cells with satisfactory results.
关键词： Fluorometric sensor,Cyanide,Oligothiophene-indenedione,Colorimetric sensor,Real sample,Live-cell imaging
更新于2025-09-23 15:23:52
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A genetically encoded FRET biosensor for visualizing EphA4 activity in different compartments of the plasma membrane
摘要： The EphA4 receptor tyrosine kinase is well known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on F?rster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensor can produce a robust and specific FRET response upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRET responses for the non-raft targeting biosensor than for the raft targeting biosensor, suggesting that stronger EphA4 activation may occur in non-raft regions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensor could serve as a powerful tool to visualize and investigate EphA4 activation and signaling in specific subcellular compartments of single live cells.
关键词： EphA4,Membrane microdomain,cytoskeleton,FRET biosensor,Live cell imaging
更新于2025-09-23 15:22:29
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European Microscopy Congress 2016: Proceedings || Germ-line cysts organisation in clitellate annelids - from electron microscopy to live cell imaging
摘要： (cid:42)(cid:72)(cid:85)(cid:80)(cid:16)(cid:79)(cid:76)(cid:81)(cid:72)(cid:3)(cid:70)(cid:92)(cid:86)(cid:87)(cid:86)(cid:3)(cid:82)(cid:85)(cid:74)(cid:68)(cid:81)(cid:76)(cid:86)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:76)(cid:81)(cid:3)(cid:70)(cid:79)(cid:76)(cid:87)(cid:72)(cid:79)(cid:79)(cid:68)(cid:87)(cid:72)(cid:3)(cid:68)(cid:81)(cid:81)(cid:72)(cid:79)(cid:76)(cid:71)(cid:86)(cid:3)(cid:98)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)(cid:72)(cid:79)(cid:72)(cid:70)(cid:87)(cid:85)(cid:82)(cid:81)(cid:80)(cid:76)(cid:70)(cid:85)(cid:82)(cid:86)(cid:70)(cid:82)(cid:83)(cid:92)(cid:3)(cid:87)(cid:82)(cid:3)(cid:79)(cid:76)(cid:89)(cid:72)(cid:3)(cid:70)(cid:72)(cid:79)(cid:79)(cid:3)(cid:76)(cid:80)(cid:68)(cid:74)(cid:76)(cid:81)(cid:74)
关键词： live cell imaging,microcopy,germ-line cells
更新于2025-09-23 15:22:29
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An ESIPT-based fluorescent probe for Hg2+ in aqueous solution and its application in live-cell imaging
摘要： A new Excited-State Intramolecular Proton Transfer (ESIPT) based fluorescent probe for the detection of Hg2+ has been rationally designed and developed. Based on the specific reactivity of mercury-promoted hydrolysis, the probe exhibits high selectivity and sensitivity for mercury ions in almost pure aqueous solution (containing only 1% DMSO) with a low detection limit of 1.9 ppb. Furthermore, the probe was also successfully used for fluorescence imaging of Hg2+ in live cells.
关键词： Fluorescent probe,ESIPT-based,Live-cell imaging,Mercury ions,3-Hydroxyphthalimide
更新于2025-09-23 15:22:29
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A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells
摘要： Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens.
关键词： chromobodies,live-cell imaging,cellular models,compound screening,nanobodies
更新于2025-09-23 15:22:29