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Biochimica Et Biophysica Acta Jul 2010Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular... (Review)
Review
Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used "gold standards" for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT-fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.
Topics: Amyloid; Animals; Benzothiazoles; Binding Sites; Fluorescent Dyes; Humans; Models, Chemical; Molecular Conformation; Molecular Probes; Protein Binding; Protein Conformation; Spectrophotometry; Thiazoles; Time Factors
PubMed: 20399286
DOI: 10.1016/j.bbapap.2010.04.001 -
Biochimica Et Biophysica Acta.... Jun 2018PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in...
PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.
Topics: Animals; Astrocytes; Centrifugation, Density Gradient; Exosomes; Female; Flow Cytometry; Fluorescent Dyes; Microscopy, Confocal; Nanoparticles; Organic Chemicals; Rats; Staining and Labeling; Ultracentrifugation
PubMed: 29551275
DOI: 10.1016/j.bbamem.2018.03.013 -
Cell Death & Disease Nov 2012Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with...
Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.
Topics: Animals; Apoptosis; Carbocyanines; Cells; Flow Cytometry; Fluorescent Dyes; HeLa Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria
PubMed: 23171850
DOI: 10.1038/cddis.2012.171 -
European Journal of Biochemistry Sep 2000We show here the identity of Alamar Blue as resazurin. The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of...
We show here the identity of Alamar Blue as resazurin. The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.
Topics: Animals; Cell Survival; Culture Media; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Male; Mass Spectrometry; Oxazines; Rats; Rats, Wistar; Xanthenes
PubMed: 10951200
DOI: 10.1046/j.1432-1327.2000.01606.x -
Journal of Visualized Experiments : JoVE Oct 2010Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division. CFSE covalently labels long-lived intracellular molecules...
Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.
Topics: Animals; Cell Division; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Lymphocytes; Mice; Succinimides
PubMed: 20972413
DOI: 10.3791/2259 -
Nature Methods Mar 2015Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to...
Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
Topics: Azetidines; Chemistry Techniques, Synthetic; Coumarins; Fluorescein; Fluorescent Dyes; HeLa Cells; Humans; Microscopy, Ultraviolet; Models, Molecular; Molecular Imaging; Quantum Theory; Rhodamines; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Structure-Activity Relationship
PubMed: 25599551
DOI: 10.1038/nmeth.3256 -
BioTechniques Nov 2017Advances in fluorescent dye and protein design have led to a wider color palette for microscopy. Nathan Blow explores how this happened and what is still to come.
Advances in fluorescent dye and protein design have led to a wider color palette for microscopy. Nathan Blow explores how this happened and what is still to come.
Topics: Animals; Biosensing Techniques; Cell Tracking; Chemistry Techniques, Synthetic; Fluorescent Dyes; Luminescent Proteins; Mice; Microscopy, Fluorescence; Models, Molecular; Optical Imaging; Phytochrome; Protein Engineering; Rhodamines; Spectroscopy, Near-Infrared
PubMed: 29185919
DOI: 10.2144/000114605 -
Journal of the American Chemical Society Sep 2019Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an...
Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Fluorescence; Fluorescent Dyes; Humans; Mercury; Viscosity
PubMed: 31441661
DOI: 10.1021/jacs.9b06450 -
Current Medicinal Chemistry 2019Self-assembled fluorescent nanomaterials based on small-molecule organic dyes are gaining increasing popularity in imaging and sensing applications over the past decade.... (Review)
Review
Self-assembled fluorescent nanomaterials based on small-molecule organic dyes are gaining increasing popularity in imaging and sensing applications over the past decade. This is primarily due to their ability to combine spectral properties tunability and biocompatibility of small molecule organic fluorophores with brightness, chemical and colloidal stability of inorganic materials. Such a unique combination of features comes with rich versatility of dye-based nanomaterials: from aggregates of small molecules to sophisticated core-shell nanoarchitectures involving hyperbranched polymers. Along with the ongoing discovery of new materials and better ways of their synthesis, it is very important to continue systematic studies of fundamental factors that regulate the key properties of fluorescent nanomaterials: their size, polydispersity, colloidal stability, chemical stability, absorption and emission maxima, biocompatibility, and interactions with biological interfaces. In this review, we focus on the systematic description of various types of organic fluorescent nanomaterials, approaches to their synthesis, and ways to optimize and control their characteristics. The discussion is built on examples from reports on recent advances in the design and applications of such materials. Conclusions made from this analysis allow a perspective on future development of fluorescent nanomaterials design for biomedical and related applications.
Topics: Fluorescent Dyes; Molecular Conformation; Nanostructures; Optical Imaging; Organic Chemicals; Small Molecule Libraries
PubMed: 29484973
DOI: 10.2174/0929867325666180226111716 -
RNA (New York, N.Y.) Dec 2018Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes....
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Topics: Blotting, Northern; DNA Probes; Fluorescent Dyes; Nucleic Acid Hybridization; RNA; RNA Probes
PubMed: 30201850
DOI: 10.1261/rna.068213.118