Search results for "Fluorescence"
showing 10 items of 2463 documents
Nondestructive fluorescence-based quantification of threose-induced collagen cross-linking in bovine articular cartilage.
2012
Extensive collagen cross-linking affects the mechanical competence of articular cartilage: it can make the cartilage stiffer and more brittle. The concentrations of the best known cross-links, pyridinoline and pentosidine, can be accurately determined by destructive high-performance liquid chromatography (HPLC). We explore a nondestructive evaluation of cross-linking by using the intrinsic fluorescence of the intact cartilage. Articular cartilage samples from bovine knee joints were incubated in threose solution for 40 and 100 h to increase the collagen cross-linking. Control samples without threose were also prepared. Excitation-emission matrices at wavelengths of 220 to 950 nm were acquir…
Direct determination of intracellular daunorubicin in intact confluent monolayers of AT1 prostate carcinoma cells using a multiwell–multilabel counter
2008
The cytostatic drug daunorubicin exerts its toxic action by intercalating into the DNA. The efficacy of daunorubicin depends on the intracellular amount in the tumor cell. Here we have evaluated the use of a multiwell-multilabel reader for the direct determination of the fluorescent cytostatic drug daunorubicin in a prostate carcinoma cell line (AT1 R-3327 Dunning prostate carcinoma cells) grown on 24-well plates. We present evidence that this simple fluorescent parameter is a good measure for the toxicologically relevant amount of the drug intercalated into the DNA and, therefore, is a good predictor for the drug's cytotoxicity. The amount of cationic cytostatics in a tumor cell is primari…
Staphylococcal alpha-toxin: repair of a calcium-impermeable pore in the target cell membrane
2000
Staphylococcal alpha-toxin forms heptameric pores that render membranes permeable for monovalent cations. The pore is formed by an amphipathic beta-barrel encompassing amino acid residues 118-140 of each subunit of the oligomer. Human fibroblasts are susceptible to alpha-toxin but are able to repair the membrane lesions. Thereby, toxin oligomers remain embedded in the plasma membrane and exposed to the extracellular medium. In this study, we sought to detect structural changes occurring in the pore-forming sequence during lesion repair. Single cysteine substitution mutants were labelled with the environmentally sensitive fluorochrome acrylodan and, after mixing with wild-type toxin, incorpo…
Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: A guide for prokaryotic and eukaryotic investigation
2008
International audience; Many fluorescent techniques are employed to evaluate the viability and activity of microbial cells used in biotechnology. These techniques are sometimes complex and the interpretation of results opened to misunderstanding. Moreover, new developments are constantly proposed especially concerning a more accurate evaluation of the state of the cells including eukaryotic microorganisms. This paper aims at presenting to biotechnologists unfamiliar with fluorescence the principles of these methods and the related possible pitfalls. It focuses on probes of the physical (integrity and fluidity) and energetical (intracellular pH and membrane potential) state of the cell membr…
Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature
2007
ABSTRACT The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the c…
Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles
1996
The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and ca…
Live cell imaging of duplex siRNA intracellular trafficking.
2015
Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging …
High-content imaging technology for the evaluation of drug-induced steatosis using a multiparametric cell-based assay.
2012
In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver ste…
Unraveling In vivo brain transport of protein‐coated fluorescent nanodiamonds
2019
The blood–brain barrier is the biggest hurdle to overcome for the treatment of neurological disorders. Here, protein‐coated nanodiamonds are delivered to the brain and taken up by neurovascular unit cells after intravenous injection. Thus, for the first time, nanodiamonds with their unique properties and a flexible protein coating for the attachment of therapeutics emerge as a potential platform for nanotheranostics of neurological disorders.Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood–brain barrier (BBB) preventing systemic…
Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes
2021
Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn2+ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn2+ metal complexes with duple…