Fluorescence spectrometry. A review

Parker, C. A.; Rees, W. T.

doi:10.1039/an9628700083

Cited by 192 publications

(94 citation statements)

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“…The reagents were added in a ratio of 5-20 ,umol of reagent per ml of packed red cells (1-4 moles of reagent per mole of hemoglobin). Usually [10][11][12][13][14][15][16][17][18][19][20] Aul of the stock solution was used per ml of cell suspension of 5-10% packed cell volume. The reagent solution was placed in a dry test tube and the-sample (cell suspension, hemoglobin solution) was added with rapid mixing.…”

Section: Methodsmentioning

confidence: 99%

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower¹,

Kosower²,

Newton³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

238

142

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The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials. syn-9,10-Dioxabimanes, a new class of compounds, are highly fluorescent (1). Exceptions to this generalization are the essentially nonfluorescent bromoderivatives, illustrated by a monobromobimane (mBBr) (I), a dibromobimane (bBBr) (II), and a monobromotrimethylammoniobimane (qBBr) (III). We have now found that these three bromobimanes are highly efficient labeling agents for cells, proteins, and small molecules like glutathione, giving rise to highly fluorescent derivatives. The fluorescent derivatives are very stable in air and under irradiation. The labeling procedures are simple and can be carried out rapidly under physiological conditions. We shall describe in the present article the use of these reagents on normal human red cells. MATERIALS AND METHODSBromobimanes. The synthesis of the bromobimanes is described elsewhere (1). mBBr and bBBr were dissolved in acetonitrile and the 50 mM stock solutions were stored in the dark; the solutions are stable for at least 2 months. The salt, qBBr, was dissolved in aqueous buffer (pH 7.4) shortly before use; the 1-4 mM solutions have a half-life at 220C of approximately 4 hr.Red Cells. Human blood, anticoagulated with heparin, was obtained from normal individuals. The buffy coat was removed after centrifugation; the red cells were washed twice with 135 mM NaCI/10 mM phosphate buffer, pH 7.4, and resuspended in the same buffer to a packed cell volume of 5-10%. Ghosts were obtained from the cells by the procedure of Steck and Kant (2). Hemoglobin solutions were prepared by the centrifugation of lysed red cells at 22,000 X g for 30 min, followed by dialysis of the membrane-free supernatants against 10 mM phosphate buffer, pH 7.4.Reactions of Bromobimanes with Intact Cells, Lysates, and Hemoglobin Solutions. The reagents were added in a ratio of 5-20 ,umol of reagent per ml of packed red cells (1-4 moles of reagent per mole of hemoglobin). Usually 10-20 Aul of the stock solution was used per ml of cell suspension of 5-10% packed cell volume. The reagent solution was placed in a dry test tube and the-sample (cell suspension, hemoglobin solution) was added with rapid mixing. A stock solution (50 mM) of the mBBr reagent could be diluted with buffer to 1-2 mM before it was mixed with a sample. The bBBr compound, which is less soluble in water, was not diluted. qBBr was dissolved in buffer immediately before use...

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Section: Methodsmentioning

confidence: 99%

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Kosower¹,

Kosower²,

Newton³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

238

142

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“…All photometric measurements were performed using a Varian DMS-90 spectrophotometer. [10], with f,-carboline as the standard [11].…”

Section: Protein and Ans Estimationsmentioning

confidence: 99%

Spectrofluorimetric assessment of the surface hydrophobicity of proteins

Cardamone

Puri²

1992

Biochemical Journal

462

305

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The equilibrium binding of the apolar fluorescent dye l-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard-and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as:Bacteriorhodopsin > BSA > ovalbumin > PST > lysozyme > RNAase

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“…At higher culture concentrations, the fluorescence curve displays non-linearity with increasing CFUs. This is most likely due to the inner filter effect, in which excitation light is depleted before penetrating the entire sample due to absorbance by highly concentrated chromophores [59]. This effect is exacerbated in this case due to the locally high concentration of the EGFP within the cells, and therefore, the samples would need to be diluted within the linear range for quantification.…”

Section: Discussionmentioning

confidence: 99%

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

et al. 2018

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A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP). The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD 600 ) and plate counting (colony-forming units (CFUs)). While both OD 600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

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Fluorescence spectrometry. A review

Cited by 192 publications

References 0 publications

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

Spectrofluorimetric assessment of the surface hydrophobicity of proteins

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

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