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• W2148535169 abstract "Altered intracellular Ca 2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca 2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca 2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca 2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca 2+ concentrations. This study describes methodology to directly measure intracellular Ca 2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca 2+ concentration standards. Titration of Ca 2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca 2+ concentrations. These standard plots can be used to convert transient Ca 2+ fluorescence obtained with experimental cells to Ca 2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca 2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca 2+ concentrations in live cells using Ca 2+ -chelating dyes exhibiting variable fluorescence intensity." @default.
• W2148535169 created "2016-06-24" @default.
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• W2148535169 date "2005-05-12" @default.
• W2148535169 modified "2023-10-17" @default.
• W2148535169 title "Impaired Intracellular Ca²⁺Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy" @default.
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• W2148535169 doi "https://doi.org/10.1017/s1431927605050488" @default.
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