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Influencing factors in the measurement of intracellular free calcium

One of the popular methods for measurement of intracellular free calcium ([Ca2+]i) is ratiometric fluorescence technique. Rmax and Rmin play an important role in the calibration of [Ca2+]i measurement. The purpose of this study was to analyze influencing factors in the [Ca2+]i measurement. Rmax and Rmin deduced from theory had a hyperbolic relation with the ratio of Cf and Cb (Cf: free indicator concentration; Cb: Ca2+-bound indicator concentration). In experiments, using radiometric fluorescence imaging system, they were measured separately in two types of cell lines in concentration gradient (2×105cells/ml, 5×105cells/ml, 1×106cells/ml and 2×106cells/ml). By Triton method, Rmax and Rmin of K562 cells were 1.21 and 0.20, respectively, and that of Hepa1-6 cells were 2.02 and 1. These results indicated that Rmax and Rmin were not influenced by concentration gradient in a certain range of cell concentration, but they were different in various types of cell lines.