Fluoresceins diacetate

B. Lundgren, Fluorescein diacetate as a stain of metabolically active bacteria in soil, Oikos 56 17 (1981). 

The determination of intercellular pH is based on measurement of fluorescence emitted by the fluorescence indicator, fluorescein. The starting compound used for this purpose is fluorescein diacetate ... 

Measurements other than respiration rate can also be used as indicators of soil microbial activity. These include measurements of the rate of multienzyme processes such as arginine ammonification rate (Alef and Kleiner 1995) fluorescein diacetate (FDA) hydrolysis rate (Alef 1995) and measurement of key endocellular enzymes such as dehydrogenase (Tabatabai 1994). 

Alef K (1995) Estimation of the hydrolysis of fluorescein diacetate. In Alef K, Nannipieri P (eds) Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, pp 232-238... 

Enzyme activity (urease, amidase, dehydrogenase, pl-glucosidase, phosphatase, arylsulfatase, fluorescein diacetate hydrolysis) Laboratory incubation Indicates potential microbial activity and nutrient cycling reactions determined in nonstandard laboratory with specialized equipment highly spatially and temporally variable dependent upon organic inputs Dick et al. (1996) Parham et aL (2002)... 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

Determined on the basis of loss of cell membrane permeability in response to a deliberate modification in culture conditions. Cell viability can be determined by either neutral red or fluorescein diacetate retention assays. Volume 1(14,15). 

Peng, X.Y., Li, P.C.H., A three-dimensional flow control concept for single-cell experiments on a microchip. 2. Fluorescein diacetate metabolism and calcium mobilization in a single yeast cell as stimulated by glucose and pH changes. Anal. Chem. 2004, 76, 5282-5292. 

Breeuwer, P., Drocourt, J. L, Bunschoten, N., Zwietering, M. H., Rombouts, F. M. and Abee, T. Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluores-... 

The fate of nanoparticles has also been investigated in vivo. Albrecht et al. studied the in vivo mucoadhesive properties of thiomer formulations using magnetic resonance imaging and fluorescence detection (Albrecht et al. 2006). Following the hypothesis that unhydrated thiomers provide better mucoadhesion in vivo the group developed polycarbophil-cysteine microparticles loaded with fluoresceine diacetate (FDA) in Eutex capsules to maintain them in the dry... 

Fig. 9.1 Mucoadhesion studies amount of FDA remaining on the intestinal mucosa when applying fluoresceine diacetate (FDA) alone (grey bars), incorporated into chitosan nanoparticles (white bars) and into thiolated chitosan nanoparticles (black bars). Adapted from Bernkop-Schnurch et al. (2006)...

More recent staining procedures largely use fluorescent dyes to characterize the physiological and biochemical states of cells. Fluorescein Diacetate (FDA), a non-polar substance which crosses the membrane and is hydrolyzed by intracellular esterases in viable cells to produce fluorescein, exhibits yellow-green fluorescence when excited at 490 nm. Damaged or non-viable cells in general are unable to hydrolyze FDA or to retain fluorescein within the cell [172,173]. In combination with Ethidium Homodimer or Propidium Iodide, a similar esterase substrate, calcein acetoxy methyl ester (CAM) has been found to be reliable for viability assessment of protozoans, but not on Candida yeast, neither on bacteria such as Bacillus cereus and Escherichia coli [174]. 

Figure 17.7 S-91 cells stained with fluorescein diacetate and propidium iodide (left) the control and (right) cells irradiated with visible light (X> 455 nm, 15 min) in the presence of [Ti02]-0-PtCI4(H20) (SOmglr1). Creen-alive cells, red-dead cells...

Jochem, F. J. (1999). Dark survival strategies in marine phytoplankton assessed by cytometric measurement of metabolic activity with fluorescein diacetate. Mar. Biol. (Berl.) 135, 721—728. 

Schnurer, J., and RosswaU, T. (1982). Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. Appl. Environ. Microbiol. 43, 1256—1261. 

Fluorescence microphotolysis can be used to measure diffusion in single cellsand nuclear envelope permeability. Pyranine probes have been used to measure internal pH changes in Escherichia coli membrane vesicles. Probe measurements of intralysosomal pH in living cells and perturbation of pH has been described by Ohkuma and Poole.Fluorescence polarization of six membrane probes in embryonal carcinoma cells have been measured by a cell sorter,Fluorescence of pancreatic islets labelled with fluorescein diacetate has been used to show the effects of cations, ionophores, and hypoglycaemic sulphonylureas. ... 

Permeabilization with carboxyfluorescein-diacetate. 6-carboxy-fluorescein-diacetate (CFDA) is a nonfluorescent, nonpolar reagent which enters the cell freely, where it becomes entrapped following enzymatic conversion to the hydrophilic fluorophore 6-carboxyfluorescein (CF, mw 370). Thus, labeled cells can then be replated onto unlabeled test cells to measure dye transfer via gap junction. This method has been successfully used to measure gap junction communication in early embryo cells (Goodall and Johnson, 1982 Kidder et al., 1987), and also to label tumor cells in culture (Price et al., 1995). We tried to extrapolate it for detection of gap junction formation among cultured cells. 

Epifluorescent techniques use fluorescent dyes that either exhibit different colours in living and dead cells (e.g. acridine orange) or appear colourless outside the cell but become fluorescent when absorbed and subjected to cellular metabolism (e.g. fluorescein diacetate). 

EPTC and Butvlate Fluorescein Diacetate Assay. Spectrophotometric determinations of the hydrolysis of fluorescein diacetate have been shown to be simple, rapid, and sensitive methods for determining microbial activity in soil (18). Essentially, the hydrolytic cleavage of diacetate from fluorescein is responsible for the reaction products including fluorescein, which may be detected spectrophotometrically at 490 nm. This method is somewhat nonspecific in that it is indicative of overall activity of several enzymes (protease, lipase, esterase) rather than of a specific class of enzymes. Enzyme activity may be influenced by subtle pH changes in the sample since abiotic hydrolysis of fluorescein diacetate may occur. Also, an associated lag phase in soil hydrolytic activity must be accounted for in each assay. 

Reed et al. (.19.) adapted the fLuorescein diacetate assay to qualitatively measure the potential ability of soil microorganisms to degrade carbamothioate herbicides in enhanced soils. They... 

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