The Experts below are selected from a list of 99 Experts worldwide ranked by ideXlab platform
Michael L Johnson - One of the best experts on this subject based on the ideXlab platform.
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fluorescence lifetime imaging of intracellular calcium in cos cells using Quin 2
Cell Calcium, 1994Co-Authors: Joseph R Lakowicz, Henryk Szmacinski, Kazimierz Nowaczyk, W J Lederer, M S Kirby, Michael L JohnsonAbstract:We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca2+ in COS cells based on the Ca(2+)-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-radiometric probes and appears to provide new opportunities for chemical imaging of cells.
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fluorescence lifetime imaging of calcium using Quin 2
Cell Calcium, 1992Co-Authors: Joseph R Lakowicz, Henryk Szmacinski, Kazimierz Nowaczyk, Michael L JohnsonAbstract:We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.
H.k. Breddin - One of the best experts on this subject based on the ideXlab platform.
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Influence of Quin 2, fura 2 and indo 1 on platelet function and on the measurement of cytosolic free calcium ions.
Platelets, 1991Co-Authors: O.k. Bellinger, C.m. Kirchmaier, A. Schirmer, H.k. BreddinAbstract:Two fluorescent calcium indicators, Fura 2 and Indo 1 were compared with Quin 2 for the measurement of cytosolic free calcium concentrations (Ca2+)1 in intact platelets. The free calcium ions were quantitatively evaluated using a single excitation wavelength fluorimeter before and after stimulation with the Ca2+ ionophores ionomycin and A23187, platelet activating factor (PAF) and arachidonic acid (AA). Using Quin 2 no rise in (Ca2+)i could be registered by stimulation with the weak agonists PAF and AA in subthreshold concentrations for aggregation in contrast to Indo 1 and, to a lesser extent, Fura 2. Stimulation with ionomycin and A23187 led to a significant rise in (Ca2+)i detected by all three indicators. The reproducibility was best for Indo 1. The three fluorescence indicators differed in their influence on platelet function and in their physical properties. Higher fluorescence intensities for Fura 2, and especially Indo 1, allowed a much lower indicator concentration of about 2–10% with a 10-fold i...
D J Reed - One of the best experts on this subject based on the ideXlab platform.
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involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes
Journal of Pharmacology and Experimental Therapeutics, 1991Co-Authors: L Carpenterdeyo, D J ReedAbstract:When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by Quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and Quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.
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toxicity to isolated hepatocytes caused by the intracellular calcium indicator Quin 2
Journal of Pharmacology and Experimental Therapeutics, 1991Co-Authors: L Carpenterdeyo, J R Duimstra, O Hedstrom, D J ReedAbstract:To determine whether incubation for several hours with intracellular Ca++ indicators caused toxicity to freshly isolated hepatocytes from rats, cells were incubated under 95% O2-5% CO2 in medium containing 2 mM Ca++ and the acetoxymethyl (AM) esters of Quin 2, Indo 1, Fluo 3, 5,5'-Dimethyl BAPTA, 4,4'-Difluoro BAPTA or Fura 2 for up to 5 hr. Quin 2-AM and Indo 1-AM (2.5 microM) induced lipid peroxidation in the cells after 1 or 3 hr of treatment, respectively. Additional experiments with Quin 2-AM (25 microM) revealed that it also caused lactate dehydrogenase leakage, cell blebbing and vitamin E loss in cells, but did not affect reduced glutathione or intracellular Ca++ content. The ability of Quin 2-AM to cause toxicity was dependent on the amount of Quin 2 which was present in the cell. Ca++ appeared to be involved in the mechanism of Quin 2-AM toxicity, for modulation of the extracellular Ca++ concentration partially inhibited lipid peroxidation, vitamin E loss, cell blebbing and lactate dehydrogenase leakage.
Joseph R Lakowicz - One of the best experts on this subject based on the ideXlab platform.
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fluorescence lifetime imaging of intracellular calcium in cos cells using Quin 2
Cell Calcium, 1994Co-Authors: Joseph R Lakowicz, Henryk Szmacinski, Kazimierz Nowaczyk, W J Lederer, M S Kirby, Michael L JohnsonAbstract:We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca2+ in COS cells based on the Ca(2+)-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-radiometric probes and appears to provide new opportunities for chemical imaging of cells.
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fluorescence lifetime imaging of calcium using Quin 2
Cell Calcium, 1992Co-Authors: Joseph R Lakowicz, Henryk Szmacinski, Kazimierz Nowaczyk, Michael L JohnsonAbstract:We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.
O.k. Bellinger - One of the best experts on this subject based on the ideXlab platform.
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Influence of Quin 2, fura 2 and indo 1 on platelet function and on the measurement of cytosolic free calcium ions.
Platelets, 1991Co-Authors: O.k. Bellinger, C.m. Kirchmaier, A. Schirmer, H.k. BreddinAbstract:Two fluorescent calcium indicators, Fura 2 and Indo 1 were compared with Quin 2 for the measurement of cytosolic free calcium concentrations (Ca2+)1 in intact platelets. The free calcium ions were quantitatively evaluated using a single excitation wavelength fluorimeter before and after stimulation with the Ca2+ ionophores ionomycin and A23187, platelet activating factor (PAF) and arachidonic acid (AA). Using Quin 2 no rise in (Ca2+)i could be registered by stimulation with the weak agonists PAF and AA in subthreshold concentrations for aggregation in contrast to Indo 1 and, to a lesser extent, Fura 2. Stimulation with ionomycin and A23187 led to a significant rise in (Ca2+)i detected by all three indicators. The reproducibility was best for Indo 1. The three fluorescence indicators differed in their influence on platelet function and in their physical properties. Higher fluorescence intensities for Fura 2, and especially Indo 1, allowed a much lower indicator concentration of about 2–10% with a 10-fold i...
