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  • Title: Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2.
    Author: Xu T, Yang W, Huo XL, Song T.
    Journal: J Biochem Biophys Methods; 2004 Mar 31; 58(3):219-26. PubMed ID: 15026208.
    Abstract:
    We compared two commonly used calibration methods for measuring the concentration of intracellular free calcium ([Ca2+]i) by ratiometric fluorescence dye, fura-2 in mouse neuroblastoma-rat glioma hybrid cells (NG108-15). One calibration method, the Triton method, employs detergent Triton X-100, while the other, the Ionomycin method, uses a calcium-specific ionophore, Ionomycin. In the Triton method, we observed that at excitation 380 nm, the fura-2 fluorescence intensity of steady-state cells abnormally situated beyond the limiting intensity for calibration. By excitation scan, we demonstrated that this abnormality was caused by the change of fura-2 isosbestic points, which in turn was due to cell lysis after the addition of Triton X-100. This problem was resolved in the Ionomycin method by avoidance of cell lysis. Our results showed the correlation between inconsistent isosbestic points and cell lysis. As the basis for [Ca2+]i calibration, the proportionality between the fluorescence intensity and the concentration of dye species was impaired because of inconsistent isosbestic points. This inconsistency can be eliminated by a preliminary experiment of excitation scan to test the feasibility of different calibration methods.
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